Advances in Immunology Volume 48

ADVANCES IN

Immunology EDITED BY FRANK J. DIXON Scripps Clinic and Research Foundation La Jolla, California

ASSOCIATE EDITORS

K. FRANKAUSTEN LEROYE. HOOD JONATHAN W. UHR

VOLUME 48

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishes San Diego New York Boston London Sydney Tokyo Toronto

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ADVANCES I N IMMUNOLOGY, VOL. 48

Internal Movements in Immunoglobulin Molecules ROALD NEZLIN Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

I. Introduction

Protein molecules are dynamic structures, exhibiting a variety of internal motions, ranging from atom fluctuations and oscillations of amino acid side groups to the movement of large portions of the molecules, such as their domains and subunits (Karplus and McCammon, 1983). This mobility has important functional implications; for example, large scale motion is significant for the regulation of catalytic activity or for the independent movement of subunits, allowing them to react with different ligands (Huber and Bennett, 1983). The latter motion is especially important in the case of multifunctional molecules, in which each type of activity links with a particular subunit. Immunoglobulins (Ig’s)are among the most thoroughly studied multifunctional protein molecules. They unite the recognition functionnamely, the ability to form complexes with a vast array of antigen molecules-with effector functions, such as complement activation and the capacity to react with different cell receptors. The functional sites responsible for all of these reactions are located in various domains, linked together with greater or lesser, flexible segments of peptide chains. T h e hypothesis that Ig’s are flexible molecules was proposed about 25 years ago, based on experiments performed using classical hydrodynamic methods (Noelken et al., 1965) and by electron-microscopic observations (Feinstein and Rowe, 1965; Valentine and Green, 1967). Since these are indirect techniques for the study of molecular dynamics, and considering the novelty of the ideas at that time, tribute should be paid to these scientists. In the years following, a complete arsenal of physicochemical methods was implemented to study the different types of internal motion in Ig molecules. Each new approach o r modification of classical methodology was used by different groups at once, and over the last few decades interest in the problem has remained at a constantly high level. As a result, many new concepts and discoveries in the field of immunology 1 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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have been applied by investigators interested in Ig flexibility. When myeloma proteins were recognized as homogeneous Ig’s, they were widely implemented in experimentation in this field. Later, monoclonal antibodies were successfully used, in particular, those against different reporter groups, such as fluorescent dyes or spin labels. Most recently, due to the enormous progress in genetic engineering, artificially constructed chimeric antibodies have become the object of investigations of the structural basis for flexibility and the functional importance of the different modes of subunit movements. T h e purpose of this chapter is to summarize the various studies of the movement of Ig subunits (i.e., segmental flexibility) as well as internal motion in the subunits themselves, and to discuss the functional implications of these motions. II. Methods for the Study of Ig Flexibility

A. HYDRODYNAMIC METHODS Classic hydrodynamic methods-such as sedimentation analysis, determination of the frictional coefficient ratio, and viscosity measurements-provide valuable information regarding the compactness of protein molecules. If the sedimentation coefficients and the molecular weights are known, it then becomes possible to calculate the frictional coefficient ratio (f :fmin),which characterizes both hydration and shape. For typical globular proteins the values of the ratio are 1.10- 1.25. Larger values may signal molecular asymmetry, as well as the possibility that certain regions of the polypeptide chains are flexible. Similarly, the viscosity data provide information which helps to reveal whether the investigated molecule is close to typical globular proteins. If the isolated subunits of a protein molecule resemble globular proteins, but the whole molecule does not, we can assume that the structure of the latter is not compact (Noelken et al., 1965). B. ELECTRON MICROSCOPY Contemporary electron microscopy enables recognition of the finest structural details of protein molecules (Green, 1969). Despite the fact that this technique is limited to the study of dried and fixed protein preparations, valuable information on the dynamic structure of protein molecules has been gained. For example, if we are able to alter the arrangement of some part of a molecule, we may assume that this molecule does not have a rigid, but has a mobile, conformation. Indeed, it was found that, after reaction with antigens, antibody configuration can

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change significantly and electron micrographs clearly differentiate the changes in the relative positions of antibody subunits (Feinstein and Rowe, 1965; Valentine and Green, 1967). C. X-RAYCRYSTALLOGRAPHY The most powerful method of obtaining high-resolution structural pictures of protein molecules is by X-ray crystallography, which enables us to determine the average position of atoms in a protein crystal. In addition, important information regarding the dynamic properties of protein molecules can also be derived from X-ray diffraction experiments (Bennett and Huber, 1984). First, if one or another part of the protein molecule is disordered-that is, it has a poorly defined electron density-we can assume that the corresponding subunit has some freedom of rotation and that the molecule under investigation is flexible. However, this assumption is correct only if the observed variation in electron density is unrelated to methodological problems, for example, peculiarities in crystal packing. Second, in some cases a protein can form different, but closely related, types of crystals. In each instance the molecule has a different general conformation-for example, a variation in the angle between subunits. Such an observation may be, primarily, the result of relatively free mobility of the subunits, which permits them to accommodate different positions during crystal formation. Third, the lack of close contact between subunits provides further indirect evidence that they could have greater or lesser freedom of rotation. Finally, difficulties in generating the crystallization of a protein molecule can also provide evidence of its flexible structure. D. FLUORESCENCE POLARIZATION Fluorescence polarization, introduced by Weber (1953), is the most direct technique for investigating the rotational properties of protein molecules. Initially, a protein molecule is labeled using a fluorescent dye, either covalently or specifically, by combination with the combining site of the corresponding antibody. A solution of labeled protein is then excited by vertically polarized light, which results in a set of excited dye molecules becoming arranged primarily in the vertical plane. Due to Brownian thermal motion, the initial orientation of the excited dye molecules becomes randomized. Depolarization of the emitted light gives information regarding the size and shape of the macromolecule, as well as its flexibility. The rotational correlation time (4 or T ) , ~which can be calculated from such experiments, provides information about the rota-

' + is used in the fluorescencepolarizationexperiments;

T,

in the spin-label experiments.

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KOALD NEZLIN

tional properties of the macromolecule under investigation. If the experimental values of the rotational relaxation time are significantly lower than those calculated, assuming the rigidity of the macromolecule, the macromolecule is flexible and its separate parts are capable of more or less independent motion. Two types of fluorescent depolarization measurements have been used: steady state (Weber, 1953) and nanosecond, o r time resolved (Stryer, 1968; Yguerabide, 1972). Reliable data on the rotational correlation times can be obtained using the steady-state approach if the following considerations are taken into account: First, to calculate the rotational correlation time, it is necessary to estimate the values of the fluorescence lifetime of the bound dye molecule. This parameter has to be measured for each labeled protein, since there are significant differences for the various conjugates. If the macromolecule has several rotational correlation times related to the different types of motion, then in the experiments using short-lived dyes, the higher values of the rotational correlation time could be not determined. Second, fluorescence depolarization could not be assumed to be due solely to Brownian motion of the macromolecule, but could also be due to independent thermally activated rotation of the covalently linked dye molecule. This problem, however, can be partially overcome, and satisfactory results have been obtained. For example, values of the rotational correlation time found for the Fab fragment in experiments with a covalently bound label, o r with a label firmly bound in the antibody combining site, coincide well (Tumerman et al., 1972a,b).The steady-state method, however, has some limitations: The method is limited to determination of the averaged rotational correlation time only. Further, in order to determine the rotational correlation time by this technique, it is necessary to perform a series of measurements, in which increasing amounts of sucrose are added to the protein solution, to extrapolate the average anisotropy to infinite viscosity. The nanosecond pulse technique permits direct measurement of the rotational correlation time from a single experiment. The timedependent anisotropy curves for whole Ig molecules obtained in these experiments are curved, showing the presence of two main depolarizing motions with two correlation times. In initial experiments performed using the nanosecond fluorescence technique (Yguerabide et al., 1970), the dansyl dye was firmly bound in the combining sites of antidansyl antibodies; thus, the independent movement of the dye molecules was prevented. This method was later refined, and previously obtained data were reinterpreted (Hanson et al., 1981). Further progress was made by using homogeneous monoclonal anti-dansyl antibodies, which eliminates the problem of random orientation of the dye molecules within the

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combining sites when heterogeneous antibodies are used; however, antidye antibodies cannot be used in all experiments. Another problem is linked with the difficulty of interpreting the results obtained in real physical terms. E. SPIN-LABEL APPROACH The spin-label method introduced by McConnell was used relatively recently to determine the rotational relaxation time. This method is based on the fact that the distance between the outer wide extrema of electron spin resonance (ESR) spectra is related quantitatively to the rotational correlation motion. Shimshick and McConnell(l972) used this dependence when the spin label has no motional freedom relative to the macromolecule. Initially, the theoretical dependence of the shift of outer wide extrema (2A’) as a function of the rotational correlation time is determined; then the shift of outer wide extrema as a function of viscosity is determined experimentally and extrapolated to infinite viscosity. The difference between the position of extrema for a solution of infinite viscosity and that for an aqueous solution is calculared and used to determine the value of the rotational correlation time from the theoretically calculated dependence. If the spin label is able to rotate relatively independently of the macromolecule, it would be possible to find conditions (e.g., by increasing the viscosity by adding sucrose) in which the macromolecule is immobolized completely, while the label is still mobile. With increasing viscosity, the rotation of the spin label would eventually cease. Experimentally, the dependence of 2A’ versus the temperature : viscosity ratio has to be plotted (an isotherm). The linear part of such an isotherm, which characterizes the hindrance of macromolecular rotation, is extrapolated to infinite viscosity and the value found is used to calculate the rotational correlation time, as in the previous case. The values of the rotational correlation time obtained using this method are in good accordance with the data obtained using other techniques, particularly those by fluorescence polarization. 111. Segmental Flexibility of Ig Molecules

A. IgG 1. Early Studies

One of the important findings of classical studies by R. R. Porter on the papain fragmentation of IgC was that Fab and Fc fragments retain their principal biological activities. This observation could only be possible if

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the general structural features of the isolated fragments were preserved during papain cleavage. The hydrodynamic studies by Noelken et al. (1965) supported this notion. It was found that the papain Fab and Fc fragments of IgG behave ’ as typical globular proteins: Their frictional coefficient ratios at f : f M l N are 1.10-1.25 and their intrinsic viscosity is 4.0 ml/g. In the same experiments, however, whole IgG appears to be a more extended molecule (f : fMIN of 1.47, intrinsic viscosity of 6.0 ml/g). Based on these observations, Noelken et al. formulated the hypothesis that the IgG molecule is built from three compact subunits resembling papain fragments, linked by flexible parts of the heavy peptide chains. At about the same time the results of two electron-microscopic studies were published which also demonstrated the free movements of the Fab subunits. In both investigations the complexes of IgG antibodies with antigens were studied using the negative contrast method. Feinstein and Rowe (1965) found two main types of antibody complexes by using ferritin, a molecule which has many identical epitopes. In soluble complexes antibody molecules bind by combining sites to nearby epitopes and have a compact structure. But after cross-linking of two antigen molecules, these researchers noted that ‘the antibody molecule “clicks open” to varying degrees about a “hinge point”.’ Valentine and Green (1967), in their elegant studies on antibodies complexed with a small bivalent hapten, also found that the angle between antibody subunits bearing the combining site varies from nearly 0” in antibody dimers to 180” in ring complexes built from several antibody molecules. These observations form the experimental basis for a flexible model of the IgG molecule, according to which antibody arms could rotate more o r less freely and independently, due to the existence of a flexible hinge region, a stretch of heavy chain between Fab and Fc. T h e methods used in these studies, however, do not provide unequivocal evidence that protein subunits rotate freely in solution. The most direct ways to prove unambiguously that a protein molecule is constructed from independently rotating subunits is by measuring the rotational correlation time, using relaxation methods such as fluorescence polarization or the spinlabel method, and comparing the data obtained with the computed values for rigid models.

2 . Fluorescence Polarization Studies Determination of the rotational correlation time of Ig molecules by using measurements of fluorescence polarization of dye-protein conjugates has a long history. For nearly 30 years this approach has been used in many laboratories, and the quality of the results was correlated with the

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7

progressive development of the methodology. In early studies (reviewed by Dorrington and Tanford, 1970; Cathou et al., 1974) the steady-state method was used, which enables the determination of an average rotational correlation time, but the contradictory results obtained from different experiments were due to some methodological problems (see Section 11,D). In 1969 Zagyansky st al. performed experiments with dansyl-human IgC conjugates, trying to avoid possible methodological errors. First, the values of the lifetime of the excited state of conjugated dansyl groups were measured directly by phase fluorometry. This parameter was necessary to calculate the rotational correlation time. The values obtained (about 7.3 nsec) were significantly lower than the previously used values of the lifetime of the excited state of dansyl-albumin conjugates (i.e., 12 nsec). Also, immediately prior to commencing polarization measurements, the conjugates were freed, using gel filtration, from aggregates which are always present in Ig solutions and which could lead to overestimation of the values obtained. The results of this study were clear-cut: The experimentally found rotational relaxation time for IgC was several times lower (i.e., 20 nsec) than that calculated on the assumption that the IgC molecule is rigid (about 70 nsec) (Table I). This indicates that the observed polarization is determined by Brownian rotation not of the whole IgC molecule, but of its parts, which are substantially smaller, being interconnected by flexible bonds. In further experiments it was found that the correlation time for the Fab fragment is close to that in experiments with intact IgC (Nezlin et al., 1970). Due to the short lifetime of the excited state of the dansyl dye in these experiments, only Fab movements were registered, not the tumbling of the whole molecule. The contribution of thermally activated free rotation of the covalently bound dansyl groups was not very significant, since the values of the rotational correlation time for Fab were nearly the same, whether the dye molecule was fixed in the combining site of antidansyl Fab or was attached nonspecifically elsewhere (Tumerman et al., 1972a,b). It was later found that the values of the rotational correlation time for the whole IgC molecule could be slightly higher. Dudich et al. (1978) showed that this parameter for IgC from various species is concentration dependent: Determinations performed at protein concentrations below 2 phi give values about 20% lower than those from higher concentrations. This phenomenon is clearly dependent on Fc. It was found only with IgC and Fc, not with Fab or with complexes of dansyl with the specific antidansyl antibody. As was later established (Dudich and Dudich, 1983),the concentration dependence was due to the dissociation of

ROALD NE%I.IN

8

TABLE I ROTATIONAL CORRELATION TIMES OF IgCs A N D THEIR FRAGMENTS DETERMINED BY FLUORESCENCE POLARIZATION" Rotational correlation time (nsec), fluorescent dye localized Protein

Inside the Outside the combining site combining site

Steady-state fluorescent polarization (dansyl dye) 20-30 IgCl, human

IgG, rabbit Fab from human, rabbit, pig IgC Fab', F (ab'):! from human IgG Fc from human IgG IgC, rat antidansyl

27 21 20-22 12-16 33

Fab, Fab', rat 22 an tidansyl IgC, Pig. precipitating 41 anti-DNP IgG, pig 63 nonprecipitating anti-DNP Time-resolved fluorescence polarization' 4s 13.7-18.0 IgG, rabbit antidansyl 4' 82.7-105.1 IgC, rabbit 4s 24 antipyrene 4 L 131 Predicted global rotational correlation time IgG 75 nsec IpG 167 nsec

Reference Zagyansky et al. (1969), Dudich et al. (1978) Dudich et al. (1978) Nezlin el al. (1970), Dudich et al. (1978) Nezlin et al. (1970) Nezlin et al. (1970) Dudich et al. ( 1 978) Tumerman ct al. ( 1 972a,b) Tumerman et al. (1972a,b) Dudich et al. ( 1 978) Dudich et al. (1978)

Hanson et al. (1981) Hanson et al. ( 1 98 1) Lovejoy et al. (1977) Weltman and Edelman (1967) Hanson el al. (1981)

DNP, Dinitrophenyl. The short and long rotational correlation times characterizing two main depolarizing motions. a

small-molecular-weightpeptides also labeled by the dansyl dye. They usually adsorb on the Fc portion of the IgG molecule (not IgM or IgA) and dissociate after dilution. In 1970 Yguerabide et al. reported the results of direct measurements of fluorescence polarization of dansyl-antibody complexes using nanose-

INTERNAL MOVEMENTS IN

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cond fluorescence spectroscopy. According to the time-dependent anisotropy curves of the whole IgC antibody, there are two main depolarizing motions: one with a correlation time of 33 nsec; the other, 168 nsec. The shorter value was interpreted in terms of Fab segmental flexibility, and the longer time was attributed to rotation of the whole molecule. The dansyl-Fab complex had only a single correlation time equal to the shorter time value for the dansyl-IgG complex, indicating that this fragment rotates as a rigid unit. Later Chan and Cathou (1977) repeated these experiments and confirmed that the rotation of IgG does not correspond to that of a compact sphere. The values of the correlation times were smaller (i.e., 26 and 110 nsec), due to the removal of protein aggregates by gel filtration. The results were not significantly different when, instead of the short-lived dansyl groups, the long-lived flourophore (pyrene) was used (Lovejoy et al., 1977). This permits the study of depolarization up to 300 nsec, whereas, in experiments with dansyl groups, measurements of the correlation times were limited to only 150 nsec. Two correlation times were found for rabbit antipyrene antibodies: 24-33 nsec and 13 1- 140 nsec. Hanson et al. (198 l), using new methodological approaches, carefully reinvestigated the fluorescence polarization of antidansyl rabbit antibodies freed from aggregates. It was found that the decay of fluorescence anisotropy can also be described by two rotational correlation times. The values, however, were significantly lower: 14- 17 nsec and 83- 104 nsec. The authors reinterpreted the data by Yguerabide et al. (1970) and concluded that the long correlation time reflects wagging or wobbling of the Fab subunits, rather than the global tumbling of the entire IgG molecule. The short correlation time was ascribed to the movement of variable parts of Fab, or Fab's twisting around its long axis. Steady-state and time-resolved fluorescence polarizations were also used to study the soluble complexes of antidansyl antibodies with staphylococcal protein A (Hanson et al., 1985). Protein A is capable of forming complexes of different sizes, with IgG molecules binding to their Fc portion, If, in the free IgG molecule, the Fab portion has considerable freedom of rotation, the formation of protein A-IgG complexes should not influence the rotational freedom of Fab, and the correlation time has been found to change only insignificantly. It was found that, despite fixation in complexes, IgC molecules retain their segmental flexibility. This also implies that IgC antibodies anchored to cells by their Fc portions also retain freedom of rotation of their Fab fragments. The finding by Slattery et al. ( 1985) that the segmental flexibility of IgE molecules does not undergo significant change after binding to cell Fc, receptors is in agreement with the data by Hanson et al. (1985). All of the data discussed above provide unequivocal proof of the

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ROALD NEZLIN

existence of the segmental flexibility of IgG antibody molecules in solution. Indeed, the experimentally found rotational correlation times are lower than the correlation times calculated for a rigid sphere of hydrodynamically equivalent dimensions: 75 nsec, according to one method of calculation, and 155- 167 nsec to another (Hanson et al., 1981; Slattery et al., 1985). There is no obvious energy restriction for rotation of the Fab arms from the fully opened position at an angle of 180" and a closed position of less than 60" (Schumaker et al., 1980). The lack of energy constraints allows both Fab arms to move relatively freely to wide angles, and this of course has crucial functional significance. However, it is difficult to discuss the real physical meaning of the short and long rotational correlation times obtained using the nanosecond polarization technique. Due to methodological problems, the emission anisotropy kinetics of these experiments were usually fitted to the sum of two exponentials. However, the rotational motions of nonspherical particles are complex, and to describe them properly, more correlation times are needed (Oi et al., 1984). Therefore, the short and long correlation times obtained in time-resolved fluorescence experiments, in fact, are weighted averages of several values (Chan and Cathou, 1977). In the latest series of nanosecond polarization experiments, the mean rotational correlation time was used for comparison among the flexibilities of different Ig molecules (Dangl et al., 1988; Schneider et al., 1988). This parameter is calculated as a weighted average of the two experimentally obtained correlation times and does not enable the direct comparison of the correlation times derived from steady-statepolarization experiments. 3 . Spin-Label Approach

The possibility of using the spin-label method to determine the rotational correlation time of protein molecules is based on initial findings by Shimshick and McConnell(l972). They found that the differences among positions of the extrema of ESR spectra of spin-labeled protein, which had been immobilized and freely rotated in aqueous solution, could be attributed to the rotational motion of the macromolecule. Several modifications in the method were used in studies of rotational motion in Ig's (Kaivarainen, 1975; Dudich et al., 1977). As in the steady-state polarization technique, the rotational correlation times obtained using the spin-label technique represent mean values. The results were not significantly different if the label was bound either noncovalently in the combining site of the antilabel antibody or covalently elsewhere on the macromolecule. In our experiments for covalent labeling, the TEMPO-trichlorotriazine spin label was used (Kaivarainen and Nezlin, 1976), which distributed relatively randomly within the IgC molecule (with some preference to Fab) (Nezlin, 1986).

INTERNAL MOVEMENTS IN

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Table 11 lists the values of the rotational correlation times obtained using the spin-label method. In general, they correspond well with the correlation times found in the steady-state polarization experiments (Table I), despite the obvious methodological differences between both approaches. Thus, the results obtained by two relaxation techniquesthe spin-label method and fluorescence polarization-coincide well and point to significant flexibility of the IgC molecules. 4 . X-Ray Crystallography

During the past two decades the three-dimensional structures of Fab and Fc fragments have been described in detail (Alzariet al., 1988; Davies et al., 1975; Huber et al., 1976; Davies and Metzger, 1983). X-Ray crystallography has also provided valuable information relating to the dynamic aspects of the IgG structure, as well as giving additional evidence for the existence of segmental flexibility in these molecules (Huber and Bennett, 1983; Bennett and Huber, 1984). Attempts by researchers to crystallize intact Ig’s however, have encountered difficulties. To date, only four IgC molecules have been crystallized, but two of them (i.e., Dob and Mcg) have deletions in the hinge region (Steiner and Lopes, 1979; Fett et al., 1973), resulting in considerably reduced freedom of Fab movement. The significant freedom of IgC subunit rotation is an obstacle for the formation of a crystal lattice. The most striking finding, however, was that, in crystals of intact IgG, the Fab fragments were well ordered, while the Fc subunit was disordered (Marquart et al., 1980; Ely et al., 1978). The Fc disorder is probably due to distribution of this part of the molecule among several different sites of the crystal lattice (Bennett and Huber, 1984). Fc is able to adopt at least four different conformations, which is why no density related to Fc was found. In contrast, no crystallographic evidence for the mobility of the Fc portion could be found in either hinge-deleted protein. In these proteins Fc is located tightly between the Fab portions, thereby limiting its mobility (Rajan et al., 1983). There are no such close contacts between subunits of the intact IgG (Huber et al., 1976). The flexibility of porcine IgC was studied using the neutron spin-echo method (Alpert et al., 1985). The data obtained are compatible with a flexible model of the molecule. 5. IgG Subclasses

Segmental flexibility depends on the structure of the part of heavy chains between the Fab and Fc subunits, the so-called hinge region. Structure of the hinge region of IgG isotypes varies greatly in the length and number of interheavy disulfide bridges; it is not surprising therefore that IgC subclasses express different degrees of flexibility.

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TABLE I1 ROTATIONAL CORRELATION TIMES OF Ig's AND THEIR FRAGMENTS ON PROTEIN MOIETY' SPIN-LABELED Rotational correlation time (nsec) Protein

Label inside the combining site

Label outside the combining site

Kaivarainen el al. (1973, 1974)

Rabbit anti-spin label antibody IgG F (ab'h Fab' Rabbit IgC antiDNP antibody IgA MOPC 3 15 an ti-DNP mouse myeloma Fab Fv Mouse monoclonal IgG anti-DNP antibody, Fab Pig IgG antiDNP antibody Precipitating Nonprecipitating Rabbit IgC F (ab')z Fab' Rabbit, human pFc'

32 30 18 39

Hsia and Piette ( 1969) Dwek et al. (1975)

44.4

23 6.5 20

19 29

Anglister et al. ( 1984)

20 28 26 25 21 6

Human IgAl myeloma

32-4 1

Human IgA2 myeloma Human IgM myeloma Human IgE Yu myeloma

45

' DNP, dinitrophenyl.

Reference

50 60-62

Sykulev et al. (1979) Timofeev et al. (1978) Nezlin et al. (1985), Nezlin and Sykulev (1984) Dudich et al. (1980) Sykulev et al. (1984) Sykulev et al. (1984) Dudich and Dudich (1980) Nezlin et al. (1973)

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In early studies it was found that human IgG2, which has an unusual hinge region structure involving four closely separated disulfide bridges between heavy chains, has a higher value in correlation time than IgG1, which has only two disulfide bridges (Nezlin et al., 1973). Differences in flexibility exist between two subclasses of porcine IgG; for example, precipitating antibodies are more flexibile than nonprecipitating ones having the same specificity (Dudich et al., 1978; Sykulev et al., 1979). Cebra et al. (1977) studied two isotopes of guinea pig antibodies against the dansyl group. These subclasses have different hinge regions (i.e., the IgGl hinge region is shorter than that of IgG2) and differing abilities to cross-link hapten-protein conjugates (i.e., IgGl is less effective). IgGl antibodies mainly formed large complexes with the bivalent dansyl hapten. Some antibody molecules in these complexes are in the “open” form with the free antibody combining sites, whereas IgG2 antibodies easily formed “closed” dimers. Using nanosecond fluorescence polarization, it was found that IgGl molecules have restricted movement of their Fab fragments and hence are more rigid than IgG2 molecules, which have a more extended hinge region. A systematic study of flexibility differences between IgG isotypes was undertaken recently by a team from Stanford University (Reidler et al., 1982; Oi et al., 1984; Dangl et al., 1988; Schneider et al., 1988). Monoclonal antidansyl mouse antibodies of different IgG subclasses were produced using the heavy-chain switch variants derived from a hybridoma cell line, which originally synthesized IgGl antidansyl antibodies. All of these molecules have the same light chain and variable heavy-chain (VH) region, but different constant heavy-chain (CH)regions. According to nanosecond polarization experiments, the IgG2b variant is the most flexible molecule, whereas IgGz, is less flexible and IgGl is relatively rigid (Table 111) (Oi et al., 1984). This study was confirmed and extended by elegant experiments by Dangl et al. (1988). A family of six chimeric IgG molecules was produced by genetic manipulations of somatic cells (Schneider et al., 1987). T h e molecules had identical light chains and VH regions, but different CH regions: among four human CH isotypes, mouse C H and ~ rabbit CH regions. A strong direct correlation among segmental flexibility, length of the hinge region, and the ability to activate complement was found (Table IV). Distribution of murine IgG isotypes according to their flexibility is as follows (mean correlation time in parenthesis): (55 nsec) > IgGn, (63 nsec) > IgG3 (84 nsec) > IgGl (120 nsec). For human isotypes the distribution is: IgG3 (50 nsec) > IgGl (69 nsec) > IgG4 (84 nsec) > IgGn (120 nsec). Rabbit IgG has an intermediate flexibility (i.e.y72 nsec).

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TABLE I11

FLEXIBILITY OF MOUSEMONOCLONAL

ANTIBODIESWITH ANTIDANSYL ACTIVIT~ Mean rotational correlation time (nsec)c Proteinb

Without With dithiothreitol dithiothreitol

124 120

88

81 60 47

58 51 42

28 28 29

’

28 28 29

~~~

From Oi et al. (1984). Spontaneous mutants of a cell line producing IgGl antibody. All molecules have the same light chains and VH regions, but different CH regions. Determined by time-resolved fluorescence polarization. Myeloma human IgE ND was covalently coupled with a dansyl group (Cathou, 1978).

In another series of experiments a number of genetically engineered IgGl-IgGn hybrids were studied (Schneider et al., 1987, 1988). All of these molecules also possess identical variable parts, having antidansyl ability but differing constant regions, in which a section of one isotype was changed with the corresponding portion of a sequence from another isotype. The main finding of these experiments was that the proper combination of the C H1 domain and the hinge region is important for the flexibility of the IgG molecule (Table V). B. I ~ M A N D I ~ A The first evidence of IgM segmental flexibility was obtained by Feinstein and co-workers (Feinstein et al., 1971; Beale and Feinstein, 1976). Electron micrographs of complexes of bacterial flagella and IgM antibodies show that the IgM molecules are often in a “staple” conformation,

TABLE IV CORRELATION OF HINGE REGIONSTRUCTURE, FLEXIBILITY, AND COMPLEMENT BINDING ACTIVITY Complement fixationb Protein

Upper hinge

Core

Mouse 1 6 , Human 1 6 , Mouse IgGzb Human I g C l Mouse IgGz, Rabbit IgG Human l g G r Mouse IgGl Human I g G Guinea pig l g C 2 Guinea pig IgGl

EPRIPKPSTPPGSS ELKTPLGDTTHT EPSGPISTINP EPKSCDKTHT EPRGPTIKP APSTCSKPT ESKYGPP VPRDCG ERK EPIRTPZBPBP QSWGHT

C P CPRCP(EPKSCDTPPPCPRCP)s CPPCKECHK CP CP CPP CPPCK CP P C CP CPS CKPCI CT CCVECPP CP CTCPK CP CPPCIP C

Upper hinge length PGNILGGP APELLGGP APNLEGGP APELLGGP APNLLGGP PPELLGGP APEFLGGP VPSEVS APPVAGP PPPENLGGP GAPZLLGGP

14(9)’ 12

11 10 9 9 7(5)’ 6 3 I1 6

Correlation times (nsec) 78 f 3 50 f 2 55 f 2 69 2 3 63 f 3 72 f 3 84 f 3

81 f 3

120 f 5 Flexible Restricted flexibility

Human

Rabbit

Guinea pig

70

15

-

100 300 250 300

30

I00 300 250 450

-

+I-

80

25 400

-

+/250

-

+I-

-

+ -

a Shown are the mean correlation times and complement fixation of mouse chimeric (Dangl et al., 1988) and guinea pig IgC, and IgG2 (Cebra et al., 1977; data on complement activity from Sandberg et al., 1971). The amino acid sequences are aligned from residues 216 to 238 (human IgCl Eu numbering). Amount of DNS26-BSA (ng) required to activate one CHso with 10 p g of antibody. Hinge length when polyproline helical structures are considered restricting elements defining upper hinge length.

16

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TABLE V DOMAIN STRUCTURE AND SEGMENTAL FLEXIBILITY OF GENETICALLY ENGINEERED ANTIBODIES~ with or without dithiothreitol

CH lc Proteinb IgG w 2 a

Interdomain hybrids Hingeless Ig& Hybrid 2 Hybrid 3 Hybrid 9 Hybrid 10 Intra-CH1 hybrids Hybrid 11 Hybrid 12 Hybrid 13 Hybrid 14

Aminoterminal

Carboxyterminal

Hinge

c H 2 cH3

Without

With

Yl Y2a

Yl

y2a

Yl y2a

Yl

Yl y2a

83 61

71 49

Y2a Yl Yl Y2a Y2Y

y2a Yl Yl Y2a y2a

Deleted Y2 Yl Yl y2a

y2a Y2 y2ad Yl Yle

y2a Y2 Yl Yl

y2a

84 84 82 108 62

87 55 61 76 52

Y2a Yl

Yl

Yl y2a Yl Y2a

Yl y2a Yl y2a

Yl y2a Yl y2a

114 89 81 64

60 49 76 47

Yl

Y2a

y2a Y2a Yl

y2a

From Schneider et al. (1988). Antidansyl IgCs with identical light- and heavy-chain variable regions. Each CH domain (CH1, hinge, c&!,and cH3) was derived from mouse IgCl or IgC2,. The crossover points in the corresponding DNA sequences were either within introns or within the coding regions at the positions coding for the amino acids, as indicated in the footnotes in Eu protein numbering. The segmental flexibility was determined from nanosecond fluorescence anisotropy kinetics. The mean rotational correlation time (4) calculated from the two exponential fits is given. The amino-terminal half of CH1 is amino acids 118-161: the carboxy-terminal half is 162-2 15. y l to residue 238; y2a from residue 239 on. y2a to residue 238; y l from residue 239 on.

when the Fab arms, linked to a thread of a single flagellum, are folded down from the Fc region, which is seen as a central disc. Later a number of studies were performed on human myeloma IgM or IgM antibodies, using fluorescence polarization and spin-label techniques (Table VI) (Cathou, 1978). All of the data provided supporting evidence for segmental flexibility of the human myeloma IgM molecules. The experimental correlation times are significantly lower than those calculated for a rigid sphere with the IgM volume. Although the IgM ~ probably serves as molecule has no typical hinge region, the C Hdomain

INTERNAL MOVEMENTS IN

Ig MOLECULES

17

TABLE VI ROTATIONAL CORRELATION TIMES OF IgA, IgE, AND IgM DETERMINED BY FLUORESCENCE POLARIZATION Correlation time (nsec)

Protein Steady-state polarization" IgA, human myeloma

147

IgA, monomer human myeloma

26-32

IgA, dimer human myeloma

30

Fab from IgA human myeloma

25

IgAl monomer human myeloma IgE, Yu human myeloma IgE, mouse antidansyl IgM, human myeloma IgM, subunit human myeloma IgM, human myeloma

33(32)b 55(60)b 54' 73(50)* 57 27-47

IgM, subunit human myeloma

22-27

Time-resolved fluorescence polarizationd Fab from equine antidansyl IgM 32 IgM, equine antidansyl IgM IgM, porcine antidansyl IgM, nurse shark

4s 4s

61 >loo0 69 568 93

+L

>loo0

f#JL

4s

4L

Reference Weltman and Davies (1970) Zagyansky and Gavrilova (1974) Zagyansky and Gavrilova (1974) Zagyansky and Gavrilova (1974) Dudich et al. (1980) Nezlin et al. (1973) Slattery et al. (1985) Dudich el al. (1980) Dudich et al. (1980) Zagyansky et al. (1972) Zagyansky et al. (1972) Holowka and Cathou (1976) Holowka and Cathou (1 976) Holowka and Cathou (1976) Holowka and Cathou (1976)

' Covalent labeling outside the combining site.

In parentheses, the values of the rotational correlation time, as determined by the spin-label method. Dansyl dye in the combining site. Short and long correlation times which characterize the two main depolarizing motions. Dansyl dye is in the combining site.

its analog (Holowka and Cathou, 1976). IgMs of other species were also studied and it was found that IgMs of lower vertebrates were less flexible than those of human proteins (see Section VII). Most of the experimental data on IgA were obtained by steady-state polarization and spin-label techniques (Table VI). The data are compati-

18

KOALD NEZLIN

ble with those of the flexible model of these molecules. Results of the nanosecond polarization experiments on IgA also provide evidence in favor of segmental flexibility of this class of Ig's (Liu etal., 1981). The data were similar for monomeric, dimeric, and secretory forms of IgA, despite the fact that monomeric IgA antibodies are poor agglutinins. There are probably other factors which may explain the poor correlation between flexibility and the ability to agglutinate antigen. The mobile part of IgA molecules are most likely their Fab fragments since the correlation time values for whole IgA and their Fab are very similar. C. IgE IgE's have specific functions which are different from those of other classes: They serve as antigen receptors on mast cells and basophils and are an important participant in the chain of events which lead to specific allergic reactions after encounter with allergens. The concentration of IgE in serum is usually low, and all experiments were initially done on two proteins isolated from myeloma patients Yu and ND. In the experiments performed by steady-state polarization and the spin-label method, the IgE (Yu) molecule was significantly less flexible than were IgC and IgM molecules (Table VI) (Nezlin et al., 1973). Time-resolved polarization measurements of IgE (ND) coupled covalently with dansyl groups also pointed to the rigidity of these molecules (Cathou, 1978). Slattery et al. (1985) used steady-state polarization measurements to study specific complexes of dansyl groups with corresponding mouse monoclonal antibodies. The average value of the correlation time for IgE in solution was found to be 54 nsec, which corresponded well with the results of the above-mentioned experiments on IgE (Yu)(i.e., 55 nsec). Similar results were found for covalent complexes of another fluorophore-pyrenylmaleimide-with the same IgE antibodies (i.e., 64 nsec). The calculated value of the correlation time for a rigid sphere with IgE parameters is 78 or 155 nsec, depending on the method of calculation. Experiments with complexes of the dansyl hapten with IgE antibodies bound to receptors on membrane vesicles from basophilic cells supported the low flexible model of IgE. Despite the large volume of such a particle with bound IgE (calculated correlation time, 1000 nsec), the experimentally found correlation time was only 64 nsec. Hence, the IgE molecule is not completely rigid, and its Fab subunits have some freedom of rotation, although it is less pronounced than that in IgG or IgM. The ability of IgE antibodies to agglutinate erythrocytes is also evidence in favor of some freedom of rotation of the Fab, subunit (Ishizaka, 1973). Among the family of monoclonal mouse antibodies with identical com-

INTERNAL MOVEMENTS IN

Ig MOLECULES

19

bining sites, but differing CH regions, IgE molecules are the most rigid (Table 111) (Oi et al., 1984). Even if one takes into account the higher molecular weight of IgE molecules (190,000 as compared to 150,000 for IgC molecules), the value of the correlation time for IgE is still higher (i.e., 98 nsec) than that for IgCl, the most rigid IgG subclass (i.e., 81 nsec). IV. Internal Motions in Fab and Fc Subunits

All Ig Fab subunits are built from variable domains of both light and heavy chains and constant domains, CL and CH1 respectively. Both variable domains, as well as two constant domains, form separate entities linked by short parts (Lea,switch peptides) of each of the chains. According to fluorescence polarization and spin-label measurements, isolated Fab fragments behave in solution as rigid particles, with rotation correlation times between 20 and 30 nsec. These values correspond well with the calculated value for a rigid ellipsoid with a volume equal to that of Fab. Crystallographic data for Fab have shown that extensive contact is nonexistent between variable and constant parts of the fragment. Thus, it is not surprising that X-ray and electron-microscopic studies have demonstrated that the angle between the pseudodyad axis of V and C parts ("elbow bend") varies significantly in Fab's from various proteins under study (Huber et al., 1976).T o date, there are crystallographic data for 15 Fab's and the elbow angle has been found to vary from 132"to 172" (Davies et al., 1988). The elbow flexibility was also demonstrated by crystallographic studies of the light-chain dimer, which imitates the structure of Fab (Edmundson et al., 1978). One of the two identical chains of the dimer have the conformation of an amino-terminal half of the heavy chain, and the other, the light chain. Despite the identity of both light chains in a dimer, their elbow angles are quite different (i.e., 70" and 110"). Further evidence for flexibility in the switch region of Fab was obtained from studies of different crystals of the light-chain dimers. The dimers of light chains of a monoclonal IgG crystallize in two forms: trigonal and orthorhombic. In two different crystal lattices the bend angle varies significantly--115" and 132" (Abola et al., 1980; Ely et al., 1983). More recently, it was found that the same Fab' from a monoclonal antibody can exist in two or more forms and they are bent differently at the switch region. Sheriff et al. (1 987) described two variants of complexes of Fab from the antilysozyme monoclonal antibody with lysozyme, which differ in switch bending, and Prasad et al. (1988) reported similar data with the

20

R0AL.D NEZLIN

Fab from an antibody against an Escherichia coli protein. In both cases the angular variation was nearly identical (i.e., 7" and 8"). Two groups presented electron-microscopic evidence of Fab bending after the reaction of antibodies with protein antigens (Roux and Metzger, 1982; Wrigley et al., 1983; ROUX,1984). This kind of flexibility is particularly evident on micrographs of closed complexes of two molecules of influenza hemagglutinin with two antibody molecules, in which the angle between the V and C portions of Fab was 90". Thus, in solution in which the Fab is either free or is part of an intact Ig molecule, it rotates as a compact structure, but the bend angle between the V and C parts can be changed significantly by some external force when it complexes with an antigen or during formation of a crystal lattice. The Fc fragment has quite different rotational properties from Fab. Measurements of the correlation time revealed that this parameter for isolated Fc is significantly smaller than that for Fab (Table I) (Nezlin et al., 1970; Dudich et al., 1978; Timofeev et al., 1978). Both fragments are built from four domains of nearly equal dimensions, and only some internal lability of Fc could be responsible for the different rotational properties of both fragments. After elucidation of the three-dimensional structure of the Fc fragment (Deisenhofer et al., 1976; Deisenhofer, 1981) it became evident that the main reason for the internal motion in the nanosecond range is the lack of lateral contact between c H 2 domains. Other structural features promoting flexibility are common to both fragments-namely, few longitudinal contacts and extended conformation of switch peptides. In intact molecules the Fc conformation is stabilized by S-S bridges on the hinge region (Seegan et al., 1979). Despite this, the Fc, even in the intact IgG molecule, has a high degree of internal rotational freedom: The correlation times of Fc and IgG spin-labeled on carbohydrates are nearly the same (Timofeev et al., 1978) and are about twice as short as the correlation time for Fab (see also Table VIII). Using the proton relaxation enhancement technique, Burton et al. (1977) not only found that the Fc of rabbit IgG is not the rigid particle, but also that significant rotational freedom is characteristic for this part of the molecule. The peculiar nonstable structure of Fc is responsible for the high sensitivity of the fragment of external influence, for example, the action of low pH (Abaturov et al., 1969). In the intact IgG molecule and in the isolated Fc fragment, the CH3 domains interact closely. The isolated pepsin pFc' fragment, which is a noncovalent dimer of c H 3 , conserves the main features characteristic for Ig C domains (Phizackerlay et al., 1979). However, in solution pFc' is flexible; the correlation times for pFc' are half of those for Fc (Table 11)

INTERNAL M O V E M E N T S IN

Ig MOLECULES

21

(Nezlin and Sykulev, 1984; Nezlin et al., 1985). The mobility of the CH3 domains in the Fc evidently is restricted by some form of contact with C H domains. ~ T h e CH2-cH.3 switch region is another flexible point of the IgG molecule. Bending in this region is clearly seen after the reduction of interheavy S-S bridges. Romans et al. (1977) found that so-called “incomplete” IgG antibodies, which are unable to agglutinate red cells, are converted into direct agglutinins after mild reduction. Such treatment cleaves S-S bonds in the hinge region, opens the upper part of the Fc region, and allows Fab arms to bridge between two red blood cells. Seegan et al. (1 979) measured the length of the Fab arms in complexes of reduced antibodies with antigens and found that it increases by 23 A. Such results could be explained by the formation of a new hinge between the cH2 and CH3domains. Nanosecond polarization experiments (Chan and Cathou, 1977) directly point to increased flexibility after the mild reduction of rabbit IgG (see also last columns of Tables 111 and V). V. Role of the Hinge Region Structure for Segmental Flexibility

The heavy-chain portion between Fab and Fc, which has a unique structure, is coded by a separate DNA segment (Sakano et al., 1979). Its susceptibility to proteolytic attack points to a noncompact extended conformation, and early electron-microscopic data have already suggested that the segmental flexibility of the IgG molecule depended mainly on this region’s action as a hinge. The principle role which the hinge region plays in the freedom of Fab and Fc movement was confirmed after crystallographic studies of the Dob and Mcg hinge-deleted proteins (Silverton et al., 1977; Ely et al., 1978). In contrast to the intact Kol and Zie proteins (in which the Fc fragment adopts more than one position in the crystal lattice), Fc of Dob and Mcg, which lack segmental flexibility, contributes fully to the diffraction pattern and has a definite position in the crystal. As a result of intensive crystallographic and proton nuclear magnetic resonance investigations, the structure of the hinge region of IgGl heavy chains, as well as its role in Ig flexibility, is now well understood (Marquart et al., 1980; Ito and Arata, 1985; Endo and Arata, 1985). This region is built from a central portion (i.e., core) and two flanking segments (Fig. 1). In crystal form as well as in solution, the central portion with the sequence -Cys-Pro-Pro-Cysforms two parallel poly-Lproline double helices, linked by S-S bridges. The segment on the amino-terminal side of the core (Cy~~~’-Thr’~~) in crystal forms a oneturn helix with little inherent stability. It is exposed to solvent and is

22

ROALD NEZLIN

L

Cys

I

I

pepsin

trypsin papain

la ProGluLeiLeuGly Pro220 I

L

I

I

I

235 IaProGluLeuLeuGly ProJ

cys

FIG. 1. The hinge region of human IgG. The residues in the box (216-230) are deleted in IgGl Dob myeloma protein (Steiner and Lopes, 1979).The central part of the hinge (core)is shadowed.

mainly responsible for the segmental flexibility of the IgC molecule. T h e segment on the carboxy-terminal side of the core ( P r ~ ~ ~ ~ - L ehas u ~an ”) extended conformation, but is not as flexible as the segment on the left side. However, after reduction and alkylation of the core S-S bonds, both segments have comparable flexibility (Endo and Arata, 1985). Another point of flexibility is the Cys229 atom, which, according to nuclear magnetic resonance studies (Ito and Arata, 1985), has considerable freedom of internal motion around the NH-C, and Cp-S bonds. The roles of different portions in flexibility of the hinge region are demonstrated in Tables IV and V, which summarize the results of polarization experiments on mutant and genetically engineered Ig molecules (Schneider et al., 1988; Dangl et al., 1988). In the first series of experiments, discussed in part above, the relationship of flexibility to the structure of the amino-terminal side segment of the core (left segment or upper hinge) is clearly established: T h e shorter the segment, the lower the correlation time. This correlation is in agreement with the abovementioned nuclear magnetic resonance findings on the role of the amino-terminal segment in flexibility. In other experiments (Table V) the rotational motion of Fab was studied on molecular hybrids of two mouse isotypes-IgGl and IgG2which possess different degrees of flexibility. T h e hybrid molecules are distinguished from each other by the structure of one or more CH domains, the hinge region, or part of the CHIdomain. It was found that, for

INTERNAL MOVEMENTS I N

Ig MOLECULES

23

flexibility, not only that the hinge region structure is important, but that both the CH1 and hinge regions must be in harmony, that is, properly matched. For example, the hybrid 13, which differed from flexible IgG2, only by the amino-terminal part of CH1, was found to be less flexible than intact IgG2,. VI. Functional Implications of Segmental Flexibility

A. PRECIPITATION AND AGGLUTINATION A condition for the formation of a precipitate or agglutinate network is the ability of the antibody molecules to bind simultaneously to epitopes of two antigen molecules or two cells. Epitope arrangement varies greatly, and freedom of Fab movement is crucial for optimal binding to antigens and the formation of a bridge between antigen molecules and cells. T h e ability of Ig molecules to change their general conformation from Y to T type (Cser et al., 1981a,b) greatly facilitates the capacity of antibodymediated linkage to antigens. Direct measurements of the flexibility of two porcine IgG antibodies having differing abilities to precipitate antigen support this view. During the early stages of the immune response to dinitrophenyl hapten, the animals synthesized antibodies which formed mostly insoluble complexes (precipitates) with heavily dinitrophenyl-substituted proteins, while antibodies generated later mostly formed soluble complexes with the same antigen molecules. Fluorescence polarization (Dudich et al., 1978) and the spin-label method (Sykulev et al., 1979) revealed that precipitating antibodies are significantly more flexible than are nonprecipitating antibodies (Tables I and 11). Spin-label experiments gave nearly identical results when the label was either in the antibody-combining site (the correlation times were 19 and 29 nsec for precipitating and nonprecipitating antibodies, respectively) or covalently bound to some residues outside the combining site (20 and 28 nsec, respectively). Neutron diffraction studies (Cser et al., 1977) and electron micrographs (Ryazantsev et al., 1989) also provided evidence for a more compact structure of the nonprecipitating antibodies. Carp IgM antibodies are a further example of the correlation between restricted flexibility and weak precipitating activity (Richter e l al., 1972). These antibodies precipitated only the presence of a large excess of antigen and, in the case of antibodies against haptens, only if a large number of hapten molecules were present on the carrier protein. According to steady-state fluorescence polarization measurements, carp

24

ROA1.D NEZLIN

antibodies are much less flexible than more recently evolved IgC and even human IgM (Table VII). The importance of flexibility in allowing antibody molecules to bridge between two cells was discussed above, as far as the ability of mild reduction to convert the incomplete agglutinins to direct agglutinins (Section IV). Another aspect of segmental flexibility is linked to the classic experiments by Karush (1978). He found that the functional affinity of antibodies is enhanced 100 times by antibody attachment to antigen with repeating determinants such as bacterial polysaccharides or synthetic antigens, not with one, but with two or more, combining sites. Although the arrangements of these repeating antigenic determinants cannot be identical for different kinds of bacteria or viruses, the gross conformations of antibody molecules of different specificities are the same. Obviously, the ability of the Fab fragments to rotate relatively independently greatly facilitates the interaction of the antibody with two or more combining sites with antigens, resulting in a significant gain in binding strength provided by bivalency . B. COMPLEMENT ACTIVATION AND BINDING TO CELLRECEPTORS Abnormal IgG proteins with a deleted hinge region (Steiner and Lopes, 1979; Deutsch and Suzuki, 1971) lack a number of important

TABLE VII FLEXIBILITY OF Ig’s FROM Low VERTEBRATES Rotational correlation time (nsec)” Species

IgM

Shark Squalis acantias

147

Carp Cyprinus carpi0 Frog R a m temporaria Tortoise Testudo horstieldi

128 135 102

Hen Galus domesticus Human, rat, rabbit

IgC

67 68

43 23-73

20-30

Reference Zagyansky and Ivannikova ( 1 974) Richter et al. (1972) Zagyansky (1975) Zagyansky and Ivannikova (1 974) Zagyansky and Ivannikova (1974) Zagyansky et al. (1969, 1972), Dudich et al. (1978, 1980)

” As determined by steady-state fluorescence polarization.

INTERNAL M O V E M E N T S IN

lg MOI.ECULES

25

biological functions, such as binding of the first component of complement (Clq) and the ability to react with different Fc cell receptors (Klein et al., 1981). Reactive sites responsible for interaction with the C l q component and a variety of Fc cell receptors are located in the CH2 domains (Burton, 1985). According to crystallographic studies of a hinge-deleted IgGI Dob protein (Silverton et al., 1977), Fab portions of the molecule are close to the C Hdomain ~ and sterically obstruct the reactive sites, making it impossible for a molecule as large as C l q to reach them (Dorrington and Klein, 1982, 1983). IgC4 molecules, which have restricted flexibility, have no complement binding capacity (Isenman et al., 1975), but the Fc fragment isolated from IgC4 is as active as Fc from I&. Clearly, the Fab fragments somehow protect the C lq binding site, and this structural property of IgC4 is obviously correlated to restricted flexibility. Analyzing the primary structure of the hinge regions of different Ig’s, Beale and Feinstein (1976) paid attention to the direct correlation between the length of the so-called upper part of the region (i.e., the amino-terminal side of the core) and complement activation (see also Feinstein et al., 1986). It is also known that the upper part of the hinge is important for the segmental flexibility of Ig molecules (Section V). Both properties-flexibility and complement activation-were recently compared on different genetically engineered chimeric mouse antidansyl antibodies, which have the same variable parts, but differ in constant parts of the heavy chain (Oi et al., 1984; Dangl et al., 1988). A clear-cut relationship was found among the length of the upper part of the hinge, the mean correlation time which reflects Fab motion, and the ability to fix complement (Table IV). The shorter the hinge, the more rigid the molecule, and the complement binding activity is corresponding lower. An extreme example is the hingeless rigid IgC2, antibody, which is unable to activate complement in all, in this respect resembling the hingedeleted human Dob protein. This study is in agreement with the results by Leatherbarrow and Dwek (1984), who studied C lq binding to immobilized mouse monoclonal Ig isotypes. The order of activity was found to be Igcg, > > IgGI. Correlation between the length of the upper part of the hinge and properties of the isotypes was also found for guinea pig Ig’s (Cebra et al., 1977). The isotype IgCl, which has restricted flexibility and a short sequence in this part of the hinge, is unable to fix complement (Sandberg et al., 1971) (Table IV). Ig’s lacking the hinge region have reduced, or even totally lack, any affinity for receptors on the surface of different kinds of cells (Klein et al., 1981). The sites involved in the binding of these receptors are probably located in the CH2 region (Burton, 1985), and it is possible that the Fab

26

KOALD NEZLIN

can also modulate the activity of these sites in the same way as a complement binding site. However, to date, no direct comparison of segmental flexibility and Fc receptor binding activity of different IgG subclasses has been made. According to the attractive hypothesis proposed by Burton ( 1986), flexibility of the IgG molecule allows the rotation of the Fc portion perpendicular to the plane of Fab subunits. This dislocation eliminates and obstruction of the Fc functional sites after immunoprecipitation and promotes interaction with C l q and cell receptors. Such movements are impossible in nonflexible molecules. VII. Evolution of Ig Flexibility

Several studies were performed in the 1970s to determine the flexibility of Ig’s isolated from sera of different species. The mean correlation times obtained by steady-state polarization measurements for Ig’s of lower vertebrates are definitely higher than those for Ig’s of the higher vertebrates (Table VII). For example, the correlation times for shark and carp IgMs are five to six times higher than those for human IgM; those for 7 S Ig’s (IgG type) from the tortoise and the hen are more rigid than those for human IgC. The nanosecond fluorescence polarization measurements also point out that nurse shark IgM is less flexible than equine or porcine IgM (Holowka and Cathou, 1976). Study of the general structure of Ig’s of low vertebrates has attracted less interest, and it is thus difficult to explain the observed differences in flexibility in real structural terms. According to Holowka and Cathou (1976), the Fab units and the whole F (ab’):! fragment of equine and porcine IgMs have some freedom for independent rotation, but for shark IgM molecules only bending of F (ab‘):!is possible. In summary, there is reason to believe that the segmental flexibility of antibody molecules appeared relatively late in evolution. VIII. Mobility of Carbohydrate Components

Ig molecules are glycoproteins, having several oligosaccharides, usually linked to heavy chains (Torano et al., 1977). In IgG about 2-3% of carbohydrates were found; in other classes their content is considerably higher (i.e., 10-12%) (Nezlin, 1977). When considering the spatial relationship between oligosaccharide chains and the protein moiety in different glycoproteins, two situations can be expected. In the first case an.oligosaccharide rotates relatively freely and links to the protein moiety by covalently binding to an amino

INTERNAL M O V E M E N T S IN

Ig MOLI:CLII.ES

27

acid residue. In the second an oligosaccharide chain interacts with several amino acid residues in a particular region of the protein surface and rotates together with the adjacent protein subunit of the molecule. In the latter case carbohydrate residues could close a part of the protein surface, preventing further interactions with some ligands. The closed area would be protected from proteolysis. Sugar residues could construct completely new antigenic o r recognition sites on the surface of a glycoprotein molecule. From the above, the importance of understanding the mode of the behaviour of carbohydrate chains becomes clear. The main carbohydrate unit of IgG linked to the Am2’’ residue of the Fc is the most studied. In crystals this branched oligosaccharide is ordered and occupies a fixed position covering part of the C2H domain surface. In human Fc it forms a few hydrogen bonds with the CH2 amino acid residues, but its main interactions are hydrophobic (Deisenhofer, 1981; Deisenhofer and Huber, 1983). The structure of the Fc carbohydrate in the rabbit is stabilizing not only by contact with amino acid residues of cH2, but also by contact with the oligosaccharide linked to the cH2 domain of the other heavy chain. Nearly all nine sugar units of the oligosaccharide core and both branches have well-defined density and are therefore fixed and immobile (Sutton and Philipps, 1983). Spatial positioning of carbohydrates in other Ig’s has not been investigated. T o study carbohydrate mobility in Ig molecules in solution, we used the spin-label approach (Nezlin and Sykulev, 1982). For spin-labeling of Ig oligosaccharides, limited periodate oxidation was performed, followed by reductive amination with TEMPO-amine spin label (Fig. 2) (Willan et al., 1977; Nezlin et al., 1978; Gnewuch and Sosnowsky, 1986). It was found that less than two spin-labeled molecules are bound to an IgG molecule. Significantly, more spin labels are incorporated in IgE (8 mol

Nal04

TEMPO amine

OH

I-

0

FIG.2. Introduction of TEMPO-amine spin label into carbohydrate residues.

28

KOALD NEZLIN

per mole of protein) and IgM (about 30 mol per mole of protein). This may be the result of the higher carbohydrate content in both IgE and IgM molecules. Using the same method, high-molecular-weight polysaccharide isolated from Pneumococczls was spin-labeled. ESR spectra of this polysaccharide are represented by three sharp lines and are similar to the spectrum of a spin label freely mobile in solution (Fig. 3). This may be the result of flexibility of the bonds between sugar units in the carbohydrate chain. However, ESR spectra of Ig’s spin-labeled at carbohydrates were quite different and reflected partially immobilized rotation of the spin label due to the attachment of oligosaccharide chains with the bound spin label to the protein moiety. In Table VIII the rotational correlation times of human Ig’s and their fragments spin labeled on carbohydrates are presented. Correlation times for IgG and Fc are similar and are about half of those for Fab, the value predicted for a rigid sphere having the same volume. Similarity of the data for IgG and Fc means that the relative mobility of oligosaccharides is the same in both isolated Fc and the intact IgG molecule. T h e correlation times obtained in these experiments closely resemble those for Fc found in the fluorescence polarization experiments (see Section IV). Apparently, the Fc oligosaccharide rotates together with an adjoining subunit (i.e., the CH2domain). The main finding of these experiments is that the close attachment of the Fc oligosaccharide to the protein surface points to the oligosaccharide B

A FREE SPIN LABEL -14OOC

20oc

PNEUMOCOCCUS POLYSACCHARIDE

IMMUNOGLOBULIN G

-

20%, OSbruuoM

O.C.0 x 8ucrose OeC,34%aucross

FIG.3. Electron spin resonance spectra. (A) Spin label free in solution (right) and immobilized (left). (B, left) Spin-labeled high-molecular-weight Pneumococcus polysaccharide without (top) and with (bottom) rabbit antipolysaccharide antibodies (Nezlin and Sykulev, 1982). (Right)Human IgC spin-labeled at carbohydrates. 2A‘, Distance between outer wide extrema (Timofeev et al., 1978).

INTERNAL MOVEMENTS IN

Ig MOI.ECULES

29

TABLE VIII ROTATIONALCORRELATION TIMES OF HUMAN Ig's A N D HLA ANTIGENS SPIN-LABELED AT CARBOHYDRATE AND PROTEIN MOIETY Rotational correlation times (nsec) Protein

IgC (mainly IgCI) Fc from IgG IgM, myeloma IgM, myeloma IgM, subunit Fc5 from IgM IgE Yu, myeloma IgAl myeloma IgAl, myeloma IgA2, myeloma HLA antigen class I, extracellular part HLA antigen class 11, extracellular part

Spin-labeling at carbohydrates

Spin-labeling at protein moiety

11 12 9.5

6 12-16" 50

7

7 6 66b 45h 2.5

55-60 41 32 45 8

2.7

14

Reference Timofeev et al. (1978) Timofeev et al. (1978) Timofeev and Lapuk (1982) Sykulev and Nezlin (1982) Sykulev and Nezlin ( 1982) Sykulev and Nezlin (1982) Sykulev and Nezlin (1982) Sykulev et al. (1984) Dudich et al. (1980) Sykulev et al. (1984) Nezlin et al. (1987, 1988) Nezlin et al. (1987, 1988)

As determined by steady-state Huorescence polarization (Nezlin et al., 1970; Dudich et al., 1978). The correlation time of the spin label bound to oligosaccharide(s) with restricted mobility.

chains being fixed not only in protein crystals, but also in solution. T h e correlation between crystallographic and spin-label data indicates that there is reason to believe that the correlation times obtained in spin-label experiments indeed reflect the mobility of the carbohydrate components of glycoproteins. Mobility of the IgCl oligosaccharide has also been investigated by ''C nuclear magnetic resonance (Rosen et al., 1979). In the nuclear magnetic resonance spectra two broad bands are seen, due to resonances from carbohydrates. T h e broad form of the bands suggests restricted mobility of the oligosaccharides relative to the protein moiety. Other Ig's have not one, but four and more, oligosaccharides per heavy chain, and the spin label can probably be incorporated in all of them. We can expect that the ESR spectra of labeled Ig's with a higher content of

30

K 0 A I . D NEZLIN

carbohydrates will reflect movement of the differing oligosaccharides with varied mobility, and the experimentally found correlation times often represent the mean values. However, the ESR spectra of IgE and IgAl spin-labeled on carbohydrates register two or more outer wide extrema. The presence of more than one wide extremum (better seen in the high field of the spectra; see Fig. 4) can be explained by the existence of differing relative mobilities of various oligosaccharide chains. T h e wide extremum, located distant from the central line of the spectrum, reflects the rotation of the spin labels bound to oligosaccharides with more restricted mobility. The correlation times calculated using these outer wide extrema are 66 nsec for IgE and 45 nsec for IgAl (Sykulev and Nezlin, 1982; Sykulev et al., 1984).The IgE and IgA oligosaccharides which possess these characteristically high correlation times are immobilized and rotated together with the whole molecule. We do not know to which part of the IgE and IgAl molecules they are bound. If they are located in the Fc portions, we could speculate that perhaps the Fc subunits of these Ig's are rigid, with no loose structure similar to the Fc of IgG. The wide extrema of ESR spectra for a spin-labeled myeloma IgM are broad and poorly resolved into subcomponents (Sykulev and Nezlin, 1982). The mean correlation times in this case were very low and similar for the whole molecule (7 nsec), the 7 S subunit (7 nsec), and the Fc5 fragment (6 nsec). A nearly identical value was obtained for another myeloma IgM: 9.5 nsec (Timofeev and Lapuk, 1982). It is difficult to

-

20°C. 0% sucrose

OOC, 38% sucrose

A part of the spectrum ol higher gain FIG. 4. Electron spin resonance spectra of human myeloma IgE Yu spinlabeled at carbohydrates.The presence of two outer wide extrema at higher gain can be explained by the existence of various relative mobilities of different oligosaccharides. Oligosaccharide(s)with more restricted rotation is characterized by longer 2A'l distance (Sykulev and Nezlin, 1982).

2%-

I N T E R N A L MOVEMENTS I N

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31

interpret the physical significance of these values; most likely they reflect the loose structure of the Fc fragment of IgM. In the IgA2 molecule the spin labels incorporated in carbohydrates rotate surprising freely. They cannot be immobilized, even when in solution with a high concentration of sucrose (i.e., about 30%),when the whole glycoprotein molecule stops its rotation (Sykulev et al., 1984). Such freedom of rotation was also observed for ovalbumin oligosaccharide (Nezlin and Sykulev, 1988). More recently, we have studied the mobility of oligosaccharides of human leukocyte antigen (HLA), glycoproteins belonging to the Ig superfamily (Nezlin et al., 1987, 1988; Pankratova, 1988). In these experiments the extracellular portions of HLA classes I and I1 proteins were isolated and then spin-labeled on oligosaccharides. The correlation times obtained (i.e., 2.5-2.7 nsec) suggest a significant restriction of the mobility of spin labels bound to carbohydrates, due to interactions of the latter with the protein moiety (Table VIII). Information is still lacking regarding the exact localization of the spin label in the above-mentioned experiments; namely, to which of the several carbohydrate chains does the spin label incorporate and what sugar residues are tagged? The correlation times are the mean values and reflect the interrelationship of the oligosaccharide chains and the protein moiety in general. However, such experiments can also provide definitive information. For example, the spin-label method could be useful in investigating the mobility of Ig oligosaccharides with abnormal structure, such as those found in some pathological conditions (Roitt et al., 1988).As discussed above, the Fc, oligosaccharide, whose main function is probably to interact with cell receptors, is located on the protein surface. T h e Fc oligosaccharides of patients with such diseases as rheumatoid arthritis lack terminal galactose residues, which are important for fixation on protein surface. Such abnormal Ig's suffer a complete loss in their ability to bind to macrophage and monocyte Fc receptors and also demonstrate a reduced ability to induce cellular cytotoxicity (Rademacher et al., 1988). According to the attractive hypothesis by Parekh et al. (1989), one of the reasons for this defect could be the increased mobility of oligosaccharides lacking terminal glactose (Fig. 5 ) . This suggestion, of course, could be verified experimentally by the spin-label technique. If an oligosaccharide is firmly fixed on the protein surface and rotates together with a protein subunit, the rotational correlation time provides information regarding the flexibility or rigidity of the structure of this subunit. Even such general information can be useful in many cases, especially if X-ray crystallography data are not available. The method would, of course, be much more informative if the exact position of the spin label is known.

32

KOA1.D NEZ1.1N

Fab

Fab

Fab

Fab

0 Mannose @ N-acetylglucosamine

Galactose

FIG. 5. (Left) A schematic representation of an IgC molecule indicating the positions of conserved N-glycosylation sites ( A d g 7in CH2 domains). T h e arrow indicates the site of interaction between the a1-3 arms of two oligosaccharides. T he residues of the al-6 arm of each oligosaccharide are in contact with the surface of the protein. X-Ray crystallographic data show a well-defined galactose binding site. (Right) An IgC molecule from patient with rheumatoic arthritis. T he oligosaccharide chains terminate with N-acetylglycosarnine (Modified from Parekh el al., 1989).

IX. Conclusions

Ig’s are complex multifunctional proteins. They can react with several types of ligands, many of which are large molecules. In some cases the formation of complexes with various ligands takes place simultaneously and in different parts of the molecule. It is therefore not surprising that the studies discussed in this chapter provide strong evidence for the presence of diverse kinds of internal motion in the Ig molecule, which have evolved in favor of optimal interactions with various ligands. There is little doubt that the Fab arms of Ig molecules have enough freedom to rotate relatively independently. This so-called hinge flexibility, which is primarily dependent, in IgG, on the structure of the part of the hinge region between the hinge core and the Fab portion, greatly facilitates bivalent recognition of variably spaced antigenic determinants and permits the binding of two antigenic molecules or cells. It is not such an easy task, however, to describe the precise character of Fab movements. The rotational relaxation times obtained from fluorescence polarization measurements usually represent an average of several correlation times. We can, however, imagine that the Fab arms rotate more or less independently about the joints in the hinge region in one

INTERNAL MOVEMENTS IN

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33

of the following ways: (1) angular waggling, (2) conelike wobbling, (3) twisting about the long axis, and (4)some movements about the short axis (Fig. 6) (Cathou, 1978; Hanson et al., 1981; Bennett and Huber, 1984). Another type of flexibility is presented by F (ab’n-Fc rotation, d u e to properties of the part of the hinge situated below the core. In the nanosecond range Fab rotates as a compact particle. However, after reaction with antigens, the angle between V and C region modules changes (so-called elbow bending). Contrary to Fab, the Fc fragment is flexible, which not surprising, since the CH2 domains are spatially separated. T h e Ig molecules are glycoproteins. T h e mobility of oligosaccharide chains, usually bound to the heavy chains, varies significantly. In Fc, the oligosaccharide chain is fixed spatially, but other Ig subclasses possess oligosaccharides capable of rotation more o r less independently from the protein moiety. Investigation and understanding of the mobility of oligosaccharides are important because of their role as ligands to cell receptors; arid changes in their freedom of movement would result, obviously, in pathological conditions. In summary, one can only begin to imagine from this review the wide variety of movements common to the various Ig molecules. This exceptional property is in direct conjunction with the functional diversity of these proteins: They are able not only to recognize antigens, but also to participate in a number of other important reactions. T h e functional sites are located in different parts of the Ig molecule, and flexibility seems to be a crucial factor for optimal binding to several different ligands, often simultaneously. T h e functional importance of flexibility is obvious after comparison with findings that hinge-deleted nonflexible Ig’s lack major effector functions. Such proteins are probably not just a genetic curiosity. According to H. F. Deutsch (cited by Rajan et al., 1983), about 1-2% of all IgC

A

.

.

B

.

C

FIG.6. Possible modes of rotation of the Fab subunit in an intact IgG molecule. (A) Angular waggling. (B) Conelike wobbling. (C) Motions along short and long axes.

34

K 0 A I . D NEZLIN

antibodies have no hinges. The functions of these proteins, which probably are abnormal, are still unknown. Study of t h e properties of this nonflexible Ig population could lead t o unexpected-and interesting-

findings.

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tiev, V. V. (1984).Structural studies of human IgAl and IgA2 immunoglobulins tagged with two different spin labels. Biofizika 29, 744-748. Timofeev, V. P., and Lapuk, V. (1982).Study of the irreversible conformational transition in IgM using spin labels introduced into the carbohydrate and peptide moieties of its molecule. Mol. Biol. (Engl. Truml.) 16, 325-333. Timofeev, V. P., Dudich, I. P., Sykulev, Y. K., and Nezlin, R. (1978). Rotational correlation times of IgG and its fragments spin-labeled at carbohydrate o r protein moieties. Spatially fixed position of the Fc carbohydrates. FEBS Lett. 89,191-195. Torano, A., Tsuzukida, J., Liu, Y. V., and Putnam, F. W. (1977). Location and structural significance of the oligosaccharides in human IgAl and IgA2 immunoglobulins. Proc. Nutl. Acad. Sci. U.S.A. 74,230 1-2305. Tumerman, L. A., Nezlin, R., and Zagyansky, Y. A. (1972a). Increase of the rotational relaxation time of antibody molecule after complex formation with dansyl-hapten. FEBS Lett. 19,290-292. Tumerman, L. A., Zagyansky, Y. A., and Nezlin, R. (1972b). Experimental evidence of the flexibility of IgG molecules. Mol. Biol. (Engl. Trunsl.) 6, 135- 147. Valentine, R. C., and Green, N. M. (1967). Electron microscopy of an antibodyhapten comp1ex.J. Mol. Bid. 27, 615-617. Weber, G. (1953). Rotational Brownian motion and polarization of the fluorescence of solutions. Adv. Protein Chem. 8,415-459. Weltman, 1. K., and Davis, R. P. (1970). Fluorescence Dolarization studv of a huma: IgA myeloma protein: absence of segmentai flexibility.J. Moi. Biol. 54. 177-185. Weltman, J. K., and Edelman, G. M. (1967). Fluorescence polarization of human yG-immunoglobulins. Biochemistry 6, 1437-1447. Willan, K. J., Golding, B., Givol, D., and Dwek, R. A. (1977).Specific spin labeling of the Fc region of immunoglobulins. FEBS Lett. 80, 133-136. Wrigley, N. G., Brown, E. B., and Skehel, J. J. (1983). Electron microscopy evidence for the axial rotation and interdomain flexibility of the Fab regions of IgG. J. Mol. Biol. 169, 771-774. Yguerabide, J. (1972). Nanosecond fluorescence spectroscopy of macromolecules. In “Methods in Enzymology” (C. H. W. Hirs and S. N. Timasheff, eds.), Vol. 26, pp. 498-577. Academic Press, New York.. Yguerabide, J., Epstein, H. F., and Stryer, L. (1970). Segmental flexibility in an antibody mo1ecule.J. Mol. Biol. 51, 573-590.. Zagyansky, Y. A. (1975). Phylogenesis of the general structure of immunoglobulins. Arch. Biochem. Biophys. 166, 371-381. Zagyansky, Y. A., and Gavrilova, E. M. (1974). Segmental flexibility of human myeloma immunoglobulins A. Zmmunochemistry 11,68 1-682. Zagyansky, Y. A., and Ivannikova, E. I. (197 . T h e general structure of shark (Squalis acantis) and hen (Galus domesti us) immunoglobulins. Mol. Bio. Rep. 1,301-304. Zagyansky, Y. A., Nezlin, R., and Tumerman, L. A. (1969). Flexibility of immunoglobulin G molecules as established by fluorescent polarization measurements. Immunochemistry 6, 787-800. Zagyansky, Y. A., Tumerman, L. A., and Egorov, A. M. (1972). Segmental flexibility of IgM molecules. Zmmunochemistry 9 , 9 1-94.

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This article was accepted for publication on 27 October 1989.

ADVANCES IN 1MML:NOLOC;Y. VOI.. 48

Somatic Diversification of the Chicken Immunoglobulin Light-C hain Gene WAYNE 1. McCORMACK AND CRAIG B. THOMPSON Departments o f Micmbiology and Immunology and Internal Medicine, Howard Hughes Medical Institute, Univenity of Michigan Medical School, Ann Arbor, Michigan 48 109

I. Introduction

A central requirement for the humoral immune system is the ability to specifically recognize a wide variety of molecular antigens. It has been estimated that both humans and mice generate between lo6 and lo8 different antibody molecules during the process of creating an immunological repertoire. In these species the heterogeneity of antibody molecules results from a series of somatic recombinations which leads to the production of a functional immunoglobulin (Ig) molecule during B cell differentiation (reviewed by Tonegawa, 1983; Alt et al., 1986; Hunkapiller and Hood, 1989). A functional heavy-chain gene is assembled in each B cell from an assortment of variable (V), diversity (D), and joining (J) elements. A functional light-chain gene is assembled from an assortment of V and J sequences. These joining events themselves lead to the generation of additional diversity through variations in the precise joining point and d e nova nucleotide addition at the joint (Alt and Baltimore, 1982). Whereas mammalian light chains might be expected to have less diversity than heavy-chain genes, because they lack D segments, additional light-chain diversity has been generated in mammals by having two independent genes ( K and A), either of which can recombine to encode a functional Ig molecule in conjunction with heavy-chain gene. Not all species, however, use somatic recombination as a means of generating the primary Ig gene repertoire. The chicken Ig light-chain (IgL)locus contains a single VL gene segment capable of rearrangement (Reynaud et al., 1985). Despite this, chickens have been shown to display considerable heterogeneity in their circulating Ig light chains, as demonstrated by isoelectric focusing (Jalkanen et al., 1984). Recent experiments have suggested that'chickens create diversity within their IgL genes by a process of gene conversion, using sequence templates derived from V region pseudogene segments (QV) located 5' from the single rearranged VL gene segment (Reynaud et al., 1987; Thompson and Neiman, 1987). 41 Copyright 0 1990 by Acddenlii Press. Inc. All rights of reproduction in any form reserved.

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WAYNE T. McCORMACK A N D CRAIG B. THOMPSON

This diversification process is induced during the clonal expansion of Ig' B cells in the bursa of Fabricius and leads to the generation of a functional immunological repertoire from as few as 3 x lo4 Ig+ cell precursors. Understanding the molecular regulation of this extensive gene conversion process may have important implications for our understanding of somatic diversification of mammalian Ig genes, as well as allow us to characterize the regulation of somatic gene conversion in higher eukaryotes. In this chapter we review the current state of knowledge concerning how the chicken generates an immunological repertoire for its unique IgL gene during B cell development in the bursa of Fabricius. 11. Bursa of Fabricius Is Essential for Normal B Cell Development

The bursa of Fabricius first aroused the interest of immunologists when Glick et al. (1956) described the central role of the bursa of Fabricius in the production of antibodies in chickens. This work led to the separation of lymphocytes into B (bursal-derived) cells essential for antibody production and T (thymus-derived) cells required for delayed-type hypersensitivity and cell-mediated immunity (reviewed by Cooper et al., 1984; Cantor, 1984). This division of lymphocytes into B and T cells became a cornerstone of modern immunology, and the mammalian counterparts of both B and T cells were soon defined. Despite the apparently central role that the bursa of Fabricius plays in avian B cell development, a single mammalian counterpart for the bursa of Fabricius has never been defined. However, in the 30 years since Glick et al.'s original discovery, the role of the bursa of Fabricius in B cell ontogeny has continued to interest avian immunologists. As a result of the ease with which avian embryos can be experimentally manipulated, a cellular characterization of bursal development has been carried out in remarkable detail (for detailed reviews, see Grossi et al., 1976; Glick, 1977; Ratcliffe, 1985; Pink, 1986). In brief, the bursa of Fabricius is a lymphoepithelial organ that arises as an invagination at the base of the posterior cloaca. Beginning at day 4 of embryogenesis, the bursal epithelial anlage begins to proliferate, forming epithelial buds within the underlying lamina propria (Fig. 1). Between days 8 and 14 of embryogenesis, lymphoid cells colonize the epithelial buds to form bursal follicles (Moore and Owen, 1966; Houssaint et al., 1976, 1983). Each follicle is colonized with two to seven lymphoid cells, and subsequent growth of the follicles is due primarily to cellular proliferation (Le Douarin et al., 1975; Lydyard et al., 1976; Pink et al., 1985a; Pink, 1986). There are approximately 10,000 follicles in the bursa of a 4-week-old chick, each containing about lo5 lymphocytes (Olah and Glick, 1978).

SOMATIC DIVEKSIFICATION OF

Igl,

I Hatching

Embryogenesis

43 Posrharching

Granulocytes

9

Prebursal

Stem Cells 0 0

\

Bursa1 Stem Cells

7

-0

0 0

(Days) 5

10

15

20 I(Weeks) 1

2

3

4

FIG.1 . Schematic representation of the basic stages of B cell development in the bursa of Fabricius.

Both the lymphoid and epithelial components of the bursal follicle have been shown to be required for normal B cell development. If the bursal epithelial anlage is removed surgically at 60 hours of embryogenesis (Jalkanen et al., 1983a,b; Corbel et al., 1987) or the differentiation of the epithelial component is inhibited by testosterone treatment between days 6 and 8 of embryogenesis (Cooper et al., 1969; Huang and Dreyer, 1978),the resulting birds are profoundly immunodeficient (Eerola et al., 1983, 1984; Granfors et al., 1982; Jalkanen et al., 1984). Although these birds develop nearly normal numbers of Ig+ cells and levels of circulating Ig at 6 months of age, they are unable to mount primary or secondary immune responses. Nearly all of the surface and circulating Ig’s in these birds is IgM. T h e lymphoid component of the bursal follicle can be depleted specifically by treatment of the developing embryo with cyclophosphamide on days 15-17 of embryogenesis (Lerman and Weidanz, 1970). The grown birds not only demonstrate profound immunodeficiency, but also fail to develop significant numbers of Ig’ cells or circulating Ig’s. In these birds the epithelial component of the bursa remains intact, and, if reseeded with lymphocytes derived from an embryonic bursa, B cell immunity will develop normally (Toivanen and Toivanen, 1973).The embryonic bursal lymphocytes that can reseed the bursal epithelium and regenerate the secretory immune system have been termed “bursal stem cells.” T h e bursal stem cells are Ig’, are present at reasonable levels between day 15 and the time of hatching, and are rapidly lost posthatching (Pink et al., 1985b). They can no longer be detected by a cell transfer assay by 2-4 weeks of age. Interestingly, if the entire bursa is removed surgically

44

WAYNE

'r. M ~ C O K M A C K A N D

C R A I G B . THOMPSON

between days 17 and 18 of embryogenesis, close to 50% of the bursectomized birds will be completely deficient of Igf cells and circulating Ig (Cooper et al., 1969). It appears that all of the cells destined to generate the humoral immune system reside in the bursa during this developmental period. Posthatching, the bursa of Fabricius continues rapid growth for 2-4 weeks. Thereafter, it reaches a plateau and begins to involute between 4 and 6 months of age, as the bird reaches sexual maturity (Glick, 1977). Bursectomy after several days posthatching does not result in loss of the chicken's humoral immune system (reviewed by Pink et al., 1987; Toivanen et al., 1987). Based on the above observations, it has been concluded that the bursa of Fabricius is the normal developmental site where the precursors of mature B cells are expanded within the developing embryo and the primary immunological repertoire of the chicken is generated. Parallel studies in mammals have shown that mammalian B cell precursors are produced continuously from bone marrowderived precursors and that these cells create the primary immunological repertoire through recombination of their Ig genes from a large pool of precursor gene segments (Alt et al., 1986). 111. Structure of the Unique Chicken lgL Gene

Protein structural and sequence data (Hood et al., 1970; Grant et al., 1971) demonstrated that the circulating Ig's of the chicken all share a single A-like light-chain isotype, suggesting the presence of a single IgL gene. A chicken IgL cDNA was first cloned by Reynaud et al. (1983). T h e initial characterization of the chicken IgLgene (Fig. 2) showed that it was composed of the same functional domains as the IgL genes of mammals. A 2 1-amino-acid hydrophobic leader segment was followed by a 92amino-acid V gene segment linked to a J gene segment of 13 amino acids and a constant (C) domain of 103 amino acids. Interestingly, when the V and C region gene segments were used to probe Southern blots of DNA derived from developing polyclonal bursa1 lymphocytes, the cells were found to share a single functional rearrangement of the IgI>gene (Fig. 2) (Reynaud et al., 1985). In order to characterize this rearrangement, Reynaud et al. (1987) cloned and characterized the genomic organization of the chicken IgL gene. As shown in Fig. 3, these studies revealed that the gene contains a single functional VL gene segment separated by 1.8 kb of DNA from a single functional JI. gene segment, which is located 2 kb 5' from a single constant region segment. The functional V element, designated VL,is separated by a small 125-bp intron from the leader segment. The unique

SOMATIC DIVERSIFICAI'ION OF

S T

II

M A I

i i

E X

P N I

I

I I V REGION PROBE

M

G B

V REGION

PROBE

II

45

C

J

V

Igl.

c;

A I

' I

C REGION PROBE

M

G B

C REGION PROBE

FIG. 2. The chicken IgL cDNA structure and genomic organization. (Top) The chicken IgLcDNA structure, including germ-line-encoded restriction endonuclease sites, is depicted. (Bottom) Southern blcts containing germ-line DNA (G) derived from chicken erythrocytes and bursal cell-derived (B) DNA isolated from a polyclonal population of Ig' bursal lymphocytes at 6 weeks of age digested with the restriction endonuclease BclI and hybridized with probes specific for the Igl. V and C gene regions. (Left) The V gene region probe hybridizes to multiple homologous segments in the genome. In contrast (right), the C gene region probe hybridizes in germ-line DNA to a single band. In bursal DNA both probes identify a single new band migrating at 8 kb, which comigrates with both probes. The intensity of this rearranged band is of intensity approximately equal to that of the remaining germ-line band. Nearly 100% of the cells isolated from the bursa express surface Ig, suggesting that nearly all of the rearranged alleles present in this polyclonal population are functional. Marker bands (M) are endlabeled Hind111 fragments of A DNA (23.1, 9.4, 6.7, 4.4, 2.3, and 2.0 kb). UT, Un translated.

leader segment is 5' from a typical Ig promoter containing a conseived octamer box 32 bp upstream from a T A T A box. This organization is also characteristic of mammalian Ig V genes (Parslow et al., 1984). Within t h e 22 kb upstream from the single rearranging VL gene segment, Reynaud et al. (1987) identified 25 V gene-homologous gene segments in both

46

W A Y N E T. McCORMACK A N D C R A I G B . THOMPSON

FIG. 3. Schematic representation of the IgL locus for both rearranged and unrearranged alleles. The IgL locus is organized in the germ-line with a single functional V gene element, designated VLI,separated by 1.8 kb of intervening DNA from a single J gene segment. The J L segment is 2 kb upstream from a single C gene segment. During rearrangement, juxtaposition of V L and J L sequences occurs by deletion of the intervening DNA and creation of a V-J joint. Upstream from the single functional V1. gene element are 25 V gene-homologous segments numbered in order of their occurrence from 3' to 5' from the VL, gene and depicted as determined by Reynaud el al. (1987). Arrows designate the orientation relative to the V L gene. ~

transcriptional orientations. All 25 of these V gene segments lack leader segments, as well as recombination signal sequences, and have therefore been designated V segment pseudogenes (rjrV1-25; see Fig. 3). These data were consistent with the observation that there is a single functional rearrangement of the IgL gene which leads to the juxtaposition of the VL gene segment with the single J L gene segment in all developing bursa1 lymphocytes. The presence of single functional IgI. V and J gene segments and the resultant limited potential for functional rearrangement contradicted the hypothesis that the bursa of Fabricius induced the generation of the immunological repertoire in the chicken by generating combinatorial diversity through stimulation of Ig gene rearrangement. Therefore, in the last several years the regulation of Ig12 gene rearrangement and diversification during development has been the subject of intense investigation by several laboratories. IV. Rearrangement of the IgLGene

The chicken IgL locus, because of its small size and unique germ-line structure, has readily lent itself to studies characterizing the timing and molecular mechanism of V-J joining. Joining of the single functional V segment with the single J segment results in the deletion of the DNA between VI, and J L from the genome. This deletion was found to be accomplished by a molecular mechanism which results in the precise

SOMATIC DIVERSII;I30 clones analyzed at each time point.

Bursal antigen-induced proliferation could account for the observed exponential growth of embryonic surface Ig+ bursal cells (Lydyard et al., 1976; Reynolds, 1987). Although such a mechanism would account for the bursa’s ability to induce the large-scale amplification of B cell progenitors, it provides no explanation for how an effective immunological repertoire could be created from a single functional IgL gene (see below). VI. Bursal Stem Cell

As mentioned in Section 11, the Ig+ cells that can home to the bursal follicle and regenerate the lymphoid component of a follicle have been termed “bursal stem cells.” T h e frequency of bursal stem cells in the developing bursa can be estimated based on bursal reconstitution experiments. For example, lo6 lymphoid cells from an 18-day embryo can be used to repopulate the bursal follicles of a second 18-day embryo which has had the lymphoid component eliminated by cyclophosphamide treatment between days 15 and 17 of embryogenesis. Based on reconstitution with cells bearing congenic markers or distinct IgLalleles, it appears that individual follicles are reconstituted by one, or at the most two, stem cells (Pink et a/., 1985b; Weill et al., 1986). Because there are approximately lo4 bursal follicles, the frequency of bursal stem stems at 18 days of embryogenesis is, therefore, approximately one in 50- 100 cells. As in the previous section, we would suggest that the cell that accounts for the expansion of the lymphoid component of individual bursal follicles is a cell that expresses the unmodified Ig molecule encoded by germ-line gene segments. In order to determine the frequency of such cells, we sequenced 42 randomly chosen rearranged V-J segments from

52

W A Y N E T. Mi.