Keith J. Dorrington

Structure-Function Relationships in Human Immunoglobulin E

The current interest in IgE has evolved from the discovery that antibodies of this minor class of immunoglobulin carry the biological, immunochemical and physical characteristics of reaginic antibodies. The important property of IgB antibodies which allows them to mediate Type I immediate hypersensitivity is their ability to bind reversibly with high affinity to specific membrane receptors on basophils and mast cells. Combination of specific cell-bound antibody with antigen triggers a series of events which ultimately leads to the release of vasoactive amines and other pharmacologically active substances responsible for the clinical manifestations of hypersensitivity. The nature of these intracellular events is poorly understood. Indeed a substantial number of people may ask why these events need exist at all, particiilarly when they are suffering through the ragweed or other airborne-antigen season! In other words the physiological importance of IgE is not known, although it has been implicated in resistance to parasitic infestation.

Conformational changes accompanying the dissociation and association of immunoglobulin-G subunits

Abstract The circular dichroism spectra, between 210 and 320 nm, of human pooled IgG, two IgG myeloma proteins and their isolated heavy and light chains have been determined. Below 250 nm all spectra are dominated by a negative band at 217 nm, probably due to β-structure. Above 250 nm the spectra are complex and indicate the presence of several overlapping side-chain transitions. Three different samples of pooled IgG give similar spectra as do their heavy chains and light chains. However, the heavy chains differ significantly from the light chains. The myeloma protein spectra differ from the pooled IgG and from each other, probably reflecting variable region conformational differences. Pooled IgG and one of the myeloma proteins shows a positive band at 235 nm, contributed by the light chain and shown to be probably due to a tyrosine transition on the basis of titration data. Algebraic addition of the subunit spectra did not yield the spectrum of the parent IgG indicating unique conformational features associated with the intact protein. The differences suggest a greater contribution of certain light chain transitions in native IgG than expected. The 235-nm tyrosine band is considerably enhanced as are other bands, probably due to tryptophan residues. There is evidence of other residues experiencing altered environments on subunit interaction but assignment of these changes to either subunit or to specific side-chains is difficult. The 217-nm band is reduced in intensity in native IgG when compared to the subunits suggesting secondary structure changes. Recombination of the myeloma subunits results in the complete recovery of the native conformation. This does not occur when pooled IgG subunits are recombined. A partial recovery takes place but differences between the native and recombined proteins are apparent throughout the wavelength range. Of particular interest is the failure to fully recover the 235-nm light chain tyrosine band.

Properties of the Fc Receptor on Macrophages and Monocytes

Macrophages and monocytes possess a surface receptor specific for the Fc region of certain subclasses of IgG. The binding site on IgG is localized within the Cgamma3 homology regions of the heavy chains. The intrinsic affinity of the receptor for IgG ranges from 10(6) to 10(8) M-1 depending on species and subclass of IgG. The most definitive studies on mouse macrophages indicate that IgG2a rapidly associates and dissociates from the receptor. The overall reaction is exothermic; increasing temperature lowers the intrinsic affinity. The Fc receptor, in common with many other membrane components, may be capped by polyvalent ligands under permissive conditions and capping is inhibited by azide. Data on the chemistry of the receptor is both sparse and conflicting. Sensitivity of the receptor of proteolytic enzymes has been clearly demonstrated for mouse macrophages although rabbit and guinea-pig cells appeared to carry resistant receptors. IgG-binding may be inhibited if cells are treated with phospholipases and certain group-specific reagents. Evidence is reviewed indicating that the Fc receptors found on various cell types are different. The macrophage Fc receptor appears to play a role in mediating phagocytosis and in non-immune cytotoxicity. Whether the receptor serves only to concentrates sensitized target cells at the cell surface or whether occupation of the receptors results in modulation of effector cell function remains to be determined.

A monoclonal immunoglobulin G1 in which some molecules possess glycosylated light chains--I. Site of glycosylation.

Abstract A human monoclonal IgG1 κ protein (Hom) was found to possess two species of light chain with molecular weights of 23,000 and 28,000 respectively. The difference in mass was due to the presence of a carbohydrate prosthetic group on the 28,000 dalton species. The two species of light chain were separated by chromatography on CM-cellulose. Circular dichroism failed to detect any significant differences in conformation between the glycosylated and non-glycosylated forms and both species recombined with a heavy chain in an identical fashion as judged by difference spectroscopy. An anti-idiotypic antibody raised against the glycosylated Fab-fragment of IgGlHom was unable to distinguish between this species and the non-glycosylated Fab fragment, suggesting that the structure of the combining site is identical or very similar in both molecules. Amino-acid sequence analyses showed that both light chains had an identical sequence up to residue 41. Further studies on the glycosylated light chain, using o -iodosobenzoic acid fragments, localized the carbohydrate attachment site to asparagine 107, the penultimate residue of the J κ region which links V κ to C κ . The sequence around the attachment site was Asn-Arg-Thr which satisfies the requirements for an acceptor sequence. The non-glycosylated light chain was found to have the same sequence in this region indicating that the absence of glycosylation was not due to the lack of an acceptor sequence. A kinetic mechanism has been proposed to account for the incomplete glycosylation of the light chains of IgG1Hom in which there is a competition between the rate of folding of the nascent polypeptide and the rate of attachment of core sugars via the dolichol pyrophosphate pathway.

Structural basis for the preferential association of autologous immunoglobulin subunits: Role of the J region of the light chain☆

We have used a series of sequenced V kappa 21 L-chains produced from murine myelomas to determine whether the V or J segment of the V region is responsible for dictating preferential recombination. In each competitive recombination, equimolar amounts of two different L-chains, selected on the basis of common V or J segments, were allowed to compete for a limiting amount of H-chain. It was found that the J segment of the L-chain was primarily responsible for dictating the ability of a chain to compete and that the nature of residue 96, the first residue of the J segment, was particularly important. Specifically, charged residues caused the L-chain to compete poorly against L-chains with hydrophobic side chains at this position. Furthermore, if Phe or, to a lesser extent, Tyr were present at position 96, the L-chain competed more successfully than chains with Trp-96 or Leu-96. This suggests that both the aromaticity and size of this residue were important factors in determining preferential recombination. It was also found that all other residues in VL were secondary to residue 96 in contributing to the ability of a chain to compete. Finally, unlike all previous studies, we observed a substantial number (64%) of preferred heterologous recombinations. These results are consistent with the hypothesis that the VL and VH gene rearrangements occur independently, thus resulting in random pairing of VH and VL domains.

Proteins associated with activity of Fc receptors on isolated human placental syncytiotrophoblast microvillous plasma membranes

To identify Fc receptors from human placental microvilli, proteins that were liberated by detergents from human placental synctiotrophoblast microvillous membranes (StMPM) were characterized by their abilities to bind human IgG in immune complexes with sheep or goat anti-human IgG and to monomeric rabbit anti-dinitrophenol (DNP) IgG bound to DNP-lysine Sepharose. Three placental IgG-binding proteins coprecipitated with immune complexes (Mr = 68,000, 52,000-56,000, 40,000) and were designated pIBP68, pIBP56 and pIBP40, respectively. Of the three proteins only pIBP56 bound to immobilized monomeric rabbit IgG. It was isolated from detergent lysates of StMPM and LDS/phenol glycoprotein extracts of placental plasma membranes suggesting that pIBP56 was a glycoprotein FcR previously reported (Mikulska et al, 1982). The binding specificities of pIBP56 and pIBP40 appeared to be detergent dependent. Photoaffinity crosslinking of StMPM surface proteins in situ to monomeric rabbit derivatized with N-succinimidyl(4-azidophenyl)-I, 3-dithiopropionate identified IgG-binding proteins identical in size to pIBP56 and pIBP40. Crosslinking further suggested that monomeric IgG covalently bound to a complex of StMPM proteins with a total size of 110,000-120,000 Mr. The findings suggest that pIBP68, pIBP56 and pIBP68 are responsible for IgG binding activity of placental StMPM.

The role of the VL- and VH-segments in the preferential reassociation of immunoglobulin subunits

Competitive reassociation experiments, in which equimolar amounts of two different L-chains were allowed to compete for a limiting amount of H-chain, were performed to assess the role of the V kappa- and J kappa-segments on the ability of an L-chain to compete. Using H- and L-chains from the murine anti-phosphorylcholine (PC) myelomas, TEPC15, MOPC167 and MCPC603, and a series of V kappa 21 L-chains, it was found that the V kappa 21 L-chains competed uniformly better than the anti-PC L-chains, when the anti-PC H-chains were used, despite any differences in the J-segments of the competing L-chains. In addition, when the anti-PC L-chains, which all employ identical J kappa-segments but very diverse V kappa-segments, were in competition against each other, a hierarchy of competitive ability existed which was independent of whether the chains were autologous or heterologous and independent of antigen binding activity. Competitive reassociation experiments between the V kappa 21 and anti-PC L-chains were also performed using the heterologous anti-lysozyme monoclonal HyHEL-10 H-chain or the anti-galactan J539 H-chain, and it was found that the relative competitive ability of the V kappa 21 L-chains with respect to the anti-PC L-chains was dependent on which H-chain was employed. The results suggested that the main factor favouring preferential reassociation by any particular L-chain was the V kappa-segment and that the effects of the J kappa-segment could not be observed where a high degree of diversity in the V-segments existed. Furthermore, while the results implied that specific pairs of VH- and VL-domains had a higher affinity for each other, this was not a necessary criterion in the formation of autologous pairs of H- and L-chains as demonstrated by the preferential heterologous reassociation of the V kappa 21 L-chains over the autologous anti-PC L-chains. These results were consistent with the independent, random rearrangement of immunoglobulin H- and L-chain V-domain gene segments and predict that the hypothetical repertoire of antibodies is not limited by the selection of specific pairs of high-affinity VH-VL domains.

Conformational studies on subfragments from the Fc region of human immunoglobulin G.

Abstract The circular dichroism (CD) spectrum of Fc fragment from Immunoglobulin G is dominated by a negative band at 217 nm. In contrast, pFc′, a subfragment of Fc representing a dimer of the C H 3 domains of heavy chain, showed no minimum at this wavelength but rather at 224 nm. Marked differences were also apparent in the aromatic side chain CD region above 250 nm. A papain-produced fragment of Fc related to but smaller than pFc′ showed CD properties quantitatively and qualitatively different from pFc′. Studies with a tryptic fragment of pFc′ indicated that the conformational differences between pFc′ and Fc′ were primarily related to the proteolytic removal of a C-terminal tridecapeptide in the Fc′ sequence relative to pFc′. A comparison of the CD spectra of pFc′ with the constant region domain of light chain indicates that despite marked sequence homology between domains their conformations may be quite different.

Induced optical activity (circular dichroism) of antibody-hapten complexes

Abstract The induced optical activity (circular dichroism) originating from the interaction of dinitrophenylated and trinitrophenylated haptens with antibody has been used as a probe of the antibody combining site. A mouse IgA myeloma protein (MOPC-315) with high affinity for DNP- and TNP-ligands was employed as an example of a homogeneous antibody. The sign and the magnitude of circular dichroism bands differed when DNP- and TNP-haptens were bound, respectively, to the single homogeneous MOPC-315 protein. Differences in the relative magnitude of distinct circular dichroism bands also were seen when the DNP-group was incorporated into different haptens and complexed with the same homogeneous antibody. The circular dichroism spectra of hapten complexed with native MOPC-315 protein, mildly reduced and alkylated protein, and reassociated MOPC-315 protein were similar. The induced circular dichroism of ϵ-DNP-lysine bound to a goat anti-DNP antibody differed markedly from the circular dichroism spectrum of the same ligand bound to the MOPC-315 protein. Circular dichroism has been shown to offer a new and powerful parameter for studying and comparing the fine structure of the combining sites of antibodies of similar specificity, and to probe the reformed site resulting from the reassociation of heavy and light polypeptide chains.

Contribution of the VK4 light chain to antibody specificity for lysozyme and β(1,6)d-galactan

The VL amino acid sequence of an anti-lysozyme hybridoma protein, HyHEL-5, was determined. HyHEL-5 expresses a V region of the VK4 family and JK1. The VK4 family also includes light chains from galactan binding antibodies, although sequence comparisons suggest that a different member of this family is used to encode HyHEL-5. The HyHEL-5 light chain has a deletion of residue 96, such that L3 is one residue shorter than the majority of murine L3. Chain recombination experiments, employing H and L chains from different anti-galactan and anti-lysozyme binding antibodies, were performed to examine the contribution of the H and L chain in dictating specificity for either galactan or the lysozyme epitope recognized by HyHEL-5. The results indicate that, although the ability to bind galactan vs lysozyme is absolutely heavy-chain dependent, having the appropriate heavy chain is not sufficient for specific high affinity binding. Both the L chains from HyHEL-5 and J539 (a galactan-binding myeloma protein) were capable of supporting binding to galactan in combination with the J539 H chain, but affinity for galactan is less with the HyHEL-5 L chain. Only VK4 L chains supported binding of the HyHEL-5 heavy chain to the HyHEL-5 epitope, although binding with the J539 L chain was low affinity and relatively nonspecific.