Serologic crossreactions among primate immunoglobulins.

We have generated and characterized 50 murine monoclonal antibodies (mAb) specific for baboon IgG. We examined crossreactivity of these mAb to baboon IgM and immunoglobulin (Ig) of various other primates including human, chimpanzee, rhesus monkey, cynomolgus monkey, and African green monkey. Those mAB that crossreacted with human IgG were further examined using myeloma proteins for specificity to human Ig subclasses. One mAB crossreacted with all four human IgG subclasses and with human IgM. We further analyzed this reactivity utilizing Bence Jones proteins representative of various light (L) chain germline gene family products. This mAB reacted with Bence Jones proteins indicating the recognition of a kappa (k) L chain specificity associated with the kappa I, kappa III, and kappa IV subgroups, but not with kappa II. Based on the differences between kappa II germ line gene encoded L chains and the other kappa L chain subgroups, we ascribe this reactivity to six amino acids that define a discontinuous epitope.

Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light-chain amyloid proteins.

The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light-chain variable-region single-gene family VkIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three-dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VkIV products have tertiary structural features comparable to those of the patient-derived proteins. This well-defined set of three clinically characterized human kIV light chains, together with the capability to produce these kIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light-chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light-chain amyloidogenicity.

Pathogenic potential of human monoclonal immunoglobulin light chains: relationship of in vitro aggregation to in vivo organ deposition.

The deposition of certain Bence Jones proteins as tubular casts, basement membrane precipitates, or amyloid fibrils results in the human light-chain-associated renal and systemic diseases--myeloma (cast) nephropathy, light-chain deposition disease, and immunocyte-derived (primary or AL) amyloidosis. To determine if light-chain nephrotoxicity or amyloidogenicity is related to the propensity of these components to form high molecular weight aggregates under physiological conditions, we used a size-exclusion chromatographic system to study 40 different Bence Jones proteins. Each samples was tested over a wide range of protein concentration in three different buffers varying in pH, osmolality, and the presence or absence of low concentrations of urea. Thirty-three of the 35 proteins found clinically and/or experimentally to form in vivo pathologic light-chain deposits were shown to undergo high-order self-association and form high molecular weight aggregates. In contrast, of five nonpathologic proteins, one showed polymerization under the chromatographic conditions used. The correlation between the in vivo results achieved by size-exclusion chromatography and that found in vivo provides (i) a rapid diagnostic method to identify potential nephrotoxic or amyloidogenic Bence Jones proteins and (ii) an experimental means to gain new insight into the physicochemical basis of light-chain aggregation and the treatment of those invariably fatal disorders associated with pathologic light-chain deposition.

Novel arrangement of immunoglobulin variable domains: X-ray crystallographic analysis of the lambda-chain dimer Bence-Jones protein Loc.

We have characterized and crystallized a human lambda I light-chain dimer, Bence-Jones protein Loc, which has variable (V) region antigenic determinants characteristic for the lambda I subgroup and constant (C) region determinants of the C lambda I gene Mcg. The crystal structure was determined to 3-A resolution; the R factor is 0.27. The angle formed by the twofold axes of the V and C domains, the "elbow bend", is 97 degrees, the smallest found so far for an antibody fragment. The antigen-binding site formed by the two V domains of the Loc light chain differs significantly from those of other immunoglobulin molecules (light-chain dimers and Fab fragments) for which X-ray crystallographic data are available. Whereas, in other antibody fragments, the V domains are related by a local twofold axis, a local twofold screw axis with a translational component of 3.5 A relates the V domains in protein Loc. In contrast to the classic antigen binding "pocket" formed by V domain interactions in the previously characterized antibody structures, the V region associations in protein Loc result in a central protrusion in the binding site, with grooves on two sides of the protrusion. The structure of protein Loc indicates that immunoglobulins are physically capable of forming a more diverse spectrum of antigen-binding sites than has been heretofore apparent. Moreover, the unusual protruding nature of the binding site may be analogous to structures required for some anti-idiotypic antibodies. Further, the complementarity-determining residues form parts of two independent grooves.(ABSTRACT TRUNCATED AT 250 WORDS)

Small-angle neutron scattering study of Bence-Jones protein Mcg: comparison of structures in solution and in crystal.

Immunoglobulins fragments are composed of globular domains linked by extended polypeptide segments. The molecular flexibility inherent in this arrangement allows for significant potential differences between structures observed in the crystalline state and those attained in solution. Small-angle neutron scattering measurements in dilute solution were performed on the Mcg Bence-Jones protein dimer, for which performed on the Mcg Bence-Jones protein dimer, for which accurate atomic coordinates have been determined by crystallographic methods [Edmundson, A. B., Ely, K. R., Abola, E. E., Schiffer, M., & Panagiotopoulos, N. (1975) Biochemistry 14, 3953-3961; Schiffer, M. (1980) Biophys. J. 32, 230-232]. The measured radius of gyration (Rg) in H2O buffer is 24.0 +/- 0.4 A and in D2O buffer is 23.3 +/- 0.1 A; the calculated value of Rv (Rg in vacuo) is 24.0 A. The above values compare well with the calculated Rg value of 23.6 A when refined coordinates of the trigonal crystal form of the Mcg Bence-Jones protein are used. On the basis of a match point of 44.2% D2O concentration, the experimental partial specific volume is 0.74 cm3/g. The experimentally derived molecular weight of 47 000 is in very good agreement with that (45 500) calculated from the amino acid composition. For comparison with different Fab's (antigen binding fragments) exhibiting various "elbow bends" due to the flexibility of the switch peptide between variable and constant domains of the immunoglobulin chains, calculation of the Rg value of the Mcg dimer was performed as a function of the elbow bend. The Rg varied from 22.8 to 26.0 A as the elbow bend was opened from 100 degrees to 180 degrees; the maximum radius of gyration of the particle was 26.5 A with the switch peptide stretched by separating the variable and constant domains by an additional 1.5 A at an elbow bend of 180 degrees.

Self-association of human immunoglobulin kappa I light chains: role of the third hypervariable region.

Gel electrophoresis and molecular sieve chromatography were used to compare 17 different human kappa I type Bence Jones proteins including 5 for which the amino acid sequence is known. Although electrophoresis in the presence of NaDodSO4 showed uniformity of covalent dimer and monomer molecular weights, Sephadex chromatography under nondissociating conditions showed that monomers eluted with different apparent molecular weights. These differences were attributed to heterogeneity in light chain self-association; dimerization constants of the 17 proteins, calculated from a computer simulation of their behavior upon gel filtration, ranged from less than 10(3) to greater than 10(6) M-1. The variable region, more specifically the third hypervariable region, appears to be responsible for the variation in the dimerization constant. Association properties of light chains of known sequence suggest that the presence of an aromatic or hydrophobic residue at position 96 enhances dimer formation whereas a charged residue at that position results in light chains remaining stable monomers. The location of hypervariable residue 96 within the amino-terminal portion of the joining segment of the variable region suggests that the joining region may account for the variability of self-association of light chains and, moreover, that it has a function in determining the selective association of immunoglobulin polypeptide chains.

Interconversion of conformational isomers of light chains in the Mcg immunoglobulins.

Previous crystallographic studies in this laboratory demonstrated that immunoglobulin light chains with the same amino acid sequence can have at least two and probably three or more conformations, depending on whether the second member of an interacting pair is a light or heavy chain. If a heavy chain is not available in the assembly medium, a second light chain plays the structural role of the heavy chain in the formation of a dimer. In the present work, the lambda-type light chains were dissociated from the heavy chains of a serum IgG1 immunoglobulin from the patient Mcg and reassembled noncovalently into a dimer. The reassembly process was completed by allowing the penultimate half-cystine residues to form an interchain disulfide bond. The covalently linked dimer was compared with the Mcg urinary Bence-Jones dimer, for which an atomic model has been fitted to a 2.3-A electron density map. The assembled dimer and the native Bence-Jones protein were indistinguishable in their chromatographic and electrophoretic properties, as well as in their activity in the binding of bis(dinitrophenyl)lysine. These results indicate that the light chains can be converted into the two types of Bence-Jones conformational isomers. The procedure was also reversed: the two Bence-Jones isomers were dissociated and reassembled as the single type of isomer associating with each of two heavy chains in the IgG1 protein. The change in activity occurring when a light chain associates with a heavy chain instead of a second light chain is illustrated by the fact that the Mcg IgG1 immunoglobulin does not bind dis(dinitrophenyl)lysine in measurable amounts.