Faculty Bibliography

Multiclonal gammopathies associated with multiple myeloma may result either from a neoplastic transformation of a cell clone undergoing immunoglobulin class switching or from independent transforming events yielding proliferation of unrelated plasma cell clones. The simultaneous presence of more than one neoplastic clone may possess regulatory implications in terms of cell proliferation, clonal expansion, secretion of M-components or response to chemotherapy. We report a patient, diagnosed with multiple myeloma stage IIIa, who presented with two well-defined homogeneous IgG1-kappa components in the serum (designated WER-1 and WER-2) with striking differences in their plasma concentration and response to the classic melphalan/prednisone treatment. Immunochemical characterization and amino terminal sequence analysis of both the heavy and light chains of each M-component undoubtedly determined their biclonal origin. WER-1 was identified as IgG1(VHII)-kappaI while WER-2 was classified as IgG1(VHIII)-kappaIII. The plateau phase was characterized by very low or undetectable levels of WER-2, a high, almost constant, concentration of WER-1 and the absence of Bence Jones proteinuria, whereas these parameters were completely reversed during the escape phase with levels resembling those observed at the time of diagnosis. The statistically significant negative correlation between the biclonal components and the different susceptibility to the treatment clearly suggests regulatory interactions between the clones WER-1 and WER-2

Fibrillary glomerulonephritis is a disease of uncertain origin and pathogenesis characterized by nonamyloidotic fibrils in glomeruli. We report immunohistological, immunochemical, and biochemical studies of a serum fibrillar cryoprecipitate obtained from a patient with fibrillary glomerulonephritis, that formed on prolonged storage at 4 degrees C. By Western blot and amino acid sequence analysis, the cryoprecipitated fibril components consisted of immunoglobulins, heavy chains gamma and mu, light chains kappa and lambda, and fibronectin, similar to the proteins identified by immunofluorescence and immunoelectron microscopy in the glomerular fibrils. These findings support the hypothesis that serum precursors may be the source of the fibrillar deposits and suggest a role for immunoglobulin-fibronectin complexes in the pathogenesis of fibrillary glomerulonephritis

Fibronectin (Fn), a mosaic protein composed of multiple copies of three different module types (Fl, F2 and F3), has been found associated with circulating immune complexes (ICs) and immunoglobulin (Ig) aggregates in a variety of IC diseases and myeloproliferative disorders. We have previously shown that a proteolytic fragment of Mr = 25,900 Da, from the NH2-terminal domain of Fn, composed of five type 1 modules (1Fl -5Fl) binds to the major Ig classes under physiologic conditions, suggesting that the presence of Fn in ICs and cryoglobulins results from a physicochemical binding interaction between these two molecules. Using an ELISA, we now show that the interaction between Fn and IgG is: (1) not influenced by any other constituent of plasma; (2) unaffected by temperature; and (3) has an estimated Kd of 3.77 x 10(-9) M. In addition, we have further delineated the respective sites involved in the interaction between Fn and IgG. Recombinant type l module pairs (1Fl.2Fl and 4Fl.5Fl) from the NH2-terminus of Fn, expressed in yeast, were employed in an ELISA and affinity chromatography and compared with the 25.9 kDa (1Fl - 5Fl) fragment and intact Fn for binding to IgG. The 4Fl.5Fl and the 25.9 kDa fragment bound to immobilized IgG and inhibited Fn binding to IgG to nearly the same extent as the intact molecule (IC50: Fn = 6.77 x 1O(-9) M; 25.9 kDa fragment = 5 x 10(-9) M; 4Fl.5Fl = 7.6 x 10(-9) M). Thus, the binding site for IgG on the Fn molecule is localized to and completely conferred by the 4Fl.5Fl module pair (residues 151-244). Similar experiments using papain-generated Fab and Fc fragments of IgG localized the Fn binding site on IgG to the Fe region of the IgG molecule. Fn bound to the Fc fragment with a nearly identical Kd of 3.69 x 10(-9) M, as to intact IgG (3.77 x 10(-9) M). These studies support the hypothesis that the interaction between Fn and Ig may contribute to the pathophysiology of immune complex related disorders

The fibronectin (Fn) monomer contains two major sites of fibrin binding affinity present within the NH2-terminal and COOH-terminal domains; they consist of five (1F1-5F1) and three (10F1-12F1) consecutive type 1 modules, respectively. Recently, we have reported that the fourth and fifth type 1 module pair (4F1.5F1) of the NH2-terminal domain of fibronectin demonstrated fibrin binding ability (Williams, M. J., Phan, I., Harvery, T. S., Rostagno, A., Gold, L. I., and Campbell, I. D. (1994) J. Mol. Biol. 235, 1303-1311). In an attempt to further localize fibrin binding activity and to characterize the nature of the interaction between different type 1 modules of Fn and fibrin, we have tested a range of recombinant proteins and subtilisin generated proteolytic fragments of Fn in an enzyme-linked immunosorbent assay (ELISA) and by fibrin affinity chromatography. Of the recombinant proteins, we found that only the 4F1.5F1 exhibited significant fibrin binding activity, while 1F1, 1F1.2F1, 7F1, and 10F1 had little to no affinity for fibrin. On a molar basis, 4-5 times more 4F1.5F1 than a proteolytic fragment, corresponding to 1F1-5F1 (25.9 kDa) was required to cause 50% inhibition (IC50) of intact biotinylated Fn binding to fibrin in a competitive ELISA. This suggests that all five type 1 modules in tandem engender higher fibrin binding activity than the 4F1.5F1 alone. Furthermore, since fibrin binding activity of the intact Fn molecule was inhibited, by 70-80%, by the 4F1.5F1, the 25.9-kDa fragment, and a MoAb mapped to an epitope on the 4F1.5F1, the fibrin-binding site within the 4F1.5F1 contributes greatly to the non-covalent interaction of intact Fn with fibrin. These results provide significant insight into the Fn/fibrin interaction, a major component of the processes of wound repair and fibrin matrix assembly

The tertiary structure of the fourth and fifth type 1 module pair from the N terminus of human fibronectin, has been determined by two-dimensional homonuclear 1H nuclear magnetic resonance (NMR) spectroscopy. Comparison of each module fold with those of two other type 1 modules shows that the type 1 'consensus' structure is conserved in the pair. The modules connect end-to-end to form an elongated structure with a limited clockwise twist around the long axis, from N to C terminus. The short five residue linker sequence forms a tight loop and the relative orientation of the two modules is maintained by fixed and intimate hydrophobic contacts, dominated by a non-conserved tryptophan residue from the fourth type 1 module. The protein binds specifically to fibrin in an ELISA and surface accessible residues that may be involved in this and other protein interactions can be identified. The structure provides an insight into how chains of type 1 modules may link up in intact fibronectin