Faculty Bibliography

The partial amino acid sequence of the Fc region of an unusual monoclonal immunoglobulin molecule (Goe), which had the allotypic markers Gm (b0, b3, b5, s, t, v), rarely encountered in Caucasians, was determined. Protein Goe was previously shown to belong to the gamma 3 subclass by antigenic typing, to possess a gamma 3-like hinge region and a gamma 1-like carboxy-terminal octadecapeptide, and to bind to staphylococcal protein A. The sequence of protein Goe resembled that of gamma 3 molecules except for the presence of tyrosine at position 296, alanine at position 339, and histidine and tyrosine at positions 435 and 436. It is of interest that histidine 435 appears to play an important role in binding to Staph protein A. Since tyrosine and phenylalanine at 296 and 300 are typical of G3m(g) molecules, whereas protein Goe is G3m(g-), this may correspond to the non-b1 allotypic marker. Of the numerous explanations to account for these findings, the most likely possibilities are that protein Goe is either a hybrid molecule or the product of a germ line gene representing the G3m s allotype, which is rare in Caucasians and common in Mongoloid populations. Support for the latter alternative is provided by the isolation from normal serum of a small amount of a protein having many of the properties of protein Goe.

Amyloid fibrils were isolated from spleen and thyroid obtained at autopsy from one patient (S.K.O.) of Jewish origin with familial amyloidotic polyneuropathy. Gel filtration on Sephadex G100 after solubilization in 5 M guanidine HCl yielded three major components with 14,000, 9,000, and 5,000 mol wt, respectively. The two larger components shared antigenic determinants with human prealbumin. Amino acid analysis and amino terminal sequence studies revealed the 14,000-mol wt protein to be an intact prealbumin subunit. The 9,000-mol wt fragment obtained in highest yield encompassed the region from position 49-127 and the 5,000 mol wt fraction encompassed the amino terminal of prealbumin (position 1-48). An amino acid substitution (Gly/Thr) was detected at position 49, where enzymatic cleavage occurred. Thus, several prealbumin-derived fragments, predominantly the carboxyl end, constitute the amyloid fibrils in a heredofamilial amyloidosis syndrome of dominant inheritance.

The primary structures of the major coat proteins from If1 and Ike filmentous coliphages have been determined by automated Edman degradation before and after cyanogen bromide cleavage and by manual sequencing of certain tryptic peptides. Carboxypeptidase A and B digestion was also used to determine the sequence of the COOH termini of these proteins. A comparison of the major coat proteins from these two phages with those from other filamentous phages show that they all share several common features, namely an asymmetric distribution of positively and negatively charged amino acid residues, which are clustered with the COOH-terminal and NH2-terminal regions respectively, and a region of 19 residues which is located in the middle of the polypeptide chain. The consequences of this charge distribution for a possible mechanism of virus maturation are discussed.

Strains of Neisseria meningitidis produce two distinct extracellular IgA proteases that cleave the human IgA1 heavy chain at different points within the hinge region. Type 1 protease cleaves the prolyl-seryl peptide bond at position 237-238; type type 2 protease cleaves the prolyl-threonyl bond two residues amino terminal to that bond attacked by type 1 enzyme. Each meningococcal isolate elaborates only one of these two enzymes, and the type of protease produced correlates with certain serogroups: group A yielding only type 1, and groups X and Y only type 2 enzyme. In addition, analysis of amino acid sequences of human alpha-chain proteins reveals that the repeating octapeptide characteristic of the IgA1 hinge region is actually triplicated.

To search for human antiidiotypic antibodies, cryoglobulins from 11 patients with mixed cryoglobulinemia, 7 of whom had evidence of prior hepatitis B virus infection were separated on Sephadex G-200. Each of the IgM, 8 of which were IgM kappa, was tested with the F(ab')2 fragments prepared from each of the IgG fractions in a solid phase assay by using binding of 125I Staph A protein after the addition of rabbit anti-IgM. Unlike rabbit anti-Fab, which reacted approximately to the same extent with all F(ab')2 fragments, the IgM varied in their binding to F(ab')2 fragments, reacting with 2 to 10 antigens. Nine reacted with their autologous antigen and in 5 instances autologous reactivity exceeded that with heterologous F(ab')2 fragments. Reactivity was not related to prior exposure to HBV. Though absorption with Cohn fraction II F(ab')2 fragments generally abolished reactivity, 1 IgM protein continued to react with nine F(ab')2 fragments and 4 others with the F(ab')2 fragment from a single patient. Even when they contained only a single type of kappa-chain, the IgM appeared to contain multiple antibodies since absorption with solid phase Fc fragments or IgG removed anti-Fc rheumatoid factor activity but failed to affect binding to F(ab')2 fragments. Five of 7 IgM were able to bind the Fv region of a monoclonal IgM kappa-protein. Some of the anti-Fab antibodies were directed against idiotypes since addition of HbsAg to F(ab')2 fragments from HBsAb-positive IgG resulted in a marked decrease in binding of the IgM fraction in 6 out of 7 studies, whereas no decrease was noted in 6 experiments with F(ab')2 fragments that were HBsAb negative. The possible existence of antibodies against other idiotypes or other determinants in the Fab region cannot be excluded.

Two human IgM myeloma proteins, IgMWEA and IgMMAY, were found to react with agar and Klebsiella polysaccharides that contain pyruvylated D-galactose (DGal). Quantitative precipitin data and precipitin inhibition studies with methyl alpha- and beta-glycosides of 4,6-pyruvylated-D-galactose showed their combining sites to be different, although each was directed against the pyruvylated-D-Gal, one reacting most specifically with Klebsiella polysaccharides with terminal nonreducing beta-linked 2,4 pyruvylated-D-Gal, whereas the other reacted equally well with Klebsiella polysaccharides that contain 3,4 beta-linked and 4,6 alpha-linked terminal nonreducing pyruvylated-DGal. Inhibition studies showed that both sites are directed toward one of the two space isomers of 3,4- or 4,6-pyruvylated DGal, the form in which the methyl group of the pyruvate is equatorial, or endo, and its carboxyl group axial, or exo, to the plane of the acetal ring. Coprecipitation studies showed the combining site of IgMWEA to be located on an (Fab')2 fragment and not on the (Fc)5mu fragment. The monoclonal peak in the serum of IgMMAY was specifically precipitated by Klebsiella polysaccharide. Myeloma proteins with specificities of this type may occur with reasonable frequency in humans and may be a consequence of clonal expansion from inapparent infection, carrier states, or disease produced by various Klebsiella organisms.

The complete sequence of a gamma 3 heavy-chain disease (HCD) protein Wis is presented. The molecule is a dimer of a 289-residue chain linked by 12 disulfide bonds. Protein Wis has an unusual amino terminus, followed by a deletion of most of the VH domain. After a small stretch homologous to the VC joining region, there is a second deletion which ends at the beginning of the quadruplicated hinge. Two carbohydrate groups are linked to Asn-6 and -140. the molecule has an extra interchain disulfide bridge at position 7 in addition to the 11 normally present in the quadruplicated hinge. The previously noted homology to the gamma 1 heavy chain is striking; from positions 224 to 234, protein Wis resembles gamma 1 Nei [Ponstingl, H., & Hilschmann, N. (1976) Hoppe-Seyler's Z. Physiol. Chem. 357, 1571--1604] except for a serine which replaces Asn at position 227. The results, taken together with studies of other immunoglobulin heavy-chain deletion mutants, support the suggestion that the different domains and interdomain regions of human H chains are coded for by different gene segments and that the deleted proteins reflect alterations in the recombination of different genes and/or the splicing of heterogeneous nuclear messenger ribonucleic acid (hn mRNA)