Intradomain Disulfide Bond Research Articles

Effects of limited reduction on disulfide bonds in human IgA1 and IgA1 fragments

Human IgA occurs in body fluids as monomers, dimers and secretory IgA (sIgA). Besides the cysteine residues in intra-domain, inter-chain and inter-subunit disulfide bonds IgA molecules contain several cysteine residues with unknown function and reactivity. Limited reductions on serum IgA1 and secretory IgA1 with glutathione revealed that four cysteine residues per monomer or subunit were part of labile bonds. Six cysteine residues were reduced in F(ab')2 fragments and about three in Fc fragments, but none in Fab fragments, indicating that the labile bonds occur in the Fc fragment. By SDS-PAGE analyses of reduced proteins labile inter-α chain bond(s) were detected in F(ab')2 and F(abc)2 fragments but not in Fc fragments and intact IgA1, thus showing the importance of the CH3 domains for the structural stability of the hinge region. Nine cysteine residues per IgA1 were reduced with 0.01 M DTT and a large proportion of the IgA1 myeloma proteins formed half-molecules consisting of an α- and a light chain, but sIgA1 remained intact. This indicates a relative stability of heavy to light chain and inter-subunit bonds. Reductions in the presence of 2% SDS disrupted several intra-chain bonds. Binding studies with (CH2)2-specific monoclonal antibodies, which detect an epitope expressed only on IgA molecules with disulfide linked α chains, were in accordance with the SDS-PAGE results. A new model for the location of labile and more stable disulfide bonds is discussed.

Characterization of a cDNA encoding the heparin and collagen binding domains of human thrombospondin.

Thrombospondin (TSP) cDNA clones were isolated from a human fibroblast cDNA library by screening with degenerate synthetic oligodeoxynucleotides. The probes were designed based on the sequences of peptides isolated from tryptic digests of TSP. The largest clone identified contains an open reading frame that encodes the amino-terminal heparin binding domain of TSP and part of the immediately adjacent collagen binding domain, a region of TSP that includes the site of disulfide crosslinking responsible for trimer formation. In addition to the known amino-terminal sequence of mature TSP (which is identical to that of the heparin binding domain) and that of the collagen binding domain, the open reading frame predicts the amino acid sequences of four tryptic peptides including the one from which the probe sequences were derived. The sequence of the mature protein is preceded by an unusual signal peptide sequence of 18 residues. The heparin binding domain contains 240 amino acids including only one intradomain disulfide bond. In contrast, 80 residues beyond the start of the collagen binding domain, a cluster of 10 cysteine residues occurs within 40 amino acids corresponding to the point of interchain disulfide crosslinking in the TSP trimer. RNA blot analysis indicates a TSP mRNA size of approximately equal to 8 kilobases and Southern blots are consistent with a single gene encoding TSP.

Molecular defects in a human immunoglobulin kappa chain deficiency.

The molecular basis of a human immunoglobulin deficiency characterized by the complete absence of kappa chains has been investigated by nucleotide sequence analyses of a patient's kappa constant region (C kappa) genes. Both of his C kappa genes had a single point mutation, resulting in the loss of the invariant tryptophan from one allele and of an invariant cysteine from the other allele. These results indicate that neither of the patient's C kappa alleles encoded a kappa chain that could form a stable intradomain disulfide bond, although peculiarities in the expression of kappa chains in the patient's family suggest that other factors may be involved.

Genes encoding alpha-heavy chains of rabbit IgA: characterization of cDNA encoding IgA-g subclass alpha-chains.

cDNA molecules encoding rabbit IgA alpha-heavy chains have been synthesized and six of these have been characterized. The complete nucleotide sequence of one cDNA, p 19 (942bp), showed that it encoded all but the N-terminal 57 amino acid residues of the constant region of alpha-chains. The cDNA molecules were subcloned into the expression vector pUC8 and E. coli were transformed. Radioimmunoassay of the molecules synthesized by these clones showed that all six cDNA molecules encoded alpha-chains of the IgA-g subclass. Comparison of the amino acids encoded by the alpha-cDNA with the amino acid sequence of mouse and human alpha-chains showed that although all of the intradomain disulfide bonds appear to be conserved, some positions, probably involved in interchain disulfide bonds, are not conserved. We propose that secretory component is covalently bound to cysteine 299 and/or cysteine 301 of the CH2 domain of mouse and human alpha-chains. The results from Southern blot analysis of genomic DNA with 32P-cDNA suggests that the rabbit genome has multiple C alpha genes.

Structural Studies of Rabbit IgA Allotypes: Partial Amino Acid Sequences of α-Chain Allotypes Controlled by Alleic Genes at the Cαg Locus

Abstract Four α-chain allotypic specificities of the IgA-g subclass of rabbit IgA are controlled by allelic genes at the Cαg locus. The structural basis for three of these allotypic specificities, g74, g75, and g76, was investigated by amino terminal sequence analysis. α-Chain fragments were isolated from Fc2α fragments which were obtained by tryptic digestion of g74, g75, and g76 secretory IgA. Amino acid sequence data for the chains were obtained for 92 positions; comparison of these sequences with the sequence of human α1-chain revealed approximately 70% homology with the Cα2 domain. The results indicated that g74 α-chains were cleaved 60 residues N-terminal to the cleavage site in g75 and g76 α-chains. By comparison of sequences of 24 residues of g75 and g76 α-chains, one allotype-related difference was observed (a cys/ser interchange at position 311). Based on homologies with the human α1-chain, a model for the arrangement of disulfide bonds in rabbit α-chains is proposed; several differences between rabbit and human are indicated, as are differences among the rabbit allotypes. The most striking difference is the apparent presence of the “extra” intradomain disulfide bond in the Cα2 domain of g74 molecules and its absence in g75 and g76 molecules.