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-alpha 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 alpha- 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 alpha chains, were in accordance with the SDS-PAGE results. A new model for the location of labile and more stable disulfide bonds is discussed.
Biewenga
J
J
Department of Cell Biology, Medical Faculty, Vrije Universiteit, Amsterdam, The Netherlands.
van Run
P E
PE
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007070:Immunoglobulin A / Q000737:chemistry*; D007071:Immunoglobulin A, Secretory / Q000737:chemistry; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D008969:Molecular Sequence Data
D007070:Immunoglobulin A; D007071:Immunoglobulin A, Secretory; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D003545:Cysteine