Human IgG is substrate for the thioredoxin system: differential cleavage pattern of interchain disulfide bridges in IgG subclasses.
Thioredoxin, a 12,000 mol. wt protein with two redox-active cysteine residues, together with thioredoxin reductase and NADPH, may reduce protein disulfides and thereby act as a molecular probe of their structure and reactivity. Interchain and intrachain disulfides are structural elements in all immunoglobulins and therefore potential substrates for the reduced thioredoxin, Trx(SH)2. It was investigated whether such disulfides are cleaved in human polyclonal IgG and IgG subclass myeloma proteins by both the human and the Escherichia coli thioredoxin systems. The reactions were monitored spectrophotometrically as oxidation of NADPH at 340 nm, and by following the kinetics of the cleavage patterns with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Human IgG was a substrate for both prokaryotic and eukaryotic Trx(SH)2, which directly reduced IgG disulfides in a time and dose-dependent manner. Stoichiometric analyses indicated near-complete reduction of mainly inter-heavy light chain and inter-heavy chain disulfides, and SDS PAGE corroborated that the buried intrachain disulfides were left intact. The kinetic studies showed that IgG1, IgG3 and IgG4 were readily reduced into heavy and light chains via the formation of half-molecules with slightly slower kinetics for IgG4. In sharp contrast, IgG2 was not cleaved at all, even with increased thioredoxin concentrations or reduction times. A small but significant NADPH consumption by IgG2 myeloma proteins suggested reduction of a labile interchain or surface-exposed mixed disulfide. Consistent results were obtained for several IgG myeloma proteins within each subclass. The structural and functional importance of interchain disulfides in immunoglobulins suggests physiological implications of the thioredoxin system.
Magnusson
C G
CG
Department of Laboratory Medicine, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden.
Björnstedt
M
M
Holmgren
A
A
D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000378:metabolism; D007700:Kinetics; D009194:Myeloma Proteins / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism*
D004220:Disulfides; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D009194:Myeloma Proteins; D011506:Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase