|
Additional N-glycosylation at Asn(13) rescues the human LHbeta-subunit from disulfide-linked aggregation.
CG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit, but differ in their hormone-specific beta-subunits. Despite the considerable homology between LHbeta and CGbeta, we previously demonstrated that, when expressed in GH(3) cells, the secreted form of LHbeta showed mispaired disulfide-linked aggregation in addition to monomer, whereas no aggregation was observed in CGbeta. To determine the domains which are associated with the LHbeta-aggregation and which prevent CGbeta-aggregation, mutant beta-subunits in glycosylation and carboxy-terminus were expressed in GH(3) cells, and the occurrence of aggregation was assessed by continuous labeling with [35S]methionine/cysteine, immunoprecipitation with anti-hCGbeta serum, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a non-reducing condition. No aggregation was seen when N-linked oligosaccharides were attached to the Asn(13) of LHbeta. Removal of the carbohydrate unit at the Asn(13) of CGbeta caused aggregation, although the amount was less than 10% of monomer. The carboxy-terminal regions of neither LHbeta nor CGbeta were associated with their aggregation. Both CGbeta wild-type (WT) and CGbeta lacking N-glycosylation at Asn(13) (CGbeta-N13) showed aggregates in lysate. However, in contrast to CGbeta-N13, CGbetaWT revealed no aggregation in medium. These results indicate that the backbone structure consisting of 114 amino acids and N-linked glycosylation at Asn(30) is involved in the aggregation of LHbeta. Moreover, N-glycosylation at Asn(13) does not prevent such aggregation, but instead plays an important role in correct folding for both LHbeta- and CGbeta-subunits to be secreted as monomer.
|
