Additional N-glycosylation at Asn(13) rescues the human LH beta-subunit from disulfide-linked aggregation

CG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones th at contain a common alpha-subunit, but differ in their hormone-specific bet a-subunits. Despite the considerable homology between LH beta and CG beta, we previously demonstrated that, when expressed in GH(3) cells, the secrete d form of LH beta showed mispaired disulfide-linked aggregation in addition to monomer, whereas no aggregation was observed in CG beta. To determine t he domains which are associated with the LH beta-aggregation and which prev ent CG beta-aggregation, mutant beta-subunits in glycosylation and carboxy- terminus were expressed in GH(3) cells, and the occurrence of aggregation w as assessed by continuous labeling with [S-35]methionine/cysteine, immunopr ecipitation with anti-hCG beta serum, and sodium dodecyl sulfate-polyacryla mide gel electrophoresis in a non-reducing condition. No aggregation was se en when N-linked oligosaccharides were attached to the Asn(13) of LH beta. Removal of the carbohydrate unit at the Asn(13) of CG beta caused aggregati on, although the amount was less than 10% of monomer. The carboxy-terminal regions of neither LH beta nor CG beta were associated with their aggregati on. Both CG beta wild-type (WT) and CG beta lacking N-glycosylation at Asn( 13) (CG beta-N13) showed aggregates in lysate. However, in contrast to CG b eta-N13, CG beta WT revealed no aggregation in medium. These results indica te that the backbone structure consisting of 114 amino acids and N-linked g lycosylation at Asn(30) is involved in the aggregation of LH beta. Moreover , N-glycosylation at Asn(13) does not prevent such aggregation, but instead plays an important role in correct folding for both LH beta- and CG beta-s ubunits to be secreted as monomer. (C) 2000 Elsevier Science Ireland Ltd. A ll rights reserved.