EXPRESSION OF HUMAN THYROTROPIN IN CELL-LINES WITH DIFFERENT GLYCOSYLATION PATTERNS COMBINED WITH MUTAGENESIS OF SPECIFIC GLYCOSYLATION SITES - CHARACTERIZATION OF A NOVEL ROLE FOR THE OLIGOSACCHARIDES IN THE IN-VITRO AND IN-VIVO BIOACTIVITY

We used a novel approach to study the role of the Asn-linked oligosacc harides for human thyrotropin (hTSH) activity. Mutagenesis of Asn (N) within individual glycosylation recognition sequences to Gln (Q) was c ombined with expression of wild type and mutant hTSH in cell Lines wit h different glycosylation patterns. The in vitro activity of hTSH lack ing the Asn(alpha 52) oligosaccharide (alpha Q52/TSH beta) expressed i n CHO-K1 cells (sialylated oligosaccharides) was increased 6-fold comp ared with wild type, whereas the activities of alpha Q78/TSH beta and alpha/TSH beta Q23 were increased 2-3-fold. Deletion of the Asn(alpha 52) oligosaccharide also increased the thyrotropic activity of human c horionic gonadotropin, in contrast to previous findings at its native receptor. The in vitro activity of wild type hTSH expressed in CHO-LEC 2 cells (sialic acid-deficient oligosaccharides), CHO-LEC1 cells (Man( 5)GlcNAc(2) intermediates), and 293 cells (sulfated oligosaccharides) was 5-8-fold higher than of wild type from CHO-K1 cells. In contrast t o CHO-K1 cells, there was no difference in the activity between wild t ype and selectively deglycosylated mutants expressed in these cell lin es. Thus, in hTSH, the oligosaccharide at Asn(alpha 52) and, specifica lly, its terminal sialic acid residues attenuate in vitro activity, in contrast to the previously reported stimulatory role of this chain fo r human chorionic gonadotropin and human follitropin activity. The inc reased thyrotropic activity of alpha Q52/CG beta suggests that recepto r-related mechanisms may be responsible for these differences among th e glycoprotein hormones. Despite their increased in vitro activity, al pha Q52/TSH beta, and alpha Q78/TSH beta from CHO-K1 cells had a faste r serum disappearance rate and decreased effect on T-4 production in m ice. These findings highlight the importance of individual oligosaccha rides in maintaining circulatory half-life and hence in vivo activity of hTSH.