In recent studies, site-directed mutagenesis has been used to alter the tripeptide glycosylation recognition sequences of glycoprotein hormone subunits, thereby affecting their structure and function. However, it is not known whether these effects result from changes in glycosylation status, amino acid sequence, or both. We therefore studied the synthesis of wild-type and mutant recombinant human thyrotropins produced by transient transfection of a human cell line. Mutating the TSH-ß sub-unit glycosylation recognition sequence, Asn-Thr-Thr (codons 23–25), to either Gln-Thr-Thr or Asn-Thr-Tyr abolished subunit glycosylation, as demonstrated by the inability to incorporate 3H-carbohydrates. However, a third mutation (Asn-Thr-Ser) contained an intact glycosylation recognition sequence site, and was shown to retain glycosylation. The mutations that abolished TSH-ß subunit glycosylation resulted in greater than 90% decreases in TSH synthesis. However, the glycosylation recognition sequence mutant that retained ß subunit glycosylation exhibited a 70% decrease in TSH production. These decreases were not attributable to the intracellular accumulation of TSH or its free ß subunit. We also engineered two TSH-ß subunit mutations that did not alter the glycosylation recognition sequence. A glycine to arginine mutation adjacent to the glycosylation recognition sequence, in a region thought to be critical for heterodimer formation, abolished TSH production. In contrast, shortening the TSH-ß subunit carboxyterminus by six amino acids increased TSH synthesis. In summary, a series of mutations within and adjacent to the TSH-ß glycosylation recognition sequence significantly reduced TSH production, whether or not they abolished subunit glycosylation. These findings emphasize the dual roles of the TSH glycosylation recognition sequence: as the signal for the addition of carbohydrate, and as an amino acid sequence necessary for the efficient synthesis of the TSH dimer.
- glycoprotein hormones
- recombinant hormones
- site-directed mutagenesis
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism