Authors: R W Lash R K Desai C A Zimmerman M R Flack T Yoshida F E Wondisford B D Weintraub
Publish Date: 2014/04/04
Volume: 15, Issue: 4, Pages: 255-263
Abstract
In recent studies sitedirected 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 wildtype and mutant recombinant human thyrotropins produced by transient transfection of a human cell line Mutating the TSHß subunit glycosylation recognition sequence AsnThrThr codons 23–25 to either GlnThrThr or AsnThrTyr abolished subunit glycosylation as demonstrated by the inability to incorporate 3Hcarbohydrates However a third mutation AsnThrSer contained an intact glycosylation recognition sequence site and was shown to retain glycosylation The mutations that abolished TSHß 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 TSHß 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 TSHß subunit carboxyterminus by six amino acids increased TSH synthesis In summary a series of mutations within and adjacent to the TSHß 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
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