title: Mutations of the human thyrotropin-beta subunit glycosylation site reduce thyrotropin synthesis independent of changes in glycosylation status.
abstract: 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-beta subunit 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-beta subunit glycosylation resulted in greater than 90% decreases in TSH synthesis. However, the glycosylation recognition sequence mutant that retained beta subunit glycosylation exhibited a 70% decrease in TSH production. These decreases were not attributable to the intracellular accumulation of TSH or its free beta subunit. We also engineered two TSH-beta 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-beta subunit carboxyterminus by six amino acids increased TSH synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Division of Endocrinology, University of Maryland School of Medicine, Baltimore 21201.
authors.forename: R W
authors.initials: RW
authors.lastname: Lash
authors.forename: R K
authors.initials: RK
authors.lastname: Desai
authors.forename: C A
authors.initials: CA
authors.lastname: Zimmerman
authors.forename: M R
authors.initials: MR
authors.lastname: Flack
authors.forename: T
authors.initials: T
authors.lastname: Yoshida
authors.forename: F E
authors.initials: FE
authors.lastname: Wondisford
authors.forename: B D
authors.initials: BD
authors.lastname: Weintraub
mesh_terms: D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D004247:DNA / Q000096:biosynthesis; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D013972:Thyrotropin / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins; D013972:Thyrotropin; D004247:DNA; D005947:Glucose