Site-directed mutagenesis defines the individual roles of the glycosylation sites on follicle-stimulating hormone.
To determine the specific role of each follicle-stimulating hormone (FSH) oligosaccharide, we mutated Asn to Gln at each glycosylation site (alpha Gln52, alpha Gln78, alpha Gln52-78, beta Gln7, beta Gln24, and beta Gln7-24) to selectively inhibit oligosaccharide attachment. For wild-type and mutant FSH, we determined the binding affinity to homogenized rat Sertoli cells and the signal-transducing activity in cultured rat granulosa cells. The binding affinity of FSH lacking any one of the oligosaccharides was increased over wild-type FSH, while the signal-transducing activity of FSH lacking the oligosaccharide at alpha Asn52 (alpha Gln52 FSH) was markedly reduced, and that of FSH lacking either beta oligosaccharide (beta Gln7 and beta Gln24 FSH) was slightly reduced. At each FSH beta glycosylation site, we made a second amino acid substitution to inhibit glycosylation (beta Tyr9 and beta Tyr26) and an amino acid substitution that preserved glycosylation (beta Ser9 and beta Ser26). The amino acid sequence of the second beta subunit glycosylation site was important for signal transduction, regardless of the presence or absence of the oligosaccharide. Thus, while each FSH oligosaccharide has a similar impact on binding affinity, the alpha 52 oligosaccharide has a disproportionate role in signal transduction, and the amino acid sequence at beta Asn24 functions in both binding and signal transduction.
Flack
M R
MR
Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Froehlich
J
J
Bennet
A P
AP
Anasti
J
J
Nisula
B C
BC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005640:Follicle Stimulating Hormone / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011869:Radioligand Assay; D051381:Rats; D011962:Receptors, FSH / Q000378:metabolism; D012708:Sertoli Cells / Q000378:metabolism; D015398:Signal Transduction
D011962:Receptors, FSH; D005640:Follicle Stimulating Hormone; D005998:Glycine