Selective removal of glycosylation sites from sex hormone-binding globulin by site-directed mutagenesis.
Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein with high affinity for sex steroid hormones. It contains two N-linked carbohydrate chains and one O-linked oligosaccharide per subunit, but their functional significance is not known. Site-directed mutagenesis of a human SHBG cDNA has enabled us to selectively disrupt the known glycosylation sites individually and in various combinations. The mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells, and it was found that the presence of carbohydrates is not an absolute requirement for the secretion of SHBG from these cells, but the absence of both N-linked oligosaccharides reduced the amount of SHBG in the culture medium. In addition, the affinity and specificity of SHBG for steroid ligands was unaffected by the lack of one or more carbohydrate chains. Proportionally greater amounts (26-31%) of the mutants lacking a single N-linked carbohydrate chain failed to interact with Concanavalin-A. (Con-A) compared to normal SHBG produced by CHO cells (15%). Western analysis demonstrated that both consensus sites for N-glycosylation are used and that the typical heavy [mol wt (M(r)), approximately 51,000] and light (M(r), approximately 47,000) subunit size-heterogeneity was maintained regardless of the absence of an O-linked carbohydrate at residue 7. Furthermore, the SHBG mutants containing only one N-linked oligosaccharide comprise only a single subunit with a M(r) of approximately 47,000. This suggests that the heavy subunit contains two N-linked oligosaccharides, while only one of these sites is used on the light subunit. The M(r) of the various SHBG mutants were also examined by gel filtration, and this indicated that they are all produced as homodimers and that carbohydrates are not involved in subunit association.
Bocchinfuso
W P
WP
Department of Obstetrics and Gynecology, University of Western Ontario, London, Canada.
Ma
K L
KL
Lee
W M
WM
Warmels-Rodenhiser
S
S
Hammond
G L
GL
D000818:Animals; D016466:CHO Cells; D003208:Concanavalin A / Q000494:pharmacology; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D016297:Mutagenesis, Site-Directed / Q000502:physiology*; D009844:Oligosaccharides / Q000032:analysis; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D009844:Oligosaccharides; D012738:Sex Hormone-Binding Globulin; D003208:Concanavalin A; D004247:DNA