O-Glycosylation of human sex hormone-binding globulin is essential for inhibition of estradiol-induced MCF-7 breast cancer cell proliferation

Abstract Human sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein, and each SHBG monomer may have an O -linked oligosaccharide at Thr 7 and up to two N -linked oligosaccharides at Asn 351 and Asn 367 . In addition, a common genetic variant of SHBG exists with an extra site for N -glycosylation at residue 327. In the present study, we isolated MCF-7 derived cell lines expressing human SHBG cDNAs encoding the wild type protein or various glycosylation mutants. Estradiol (1 nM) treatment of parental (untransfected) MCF-7 cells or MCF-7 cells transfected with control expression vectors resulted in an increase in proliferation which was fully abrogated by co-incubation with an equimolar amount of human SHBG. In contrast, the same amount of purified SHBG added to MCF-7 cells expressing wild type SHBG partially inhibited the estradiol-induced cell proliferation. A high affinity binding site for SHBG was detectable on untransfected and control cells, but not on MCF-7 cells expressing wild type SHBG. Moreover, the treatment of MCF-7 cells with the conditioned medium containing wild type SHBG caused the disappearance of the SHBG plasma membrane-binding site. Media containing SHBG N -glycosylation mutants exerted the same effect, but mutants lacking the O -linked oligosaccharide at Thr 7 failed to do so. Estradiol-induced proliferation of parental MCF-7 cells was also inhibited by treatment with conditioned medium containing wild type SHBG or SHBG mutants lacking N -linked oligosaccharides, or containing an additional N -linked oligosaccharide at residue 327. However, MCF-7 conditioned medium containing SHBG mutants lacking an O -linked oligosaccharide at Thr 7 failed to exert this effect. These data suggest that O -glycosylation of SHBG is essential for SHBG binding to a membrane receptor that is responsible for inhibiting the estradiol-induced proliferation of MCF-7 breast cancer cells.