Influence of glycosylation on the clearance of recombinant human sex hormone-binding globulin from rabbit blood

Abstract Human sex hormone-binding globulin (hSHBG) is a plasma glycoprotein that binds sex steroids with high affinity. Variations in hSHBG glycosylation contribute to its electrophoretic microheterogeneity, but the functional significance of different SHBG glycoforms is unknown. Carbohydrates may influence the biological activities and half-lives of glycoproteins and we have examined how oligosaccharides at specific sites influence the plasma clearance of hSHBG in vivo. To accomplish this, fully-glycosylated hSHBG, or hSHBG mutants lacking specific oligosaccharides chains, were expressed in Chinese hamster ovary (CHO) cells and purified by immunoaffinity chromatography. The purified recombinant proteins were then biotinylated to study their plasma half-lives after intravenous injection into rabbits. When compared to hSHBG isolated from serum, recombinant hSHBG migrates with a slightly larger average molecular size during denaturing polyacrylamide gel electrophoresis. This is due to a greater proportion (33–39% vs. 3%) of more highly branched N -linked oligosaccharides on the recombinant proteins. When injected into rabbits, the disappearance of recombinant hSHBG showed two exponential components, as previously shown for natural hSHBG in the same animal model. The mean±S.E.M. plasma half-lives of recombinant hSHBG (t 1/2 α 0.11±0.03 h and t 1/2 β 18.94±1.65 h) are shorter than previously measured for natural hSHBG (t 1/2 α 3.43±0.72 h and t 1/2 β 38.18±7.22 h) and this is likely due to differences in the composition of their N -linked oligosaccharides. An O -linked chain at Thr 7 does not influence the plasma clearance of hSHBG in the presence or absence of N -linked carbohydrates at Asn 351 and Asn 367 . However, a 1.5–1.6 fold ( p N -glycosylation sites was observed and this is probably due to the fact these variants are not recognized by the asialoglycoprotein receptor-mediated clearance system. Removal of either N -glycosylation consensus site also increased ( p N -linked oligosaccharides rather than their location.