Variations in the Oligosaccharides on Free and Combined α-Subunits of Human Choriogonadotropin in Pregnancy

Abstract

The glycoprotein hormone hCG and a free alpha-subunit are secreted by trophoblastic cells during pregnancy. We have purified the alpha-subunit of hCG and the free alpha-subunit population from pregnancy urine. Dissociation of hCG with 10 M urea at 37 C, followed by chromatography on DEAE-Sephacel, resulted in separation of alpha- from beta-subunit, as hCG alpha does not bind to DEAE in the presence of urea, while beta-subunit does bind. Similar treatment of the free alpha population resulted in fractionation into two populations, a nonbinding fraction of free alpha and a population which was retained by DEAE in the presence of urea (free alpha 2). The three populations, hCG alpha, free alpha, and free alpha 2, were further purified by affinity chromatography using anti-alpha antisera linked to Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparations showed that hCG alpha consisted of a single component with an apparent mol wt of 22,000, while free alpha and free alpha 2 consisted of multiple components. Radioactive labeling of sialic acid by limited periodate oxidation and NaB[3H]4 reduction resulted in a higher specific activity for free alpha than for hCG alpha, suggesting that free alpha contains more sialic acid per immunoreactive molecule than does alpha dissociated from hCG. [3H]hCG alpha, but not 3H-labeled free alpha, was able to combine with native hCG beta-subunit. The oligosaccharide moieties were released from the different labeled subunits by alkaline hydrolysis and then compared with respect to Concanavalin A (ConA)-binding and DEAE-binding properties. Most of the oligosaccharides from dissociated hCG alpha bound to ConA-Sepharose (72%), while less material from free alpha (40%) and even less from free alpha 2 (25%) were capable of binding to ConA. DEAE chromatography of the oligosaccharides suggested that hCG alpha contained primarily monosialylated forms (greater than 60%), while free alpha and alpha 2 contained primarily (greater than 70%) di- and trisialylated forms. Thus, the ConA and DEAE binding data indicated that the oligosaccharide contents of free alpha and free alpha 2 were quite different from that of hCG alpha. These results also suggest that some of the oligosaccharide structures contained on hCG alpha and most of those on free alpha and free alpha 2 differ substantially from the structures that have been previously described.