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

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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 by .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 (Con A)-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 (> 60%), while free .alpha. and .alpha.2 contained primarily (> 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.