Glycoprotein hormone ␣ subunit, in its free form (free ␣), is a major placental product. Its glycosylation was found to change dramatically during the advancement of pregnancy. In this study, we have analyzed these glycosylation changes in five normal pregnancies. Binding to Lens culinaris lectin increased dramatically in all subjects between weeks 14 and 17 from the last menstrual period, indicating more core fucosylation as well as possible changes in branching of glycans. Studies using Datura stramonium agglutinin confirmed that the type of triantennary branching changed in this period of pregnancy. The precise structural nature of these changes was determined by high-pH anion-exchange chromatography and electrospray ionization mass spectrometry. Amounts of core fucosylation and of triantennary glycans increased substantially from early to late second trimester, and a shift was observed from 134/ 133-toward predominantly 136/136-branched triantennary structures. The glycosylation changes occurred in all five individuals at the same time period in gestation, suggesting developmental regulation of N-acetylglucosaminyltransferases IV and V and ␣6-fucosyltransferase during normal pregnancy. These enzymatic activities also appear to be affected in malignant transformation of the trophoblast. Our findings have important implications for the proposed use of specific forms of glycosylation as markers for cancer, as the relative amounts of these glycans in normal pregnancy will be determined by gestational age.Glycoprotein hormone ␣ subunit is common to the heterodimeric hormones chorionic gonadotropin, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone. However, in its free form (free ␣ subunit), it is an important placental (1, 2) and pituitary (3) product, and it has been shown to have functions that are independent of the dimeric hormones (4 -7). Glycosylation of free ␣ differs from glycosylation of the combined form (8, 9). The combination of ␣ and ␤ for heterodimer formation takes place in the endoplasmic reticulum prior to processing of the immature glycans. In ␣ subunits that have not combined with a ␤ subunit, enzymes from the post-translational glycosylation machinery have access to substrate sites that are normally protected by the ␤ subunit of the heterodimer. As a result, the free form of ␣ subunit generally contains more elaborate oligosaccharide branching as well as higher amounts of core fucosylation than ␣ subunit obtained from dissociated hormone (8, 9). These characteristic glycosylation patterns prevent secreted free ␣ subunits from combining with ␤ subunits that might be encountered extracellularly, thus ensuring a population of free ␣ molecules (9, 10).The structural diversity of complex-type N-linked glycans is initiated by GlcNAc branching of the trimannosyl core and continues with the action of different glycosyltransferases that further extend these antennae (11). Specifically, the activity of N-acetylglucosaminyltransferase IV initiates the 134/133-branch of c...

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Bovine colostrum CMP-NeuAc:Gal beta(-->4)GlcNAc-R alpha(2-->6)-sialyltransferase (alpha 6-sialyltransferase) appears to be capable of catalysing alpha 6-sialylation of the disaccharide GalNAc beta(1-->4)GlcNAc to yield the trisaccharide NeuAc alpha(2-->6)GalNAc beta(1-->4)GlcNAc. This provides an enzymic basis for the occurrence of this sialylated structure on the N-linked glycans of a number of bovine milk glycoproteins. Competition experiments using Gal beta(1-->4)GlcNAc and GalNAc beta(-->4)GlcNAc as acceptors indicate that both substrates are recognized by a single active site on the alpha 6-sialyltransferase. Extrapolation of these results suggests that the NeuAc alpha(2-->6)GalNAc beta(1-->4)GlcNAc structural element occurring on the N-linked glycans of several human glycoproteins are similarly synthesized by the action of a Gal beta(1-->4)GlcNAc-R alpha(2-->6)-sialyltransferase.

The activity of bovine colostrum CMP-NeuAc: Gal/31--~4GIcNAcl3-R c~2-->6-sialyltransferase (a6-NenAcT) toward oligosaccharides that form part of complex-type, Nlinked glycans appears significantly reduced when a bisecting GlcNAc residue or additional branches are present, or when core GIcNAc residues are absent. By contrast human placenta CMPNeuAc: Gall~l -->4GicNAc/]-R a2--> 3-sialyltransferase (a3-NeuAcT) is much less sensitive to structural variations in these acceptors. Furthermore the a3-NeuAcT shows a much higher activity than the c~6-NeuAcT with oligosaccharides that form part of linear and branched iactosaminoglycan extensions. These results indicate that, in tissues that express both enzymes, branching and lactosaminoglycan formation of N-linked glycans will cause a shift from termination with ~2-->6-1inked sialic acid to termination with a2-->3-1inked sialic acid residues. These findings provide an enzymatic basis for the sialic acid linkage-type patterns found on the oligosaccharide chains of N-glycoproteins.

The effects of altered terminal sequences in human chorionic gonadotropin (hCG) N- and O-linked glycans on receptor binding and signal transduction were analyzed using forms of hCG with remodelled carbohydrate chains. hCG derivatives were obtained by enzymic removal of the alpha 3-linked sialic acid residues followed by alpha 6-sialylation, alpha 3-galactosylation or alpha 3-fucosylation of uncovered Gal beta 1-->4GlcNAc (LacNAc) termini, or alpha 3-sialylation of Gal beta 1-->3GalNAc sequences. Also a form that carried GalNAc beta 1-->4-GlcNAc units, which are typical for pituitary hormone oligosaccharides, was derived by enzymic desialylation and degalactosylation followed by beta 4-N-acetylgalactosaminylation. The potency to stimulate testosterone production and the binding to the lutotropin/choriogonadotropin receptor of the preparations were compared with those of native and desialylated hCG (as-hCG). The decrease in bioactivity caused by desialylation of hCG was only restored upon alpha 6-sialylation of the Gal beta 1-->4GlcNAc beta 1-->-2Man alpha 1-->3Man branch of the N-linked glycans. This was without a major effect on receptor binding. Further alpha 6-sialylation, occurring at the Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6Man branch, resulted in a bioactivity below a level found with as-hCG, concomitant with a decreased receptor binding affinity. Similarly alpha 3-galactosylation of the Gal beta 1-->4GlcNAc beta 1-->2-Man alpha 1-->6Man branch yielded a hCG derivative that showed decreased bioactivity and receptor binding. alpha 3-Fucosylation of native as well as as-hCG also led to a decreased activity. Re-alpha 3-sialylation of the O-linked chains on as-hCG had little effect on the bioactivity and receptor binding. Hormone preparations with GalNAc beta 1-->4GlcNAc termini showed lower bioactivity and receptor affinity than as-hCG. It is concluded that the Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->3Man- rather than the Gal beta 1-->4GlcNAc beta 1-->2-Man alpha 1-->6Man branch of the N-linked glycans on hCG plays an essential role in signal transduction, whereas the latter branch can potentially interfere with receptor binding. Furthermore attachment of sialic acid, but not of other sugars, to the first branch fulfils the requirement for the full expression of bioactivity, while sialylation of the O-linked chains is of minor importance.