Enzymic Remodelling of the N- and O-Linked Carbohydrate Chains of Human Chorionic Gonadotropin. Effects on Biological Activity and Receptor Binding

Abstract

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.

Exploring the Acceptor Substrate Recognition of the Human β-Galactoside α2,6-Sialyltransferase

“…It was concluded that like the plasma proteins tested above, these glycoprotein acceptors do not appear to contain any structural features governing sialic acid transfer in the ␣2,6-position to specific lactosaminyl groups. These data are in total agreement with previous structural work on hCG remodeling using soluble bovine ST6GalI (41).…”

“…Structure-function relationships of the rat liver enzyme ST6GalI have been well advanced, especially the role of the two sialyl motifs in catalysis (6,8,9). In this regard, it was shown that LacNAc was less effective than glycoprotein acceptors, partly because enzyme specificity extends over glycan branching (35,36,41). Although the S sialyl motif is involved in the recognition of both the donor and the lactosaminyl substrates (9), it is anticipated that regions located elsewhere in the protein should also contribute to the display of glycan specificity through more distal interactions.…”

Sialylated Complex-type N-Glycans Enhance the Signaling Activity of Soluble Intercellular Adhesion Molecule-1 in Mouse Astrocytes

“…The polysialoganglioside GT1b was found to suppress the induction of major histocompatibility complex I molecules by interferon-␥ in mouse astrocytes, and this suppressive action was critically dependent on the presence of sialic acid in the ganglioside (54). Another study (55) showed that proper sialylation of human chorion gonadotropin is necessary for optimal in vitro bioactivity.…”

Glycosylphosphatidylinositol (GPI) Anchored Proteins of Plasmodium falciparum:Antigenic Determinants and Role of Sugar Moieties in the GPI Anchor

“…Terminal galactose in glycans was shown to increase receptor binding of the human chorionic gonadotropin hormone [109]. Furthermore, replacement of galactose by GalNAc resulted in decreased bioactivity and lower receptor binding of human chorionic gonadotropin hormone.…”