title: 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.
authors.affiliation: Department of Medical Chemistry, Vrije Universiteit, Amsterdam, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Nemansky
authors.forename: R
authors.initials: R
authors.lastname: de Leeuw
authors.forename: R A
authors.initials: RA
authors.lastname: Wijnands
authors.forename: D H
authors.initials: DH
authors.lastname: van den Eijnden
mesh_terms: D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000737:chemistry; D006026:Glycoside Hydrolases; D006031:Glycosylation; D016695:Glycosyltransferases; D006801:Humans; D066298:In Vitro Techniques; D007985:Leydig Cells / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019158:N-Acetylneuraminic Acid; D011134:Polysaccharides / Q000737:chemistry; D011974:Receptors, LH / Q000378:metabolism; D012794:Sialic Acids / Q000737:chemistry; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013739:Testosterone / Q000096:biosynthesis
chemical_list: D006063:Chorionic Gonadotropin; D011134:Polysaccharides; D011974:Receptors, LH; D012794:Sialic Acids; D013739:Testosterone; D016695:Glycosyltransferases; D006026:Glycoside Hydrolases; D019158:N-Acetylneuraminic Acid; D005690:Galactose