ENZYMATIC REMODELING OF THE N-LINKED AND O-LINKED CARBOHYDRATE CHAINSOF HUMAN CHORIONIC-GONADOTROPIN - EFFECTS ON BIOLOGICAL-ACTIVITY AND RECEPTOR-BINDING

The effects of altered terminal sequences in human chorionic gonadotro pin (hCG) N- and O-Linked glycans on receptor binding and signal trans duction 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-->4Gl cNAc (LacNAc) termini, or alpha 3-sialylation of Gal beta 1-->3GalNAc sequences. Also a form that carried Gal-NAc beta 1-->4-GlcNAc units, w hich are typical for pituitary hormone oligosaccharides, was derived b y enzymic desialylation and degalactosylation followed by beta 4-N-ace tylgalactosaminylation. The potency to stimulate testosterone producti on and the binding to the lutotropin/choriogonadotropin receptor of th e 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-->2hMan alpha 1-->3Man branch of the N-linked glycans. This wa s without a major effect on receptor binding. Further alpha 6-sialylat ion, occurring at the Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6Ma n branch, resulted in a bioactivity below a level found with as-hCG, c oncomitant 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 bioactivi ty 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 recep tor binding. Hormone preparations with GalNAc beta 1-->4GlcNAc termini showed lower bioactivity and receptor affinity than as-hCG. It is con cluded that the Gal beta 1-->4GlcNAc beta 1-->2Man-alpha 1-->3Man- rat her 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 tran sduction, whereas the latter branch can potentially interfere with rec eptor binding. Furthermore attachment of sialic acid, but not of other sugars, to the first branch fulfils the requirement for the full expr ession of bioactivity, while sialylation of the O-linked chains is of minor importance.