Processing of O-linked Glycosylation in the Chimera Consisting of .ALPHA.-Subunit and Carboxyl-terminal Peptide of the Human Chorionic Gonadotropin .BETA.-Subunit is Affected by Dimer Formation with Follicle-stimulating Hormone .BETA.-Subunit

Abstract

Abstract. hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common a-subunit, but differ in their hormone-specific b-subunits. hCGb is unique among b-subunits due to a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharides. We previously reported that there were differences in O-glycosylation between two chimeras consisting of a-subunit and CTP, i.e. a variant with CTP at the N-terminal region (Ca) and another analog with CTP at the C-terminus (aC) of the a-subunit. To address whether O-glycosylation is influenced by the heterodimer formation, Ca and aC were expressed alone or with FSHb-subunit in Chinese hamster ovary cells. The Olinked glycosylation was assessed by continuous labeling with [ 35 S]methionine/cysteine, immunoprecipitation with anti-a or anti-FSH serum, serial digestion with endoglycosidase-F and neuraminidase, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The decrease in molecular weight of dimeric chimeras digested with endoglycosidase-F was greater in Ca than that in aC after treatment with neuraminidase, revealing that both chimeras have different numbers of sialic acids on O-linked carbohydrates. By treating with endoglycosidase-F, the dimeric aC migrated faster than its free form, whereas the mobility difference between assembled and unassembled forms of Ca was very little. These data indicate that processing of O-glycosylation is affected by the backbone polypeptide chain(s). (Fig. 1). It has been shown that the CTP does not play roles in the assembly, secretion and in vitro bioactivity of hCG [5][6][7], but is important for maintaining the longer halflife of hCG [6]. The structures of O-linked carbohydrates in hCGb-subunit have been elucidated using the urine of patients with trophoblastic diseases and healthy pregnant women [8], and the choriocarcinoma cell line, BeWo [9]. The major backbone structures identified are galactose (Gal) b1®3N-acetylgalactosamine (GalNAc) and Galb1®4N-acetylglucosamineb1 ®6 (Galb13) GalNAc, each of which has zero, one or

Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer

“…Thus, in order to improve the pharmacokinetic profile of TFF2, we added the 28 amino acid carboxyl-terminal peptide (CTP) from the human chorionic gonadotropin β subunit to the C-terminus of the TFF2 coding sequence. The addition of CTP typically prolongs the half-life of peptides without affecting biological activity, secretion or receptor binding [4][5][6]. The CTP sequence contains four O-linked oligosaccharides sites that seem to prevent plasma clearance by increasing residence in the blood [7,8].…”