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Abstract. Equine chorionic gonadotropin (eCG) consists of highly glycosylated a-and fl-subunits and belongs to the glycoprotein hormone family that includes LH and FSH. eCG is a unique member of the gonadotropin family because it elicits response characteristics of both FSH and LH in other species than the horse. To determine the biological role of the N-linked oligosaccharide at Asn 56 of the a-subunit and 0-linked oligosaccharides at the carboxyl-terminal peptide (CTP) of the fl-subunit, two mutant eCGs, in which Asn 56 of the a-subunit was replaced with Gln (eCGa56/fJ) or CTP was deleted (eCGa/ fl-CTP), were produced by site-directed mutagenesis and transfecting chinese hamster ovary (CHO-K1) cells. LH-and FSH-like activities were assayed in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells and granulosa cells, respectively.The wild type eCG showed similar LH-and FSH-like activities to native eCG in the in vitro bioassays. The LH-like activity of eCGa56/fl was greatly reduced, whereas that of eCGa/f3-CTP was unaffected, demonstrating that the oligosaccharide at Asn 56 of the a-subunit of eCG plays an indispensable role in LH-like activity. Interestingly, the FSH-like activity of eCGa56/fl was increased markedly in comparison with the wild type, and that of eCGa/f3-CTP was also considerably increased. These data indicate that the dual activities of eCG, LH-and FSH-like activities, could be separated by removal of the N-linked oligosaccharide on the a-subunit Asn 56 or CTP-associated 0-linked oligosaccharides.

Abbreviations: ACN, acetonitrile; EDTA, ethylenediaminetetraacetic acid; LC-MS/MS, liquid chromatography-tandem mass spectrometry; TFA, trifluoroacetic acid Pre-print version Takeuchi T. et al. Carbohydrate Research 346 (2011) Adsorbed C. elegans proteins were eluted with ethylenediaminetetraacetic acid (EDTA) and followed by lactose (Galβ1-4Glc), digested with trypsin, and were then subjected to proteomic analysis using LC-MS/MS. Three annexins, namely NEX-1, -2, and -3, were assigned in the EDTA-eluted fraction. Whereas, galectins, namely LEC-1, -2, -4, -6, -9, -10, and DC2.3a, were assigned in the lactose-eluted fraction. The affinity of annexins for Galβ1-4Fuc was further confirmed by adsorption of recombinant NEX-1, -2, and -3 proteins to the Galβ1-4Fuc column in the presence of Ca 2+ . Furthermore, frontal affinity chromatography analysis using an immobilized NEX-1 column showed that NEX-1 has an affinity for Galβ1-4Fuc, but no affinity towards Galβ1-3Fuc and Galβ1-4GlcNAc.We would hypothesize that the recognition of the Galβ1-4Fuc disaccharide unit is involved in some biological processes in C. elegans and other species of the Pre-print version Takeuchi T. et al.

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