Biol. Chem. Hoppe-Seyler, Vol. 376, pp. 531 -537, September 1995 · Copyright © by Walter de Gruyter & Co · Berlin · NewYork Characterization of N-Linked Carbohydrate Chains of the Crayfish, Astacus leptodactylus Hemocyanin Despina Tseneklidou-Stoeter 1 , Gerrit Johannes Gerwig 2 , Johannis Paulus Kamerling 2 , and Klaus-Dieter Spindler 1 * 1 Lehrstuhl für Hormon- und Entwicklungsphysiologie, Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf, Germany 2 Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P.O. Box 80.075, NL-3508TB Utrecht, The Netherlands * Corresponding author The primary structure of the carbohydrate chains of hemocyanin from the crayfish Astacus leptodactylus were investigated. The carbohydrate content is 0.2% (w/w) as referred to total hemocyanin content, resp. 1.8% as referred only to the one subunit which is glycosylated. Mannose and M-acetylglucosamine are present in a molar ratio of 6:2. The carbohydrate chains are N-glycosidically linked as revealed by dot blot analysis using various lectins and enzyma- tic deglycosylation. Furthermore, they are part of only one hemocyanin subunit of A. leptodactylus. After enzymatic deglycosylation with PNGase F, the oligosaccharide pool was separated by FPLC on Mono Q and subsequent HPLC on Lichrosorb-NH 2 , the subfractions were characterized by 1 H NMR spectroscopy. A total of six oligosaccharides, rang- ing from Man 4 GlcNAc 2 to Man 9 GlcNAc 2 is present, Man e GlcNAc 2 representing the most abundant one with 57% of all oligosaccharides. Key words: Deglycosylation / Mannose rich oligosaccharides. Introduction Hemocyanin is the predominant protein in the hemolymph of most molluscs, spiders and crustaceans. In crusta- ceans, hemocyanin is produced in the midgut gland (Preaux et al., 1986; Gellissen et al., 1991) and represents the major protein synthesized in this organ (Spindler et al., 1992). It is rapidly secreted in vitro. As with many secreted proteins, glycosylation could be important for processing and secretory processes, and indeed most hemocyanins are glycosylated (Van Holde and Miller, 1982; Ellerton et al., 1983; Van Kuik etal., 1985,1986,1987,1990). Sofarthe physiological meaning of this glycosylation has not been studied, and is speculative until now. As a first step to- wards more insight, knowledge of the carbohydrate struc- tures of these glycoproteins is necessary. Unfortunately, structural studies have been carried out only for a few species, amongst them one crustacean, Panulirus inter- rupfus(Van Kuik et al., 1986,1987,1990). In order to study the physiological role of glycosylation of hemocyanin we have selected the species Astacus lep- todactylus. This choice is based on the following reasons: 1) hemocyanin synthesis has been studied in this species in some detail and more extensively than in other crus- taceans (Gellissen ef al., 1991; Spindler et al., 1992; Hennecke et al., 1990,1991); 2) A. leptodactylus hemocyanin has often been used for comparative purposes, e.g. as concerns subunit com- position and aspects of the evolution of hemocyanin (Markl, 1986; Markl ef al., 1979,1983,1986,1992; Markl and Kempter, 1981; Stöcker ei al., 1988); 3) data exist on the partial amino acid sequence of one subunit (Schneider ef al., 1986); 4) in preliminary studies it has been shown that the hemocyanin of A. leptodactylus has a carbohydrate content <^ 1 % and a monosaccharide composition of Man and GlcNAc (Van Kuik et al., 1990). In this paper the /V-glycosylation pattern of the hemocyanin of A. leptodactylus is presented. Results and Discussion In order to get information with respect to the type of carbohydrate constituents/chains of A. leptodactylus hemocyanin, various lectins were used for a dot blot a b c SNA PNA DSA MAA Fig. 1 Dot Blot Analysis of Astacus leptodactylus Hemocyanin. Digoxigenin-labelled lectins (SNA, PNA, DSA and MAA) were used. In (a) each 1 of the corresponding control proteins (trans- ferrin for SNA, asialofetuin for PNA and DSA, fetuin for MAA), and in (b) 1 hemocyanin from A. leptodactylus were blotted and as- sayed. In (c) no protein was added. Brought to you by | Purdue University Libraries Authenticated Download Date | 6/18/15 6:27 PM