Polylactosamine synthesis and branch formation of N-glycans in b1,4-galactosyltransferase-1-deficient mice q Norihiro Kotani, a,1 Masahide Asano, b,2 Noboru Inoue, c Yoichiro Iwakura, b and Seiichi Takasaki a, * a Division of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan b Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan c R&D Center, Pharmaceutical Division, Kirin Brewery. 100-1 Hagiwara, Takasaki, Gunma 370-0013, Japan Received 29 December 2003, and in revised form 13 February 2004 Available online 21 March 2004 Abstract Analysis of glycans from erythrocyte membrane glycoproteins from b1,4-galactosyltransferase-1 (b4GalT-1)-deficient mice re- vealed moderately decreased galactosylation but comparable polylactosamine content compared to control b4GalT-1 þ= mice. The increased expression of more branched N-glycans was observed in b4GalT-1 = mice, and its extent was more remarkable in elder b4GalT-1 = mice (28 weeks old) than in younger b4GalT-1 = mice (6–9 weeks old). In relation to this issue, the less galactosylation of biantennary glycans was observed in the elder group, suggesting that b4GalTs actually compete with N-acetyl- glucosaminyltransferases IV and V in erythroid cells. In contrast, approximately 80% of core 2 O-glycans were not b1,4-galac- tosylated regardless of age of the knockout mice. These results suggest that b4GalT-1 expressed in erythroid cells may regulate a constant branch formation of N-glycans and plays a predominant role in b1,4-galactosylation of core 2 O-glycan. Ó 2004 Elsevier Inc. All rights reserved. Keywords: b4GalT-1; Knockout; Galactosylation; Polylactosamines; Branch formation It is now known that six members of a b1,4-galac- tosyltransferase family, b4GalT 3 -1 to 6 [1–6], can transfer Gal in a b1,4-linkage to GlcNAc or Glc. Among them, b4GalT-1 is the first mammalian enzyme charac- terized biochemically and cloned. Previous in vitro studies using human b4GalT-1 enzyme suggested that the enzyme plays an important role not only in the b1,4- galactosylation of terminal GlcNAc residues of the trimannosyl core of N-glycans but also in the synthesis of polylactosamines on N-glycans [7,8]. These structures are widely found in several tissues and involved in sev- eral physiological and pathological events. Therefore, b4GalT-1 has been considered to be indispensable to homeostasis of organism. b4GalT-1 knockout mice generated by two groups support this consideration [9,10]. The homozygous b4GalT-1 knockout mice exhibited abnormalities such as growth retardation, semi-lethality, enhanced prolif- eration of epithelial cells, abnormal differentiation of small intestine villus cells, endocrine insufficiency, de- creased fertility, and absence of lactose in milk [9–11]. Recently, it has been shown that b4GalT-1-deficient mice show impaired selectin ligand biosynthesis and reduced inflammatory responses [12]. The critical in- volvement of b4GalT-1 in physiological pathways is also supported by the recent description of a human patient with a complete deficiency of b4GalT-1 [13]. b4GalT-1 knockout mice are quite useful not only for the q This paper is dedicated to the memory of Victor Ginsburg. * Corresponding author. Fax: +81-3-5449-5417. E-mail address: takasaki@ims.u-tokyo.ac.jp (S. Takasaki). 1 Present address: Department of Biochemistry, Osaka University Graduate School of Medicine, Suita 565-0871, Japan. 2 Present address: Department of Transgenic Animal Science, Graduate School of Medical Science, Kanazawa University, Kanaz- awa 920-8640, Japan. 3 Abbreviations used: b4GalT, b1,4-galactosyltransferase; GnT, N-acetylglucosaminyltransferase; 2AB, 2-aminobenzamide; HPAEC, high-pH anion exchange chromatography; PAD, pulsed amperometric detection; MALDI-TOF-MS, matrix-assisted laser desorption ioniza- tion time of flight mass spectrometry. 0003-9861/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2004.03.001 Archives of Biochemistry and Biophysics 426 (2004) 258–265 ABB www.elsevier.com/locate/yabbi