Redefined Substrate Specificity of ST6GalNAc II: A Second Candidate Sialyl-Tn Synthase Mari Kono, 1 Tetsuro Tsuda,* Shunichiro Ogata,† Shou Takashima, 2 Hong Liu, 3 Toshiro Hamamoto, 4 Steven H. Itzkowitz,† Shinichiro Nishimura,* and Shuichi Tsuji 5 Molecular Glycobiology, Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan; *Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060-0810 Japan; and †Division of Gastroenterology, Mount Sinai Medical Center, One Gustave L. Levy Place, New York, New York 10029-6574 Received April 7, 2000 The acceptor substrate specificities of ST6GalNAc I and II, which act on the synthesis of O-linked oligosac- charides, were reexamined using ovine submaxillary mucin, [Ala-Thr(GalNAc)-Ala] n polymer (n  7–11). It has been suggested that only ST6GalNAc I can synthe- size carbohydrate structures of sialyl-Tn-antigen; i.e., NeuAc2-6GalNAc-O-Thr/Ser [Kurosawa et al., J. Biol. Chem. 269, 19048 –19053 (1994)] based on the result that ST6GalNAc I, not ST6GalNAc II, exhibited activity to- ward asialoagalacto-fetuin. In this study, we present evidence that both ST6GalNAc I and II exhibit activity toward asialo-OSM (ovine submaxillary mucin) and [Ala-Thr(GalNAc)-Ala] n polymer (n  7–11) which have only the GalNAc-O-Thr/Ser-structures. These results strongly indicate that not only ST6GalNAc I but also II are candidates for sialyl-Tn synthases. © 2000 Academic Press Key Words: sialyl-Tn; sialyltransferase; ST6GalNAc I; ST6GalNAc II. The sialyl-Tn antigen (STn; NeuAc2-6GalNAc-O- Thr/Ser) is a mucin-associated carbohydrate antigen, an epitope that can be expressed in a tumor-associated fashion in different organs (2). The addition of a sialic acid through an 2-6-linkage to GalNAc-O-Thr/Ser prevents the elongation of O-linked oligosaccharides (3). Thus, the enzyme which catalyzes this reaction is a key enzyme for O-linked oligosaccharide biosynthesis. The transfer of sialic acid with an 2-6-linkage to N-acetylgalactosamine (GalNAc) from CMP-sialic acid is catalyzed by a family of sialyltransferases, GalNAc 2-6-sialyltransferases (ST6GalNAc-family). The cDNAs of six members of ST6GalNAc-family (ST6GalNAc I–VI) have been currently cloned from chicken (cST6GalNAc I and II) (4, 5), mouse (mST6GalNAc II–VI) (6 –10, 13), rat (rST6GalNAc III) (11) and human (hST6GalNAc I, II, and VI) (12). Among them, ST6GalNAc III–VI exhibit restricted substrate specificity, utilizing only the NeuAc2- 3Gal1-3GalNAc-sequence as an acceptor. However, there are some differences in their substrate prefer- ences. ST6GalNAc III can transfer sialic acid to both NeuAc2-3Gal1-3GalNAc-O-Ser/Thr and ganglio- side GM1b. ST6GalNAc IV exhibits strong activity toward NeuAc2-3Gal1-3GalNAc and O-glycans. ST6GalNAc V and VI may be the candidate for GD1 synthase. On the other hand, ST6GalNAc I exhibits the broadest substrate specificity toward GalNAc-O- Ser/Thr, Gal1-3GalNAc-O-Ser/Thr, and NeuAc2- 3Gal1-3GalNAc-O-Ser/Thr (4, 6, 12). ST6GalNAc II was reported to exhibit activity toward Gal1- 3GalNAc-O-Ser/Thr but not toward GalNAc-O-Ser/ Thr (5). Thus, only ST6GalNAc I was considered to be a candidate for STn synthase. In this study, how- ever, we found that ST6GalNAc II also exhibits ac- tivity toward GalNAc-O-Ser/Thr, suggesting that ST6GalNAc II is a second candidate for STn syn- thase. Abbreviations used: OSM, ovine submaxillary mucin; BSM, bovine submaxillary mucin; kb, kilobase(s); PCR, polymerase chain reac- tion; STn, sialyl-Tn antigen. The abbreviated nomenclature for cloned sialyltransferases follows the system of Tsuji et al. (1). This work was supported by the following grants: Grants-in-Aid for Scientific Research on Priority Areas, Nos. 10152263, 10178104, and 10178105 and for Scientific Research, B11480184, from the Ministry of Education of Japan, and Grant RO1 CA52491 from the United States National Cancer Institute (SHI). 1 Present address: Genetics of Development and Disease Branch, Building 10, Room 9D-11, National Institutes of Health, 10 Center DR MSC 1810, Bethesda, MD 20892-1810. 2 Present address: Laboratory for Neural Regeneration, Brain Sci- ence Institute, RIKEN, Wako, Saitama 351-0198, Japan. 3 Present address: Department of Biochemistry and Molecular Bi- ology, Georgetown University, Medical Center, 3900 Reservoir Road, NW, Washington, DC 20007. 4 Present address: Department of Biochemistry, Jichi Medical School, Minamikawachi, Tochigi 329-0498, Japan. 5 To whom correspondence should be addressed. Fax: +81-463-85- 5301. E-mail: stsuji02@yhb.att.ne.jp. Biochemical and Biophysical Research Communications 272, 94 –97 (2000) doi:10.1006/bbrc.2000.2745, available online at http://www.idealibrary.com on 94 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.