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J. Biol. Chem., Vol. 280, Issue 9, 7720-7728, March 4, 2005

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A Clostridial Endo--galactosidase That Cleaves Both Blood Group A and B Glycotopes

THE FIRST MEMBER OF A NEW GLYCOSIDE HYDROLASE FAMILY, GH98^*

Kimberly M. Anderson, Hisashi Ashida¶, Karol Maskos||, Anne Dell**, Su-Chen Li, and Yu-Teh Li

From the Department of Biochemistry, Tulane University Health Sciences Center School of Medicine, New Orleans, Louisiana 70112, the ^||Coordinated Instrumentation Facility, Tulane University, New Orleans, Louisiana 70118, and the ^**Department of Biological Sciences, Imperial College London, London SW7 2AZ, United Kingdom

We have isolated an endo--galactosidase designated E-ABase from Clostridium perfringens ATCC 10543 capable of liberating both the A trisaccharide (A-Tri; GalNAc13(Fuc12)Gal) and B trisaccharide (B-Tri; Gal13(Fuc12)Gal) from glycoconjugates containing blood group A and B glycotopes, respectively. We have subsequently cloned the gene (eabC) that encodes E-ABase from this organism. This gene was found to be identical to the CPE0329 gene of C. perfringens strain 13, whose product was labeled as a hypothetical protein (Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T., Ogasawara, N., Hattori, M., Kuhara, S., and Hayashi, H. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 996–1001). Since the amino acid sequence of E-ABase does not bear detectable similarity to any of the 97 existing families of glycoside hydrolases, we have proposed to assign this unusual enzyme to a new family, GH98. We also expressed eabC in Escherichia coli BL21(DE3) and obtained 27 mg of fully active recombinant E-ABase from 1 liter of culture. Recombinant E-ABase not only destroyed the blood group A and B antigenicity of human type A and B erythrocytes, but also released A-Tri and B-Tri from blood group A⁺- and B⁺- containing glycoconjugates. The structures of A-Tri and B-Tri liberated from A⁺ porcine gastric mucin and B⁺ human ovarian cyst glycoprotein were established by NMR spectroscopy. The unique specificity of E-ABase should make it useful for studying the structure and function of blood group A- and B-containing glycoconju-gates as well as for identifying other glycosidases belonging to the new GH98 family.

Received for publication, December 15, 2004 , and in revised form, December 22, 2004.

^* This work was supported in part by National Institutes of Health Grant NS09626 (to Y.-T. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We dedicate this work to the late Dr. Karol Maskos. We will always remember his devotion to research and also his passion and aspiration for scientific excellence.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY492085.

Supported by National Research Service Award Predoctoral Training Grant 5F31 HL068296-03 from the National Institutes of Health.

^¶ Present Address: Dept. of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.

Deceased March 29, 2004.

Supported by a research professorship from the Biotechnology and Biological Sciences Research Council.

To whom correspondence should be addressed: Dept. of Biochemistry, Tulane University Health Sciences Center School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112. Tel.: 504-988-2451; Fax: 504-988-2739; E-mail: yli1{at}tulane.edu.

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