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J. Biol. Chem., Vol. 283, Issue 13, 8545-8554, March 28, 2008

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Identification of a GH110 Subfamily of 1,3-Galactosidases

NOVEL ENZYMES FOR REMOVAL OF THE 3GAL XENOTRANSPLANTATION ANTIGEN^*

Qiyong P. Liu¹, Huaiping Yuan, Eric P. Bennett, Steven B. Levery^¶, Edward Nudelman, Jean Spence, Greg Pietz, Kristen Saunders, Thayer White, Martin L. Olsson^||**, Bernard Henrissat, Gerlind Sulzenbacher, and Henrik Clausen

From the ZymeQuest Inc., Beverly, Massachusetts 01915, the Departments of Cellular and Molecular Medicine and Odontology, University of Copenhagen, Copenhagen N DK-2200, Denmark, the ^¶Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824, the ^||Division of Hematology and Transfusion Medicine and the Department of Laboratory Medicine, Lund University and University Hospital Blood Center, Lund SE-22185, Sweden, the ^**Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, and the Architecture et Fonction des Macromolécules Biologiques, UMR6098, CNRS, Universités Aix-Marseille I & II, Case 932, 13288 Marseille Cedex 9, France

In search of -galactosidases with improved kinetic properties for removal of the immunodominant 1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of -galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454–464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Gal1–3(Fuc1–2)Gal, whereas linear oligosaccharides terminated by 1,3-linked galactose such as the immunodominant xenotransplantation epitope Gal1–3Galβ1–4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific 1,3-galactosidases that act equally well on both branched blood group B and linear 1,3Gal structures. We determined by one-dimensional ¹H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known -galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant 3Gal xenotransplantation epitope.

Received for publication, November 2, 2007 , and in revised form, January 17, 2008.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AM259273.

^* This work was supported by ZymeQuest Inc. Work performed in M. L. O.'s laboratory was supported by the Swedish Research Council (project no. K2005-71X-14251), governmental ALF research grants to Lund University Hospital, and the Inga and John Hain Foundation for Medical Research and Region Skåne, Sweden. Work in H. C.'s laboratory was supported by the Danish Research Council and the Benzon Foundation. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Figs. S1 and S2, Table S1, and additional references.

¹ To whom correspondence should be addressed: ZymeQuest Inc., 100 Cummings Center, Suite 436H, Beverly, MA 01915-6122. Tel.: 978-232-8370; Fax: 978-232-8371; E-mail: pliu{at}zymequest.com.

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