A Clostridial Endo--galactosidase That Cleaves Both Blood Group
A and B Glycotopes
THE FIRST MEMBER OF A NEW GLYCOSIDE HYDROLASE FAMILY, GH98*
Received for publication, December 15, 2004, and in revised form, December 22, 2004
Published, JBC Papers in Press, December 22, 2004, DOI 10.1074/jbc.M414099200
Kimberly M. Anderson‡§, Hisashi Ashida‡¶, Karol Maskos†, Anne Dell**‡‡, Su-Chen Li‡,
and Yu-Teh Liत
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 ca-
pable of liberating both the A trisaccharide (A-Tri;
GalNAc133(Fuc132)Gal) and B trisaccharide (B-Tri;
Gal133(Fuc132)Gal) from glycoconjugates contain-
ing 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., Ya-
mashita, A., Shiba, T., Ogasawara, N., Hattori, M., Ku-
hara, 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 recombi-
nant E-ABase from 1 liter of culture. Recombinant E-
ABase not only destroyed the blood group A and B anti-
genicity 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 be-
longing to the new GH98 family.
Clostridium perfringens, commonly found in sewage, soil,
and the gastrointestinal tracts of higher animals, is known to
cause gas gangrene (clostridial myonecrosis) and food poison-
ing in man (1, 2). It is also responsible for such animal diseases
as lamb dysentery, ovine enterotoxemia, and pulpy kidney
disease in sheep (1–5). The pathogenesis of C. perfringens in-
fection has been attributed, in part, to the large number of
extracellular toxins and hydrolytic enzymes (6 – 8) capable of
destroying host tissues (9). Among them, sialidase is one of the
most extensively studied extracellular enzymes of this orga-
nism (10 –12).
Although commercially available clostridial sialidase prepa-
rations have been widely used for studying the structure and
function of sialoglycoconjugates (12), they have been shown to
contain proteolytic (13–15), glycosidic (14), and cytotoxic/hemo-
lytic (16 –18) activities. We have reported previously that the
commercial sialidase prepared from C. perfringens ATCC
10543 is contaminated with an unusual endo--galactosidase
capable of releasing a specific disaccharide glycotope,
GlcNAc134Gal-, from blood group A
+
porcine gastric mucin
(A
+
-PGM)
1
(19). From the same preparation, we have detected
another unique endo--galactosidase capable of releasing
both the blood group A trisaccharide (A-Tri; GalNAc133-
(Fuc132)Gal) and B trisaccharide (B-Tri; Gal133-
(Fuc132)Gal) glycotopes from blood group A- and B-contain-
ing glycoconjugates, respectively. The unique specificity and
the potential usefulness of this enzyme for glycoconjugate re-
search prompted us to carry out the isolation of the native
enzyme as well as the cloning and characterization of the
recombinant enzyme. Based on the substrate specificity, we
propose to name this enzyme E-ABase for blood group A- and
B-cleaving endo--galactosidase. We found that the gene
(eabC) that encodes E-ABase is identical to the CPE0329 gene
of C. perfringens strain 13, whose product was labeled as a
hypothetical protein (20). Sequence comparison of E-ABase
with other glycosidases belonging to any of the 97 established
families of glycoside hydrolases revealed no significant se-
* 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.
† Deceased March 29, 2004.
§ Supported by National Research Service Award Predoctoral Train-
ing 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.
‡‡ Supported by a research professorship from the Biotechnology and
Biological Sciences Research Council.
§§ To whom correspondence should be addressed: Dept. of Biochem-
istry, 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@tulane.edu.
1
The abbreviations used are: A
+
-PGM, blood group A
+
porcine gastric
mucin; A-Tri, A trisaccharide (GalNAc133(Fuc132)Gal); B-Tri, B
trisaccharide (Gal133(Fuc132)Gal); nE-ABase, native E-ABase;
ConA, concanavalin A; kbp, kilobase pair; rE-ABase, recombinant E-
ABase; B
+
-HOCG, blood group B
+
human ovarian cyst glycoprotein; RBC,
red blood cell(s); PBS, phosphate-buffered saline; FITC, fluorescein 5-iso-
thiocyanate; FACS, fluorescence-activated cell sorting; A-Tetra, A
tetrasaccharide (GalNAc133(Fuc132)Gal134Glc); A-Penta, A
pentasaccharide (GalNAc133(Fuc132)Gal134(Fuc133)Glc); B-
Penta, B pentasaccharide (Gal133(Fuc132)Gal134(Fuc133)Glc);
A-Hexa, A hexasaccharide (GalNAc133(Fuc132)Gal13
3GlcNAc133Gal134Glc); FRP, fucolectin-related protein.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 9, Issue of March 4, pp. 7720 –7728, 2005
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