Sulphation of galactose at the C-3 position is one of the major post-translational modifications of colorectal mucin. Thus we partially purified a Gal 3-O-sulphotransferase from porcine colonic mucosa (pGal3ST) and studied its enzymatic characteristics. The enzyme was purified 48500-fold by sequential chromatographies on hydroxyapatite, Con A (concanavalin A)–Sepharose, porcine colonic mucin–Sepharose, Cu2+-chelating Sepharose and AMP–agarose. Interestingly, the purified pGal3ST required submillimolar concentrations of spermine or basic lipids, such as D-sphingosine and N,N-dimethylsphingosine, for enzymatic activity. pGal3ST recognized Galβ1→3GalNAc (core 1) as an optimal substrate, and had weaker activity for Galβ1→3GlcNAc (type 1) and Galβ1→4GlcNAc (type 2). Substrate competition experiments proved that a single enzyme catalyses sulphation of all three oligosaccharides. Among the four human Gal3STs cloned to date, the substrate specificity of pGal3ST is most similar to that of human Gal3ST-2, which is also strongly expressed in colonic mucosa, although the kinetics of pGal3ST and human Gal3ST-2 were rather different. To determine whether pGal3ST is the orthologue of human Gal3ST-2, a cDNA encoding porcine Gal3ST-2 was isolated and the enzyme was expressed in COS-7 cells for analysis of substrate specificity. This revealed that porcine Gal3ST-2 has the same specificity as pGal3ST, indicating that pGal3ST is indeed the porcine equivalent of Gal3ST-2. The substrate specificity of mouse Gal3ST-2 was also different from those of human and porcine Gal3ST-2 enzymes. Mouse Gal3ST-2 preferred core 1 and type 2 glycans to type 1, and the Km values were much higher than those of human Gal3ST-2. These results suggest that porcine Gal3ST-2 requires basic compounds for catalytic activity and that human, mouse and porcine Gal3ST-2 orthologues have diverse substrate specificities.
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Research Article|
September 26 2005
Porcine, mouse and human galactose 3-O-sulphotransferase-2 enzymes have different substrate specificities; the porcine enzyme requires basic compounds for its catalytic activity
Akira Seko;
Akira Seko
*Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062
†CREST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan
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Jun-ichi Sumiya;
Jun-ichi Sumiya
*Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062
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Katsuko Yamashita
Katsuko Yamashita
1
*Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062
1To whom correspondence should be addressed (email yamashita@sasaki.or.jp).
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Publisher: Portland Press Ltd
Received:
March 02 2005
Revision Received:
May 19 2005
Accepted:
May 31 2005
Accepted Manuscript online:
May 31 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 391 (1): 77–85.
Article history
Received:
March 02 2005
Revision Received:
May 19 2005
Accepted:
May 31 2005
Accepted Manuscript online:
May 31 2005
Citation
Akira Seko, Jun-ichi Sumiya, Katsuko Yamashita; Porcine, mouse and human galactose 3-O-sulphotransferase-2 enzymes have different substrate specificities; the porcine enzyme requires basic compounds for its catalytic activity. Biochem J 1 October 2005; 391 (1): 77–85. doi: https://doi.org/10.1042/BJ20050362
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