| First Author | Seko A | Year | 2005 |
| Journal | Biochem J | Volume | 391 |
| Issue | Pt 1 | Pages | 77-85 |
| PubMed ID | 15926885 | Mgi Jnum | J:117579 |
| Mgi Id | MGI:3696920 | Doi | 10.1042/BJ20050362 |
| Citation | Seko A, et al. (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. Biochem J 391(Pt 1):77-85 |
| abstractText | 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 Galbeta1-->3GalNAc (core 1) as an optimal substrate, and had weaker activity for Galbeta1-->3GlcNAc (type 1) and Galbeta1-->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 K(m) 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. |
