Porcine, mouse and human galactose 3-O-sulphotransferase-2 enzymes have different substrate specificities; the porcine enzyme requires basic compounds for its catalytic activity
AUTOR(ES)
Seko, Akira
FONTE
Portland Press Ltd.
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1237141Documentos Relacionados
- Human Cytomegalovirus Stimulates Cellular IKK2 Activity and Requires the Enzyme for Productive Replication†
- Experimental infection of C3H mice with avian, porcine, or human isolates of Serpulina pilosicoli.
- Substrate Specificities of Hybrid Naphthalene and 2,4-Dinitrotoluene Dioxygenase Enzyme Systems
- Genetic relationship between acylpeptide hydrolase and acylase, two hydrolytic enzymes with similar binding but different catalytic specificities.
- The Kex2p Proregion Is Essential for the Biosynthesis of an Active Enzyme and Requires a C-terminal Basic Residue for Its Function