ABSTRACT
beta1,3-N-Acetylglucosaminyltransferase-7 (beta3Gn-T7) has been identified as an anti-migration factor for a lung cancer cell line but its enzymatic activity has not yet been characterized. Here we show that beta3Gn-T7 efficiently acts on keratan sulfate-related glycans including Galbeta1-->4(SO(3)(-)-->6)GlcNAcbeta1-->3Galbeta1-->4(SO(3)(-)-->6)GlcNAc (L2L2), while lacto-N-tetraose and lacto-N-neo-tetraose were poor substrates. Moreover, we found that among the other five beta3Gn-Ts and i antigen-producing beta3Gn-T (iGn-T), beta3Gn-T2 and iGn-T act well on L2L2, although these specific activities were lower than those for a tetraantennary N-glycan. These results indicate that beta3Gn-T7 is the one that most efficiently elongates L2L2 and may be involved in the biosynthesis of keratan sulfate.
Subject(s)
Keratan Sulfate/analogs & derivatives , N-Acetylglucosaminyltransferases/metabolism , Polysaccharides/chemistry , Polysaccharides/metabolism , Carbohydrate Sequence , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Keratan Sulfate/biosynthesis , Kinetics , Molecular Sequence Data , N-Acetylglucosaminyltransferases/genetics , Oligosaccharides/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , TritiumABSTRACT
Novel chromogenic substrates for endo-beta-galactosidase were designed on the basis of the structural features of keratan sulfate. Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (2), which consists of two repeating units of N-acetyllactosamine, was synthesized enzymatically by consecutive additions of GlcNAc and Gal residues to p-nitrophenyl beta-N-acetyllactosaminide. In a similar manner, GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (1), GlcNAcbeta1-3Galbeta1-4Glcbeta-pNP (3), Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta-pNP (4), Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glcbeta-pNP (5), and Galbeta1-6GlcNAcbeta1-3Galbeta1-4Glcbeta-pNP (6) were synthesized as analogues of 2. Endo-beta-galactosidases released GlcNAcbeta-pNP or Glcbeta-pNP in an endo-manner from each substrate. A colorimetric assay for endo-beta-galactosidase was developed using the synthetic substrates on the basis of the determination of p-nitrophenol liberated from GlcNAcbeta-pNP or Glcbeta-pNP formed by the enzyme through a coupled reaction involving beta-N-acetylhexosaminidase (beta-NAHase) or beta-d-glucosidase. Kinetic analysis by this method showed that the value of Vmax/Km of 2 for Escherichia freundii endo-beta-galactosidase was 1.7-times higher than that for keratan sulfate, indicating that 2 is very suitable as a sensitive substrate for analytical use in an endo-beta-galactosidase assay. Compound 1 still acts as a fairly good substrate despite the absence of a Gal group in the terminal position. In addition, the hydrolytic action of the enzyme toward 2 was shown to be remarkably promoted compared to that of 4 by the presence of a 2-acetamide group adjacent to the p-nitrophenyl group. This was the same in the case of a comparison of 1 and 3. Furthermore, the enzyme also catalysed a transglycosylation on 1 and converted it into GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (9) and GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-pNP (10) as the major products, which have N-acetyllactosamine repeating units.
Subject(s)
Amino Sugars/chemistry , Glycoside Hydrolases , Glycosides/biosynthesis , Glycosides/chemistry , Oligosaccharides/chemistry , beta-Galactosidase/chemistry , beta-Galactosidase/metabolism , Bacteroides fragilis/enzymology , Binding Sites , Calorimetry/methods , Carbohydrate Sequence , Citrobacter freundii/enzymology , Glycosylation , Hydrolysis , Isomerism , Keratan Sulfate/analogs & derivatives , Keratan Sulfate/metabolism , Kinetics , Molecular Sequence Data , Nitrophenols/analysis , Nitrophenols/chemistry , Nitrophenols/metabolism , Nuclear Magnetic Resonance, Biomolecular , Oligosaccharides/biosynthesis , Oligosaccharides/chemical synthesis , Substrate SpecificityABSTRACT
G(M1)-gangliosidosis and Morquio B disease are distinct in clinical and biochemical features, but both disorders are caused by genetic defects of the same enzyme, acid beta-galactosidase (beta-Gal). We analyzed the kinetic properties of mutant beta-Gals from patients with G(M1)-gangliosidosis and Morquio B disease to examine the clinical and biochemical differences between both disorders. Five skin fibroblast lines from patients with G(M1)-gangliosidosis (2 cases; R201C/R201C and I51T/I51T), Morquio B disease (2 cases; W273L/W273L and Y83H/R482C), and galactosialidosis (1 case; Y395C/S90L) were used as enzyme sources. Residual enzyme activity in the cells was subjected to kinetic analysis. Substrate analogs including Galbeta1-3GalNAc, as an analog for G(M1)-ganglioside, and Galbeta1-4GlcNAc, as an analog for keratan sulfate, were used to determine IC(50) and K(i) for beta-Gals with an artificial substrate (4-methylumbelliferyl beta-D-galactopyranoside). Enzymatic assay method was established to examine the hydrolytic activity with the mutant beta-Gal for the substrate analogs. The mutant beta-Gal activities were inhibited by Galbeta1-3GalNAc and Galbeta1-4GlcNAc in a concentration-dependent manner. Remarkable increase in IC(50) ratio and K(i) ratio (Galbeta1-4GlcNAc/Galbeta1-3GalNAc) was observed in Morquio B disease. Relative hydrolytic activity (Galbeta1-4GlcNAc/Galbeta1-3GalNAc) was markedly decreased in Morquio B disease as compared with other subjects; controls (means+/-SD, n=4), 1.00+/-0.02; galactosialidosis, 1.03; G(M1)-gangliosidosis, 1.15 and 1.00; and Morquio B disease, 0.27 and 0.32. The mutant beta-Gals from the patients with Morquio B disease exhibited lower affinity and lower hydrolytic activity toward Galbeta1-4GlcNAc rather than Galbeta1-3GalNAc. These findings suggest that imbalanced substrate specificity of the mutant beta-Gals induces predominant accumulation of keratan sulfate and a rationale for performing differential diagnostic analysis for both disorders.
