Ragupathy Madiyalakan

Cancer-associated elevation of α(1→3)-L-fucosyltransferase activity in human serum

GDP‐fucose:N‐acetylglucosaminide α(1 → 3)‐L‐fucosyltransferase activity was measured in sera of patients with various cancers using a synthetic substrate, N‐acetyl‐2′‐O‐methyllactosamine, as an acceptor. One hundred twenty‐four of the 169 patients showed significantly high levels of the enzyme activity when compared to healthy controls, irrespective of the location of their tumor. However, enzyme levels were in the normal range in patients with non‐neoplastic diseases, such as infectious disease, liver disease, and other inflammatory problems as well as in leukemic patients. The chromatofocusing profile of the enzyme using PBE‐94 gel over a pH grandient from pH 6.0 to 4.0 demonstrated that the level of the enzyme eluted at pH 5.4 was markedly elevated in the sera of stomach and ovarian cancer patients. A correlation was established between α(1 → 3)‐L‐fucosyltransferase activity and the presence of malignancy which may be used to evaluate the utility of the enzyme as a tumor marker.

Use of N-acetyl-2′-O-methyllactosamine as a specific acceptor for the determination of α-l-(1→3)-fucosyltransferase in human serum☆

A synthetic substrate, N-acetyl-2-O-methyllactosamine, was employed as a specific acceptor for alpha-L-(1----3)-fucosyltransferase from human serum. The fucosyl linkage of the product from this substrate was characterized by hydrolysis with a specific alpha-L-(1----3)/(1----4)-fucosidase. Using this acceptor, the pH optimum for the serum alpha-L-(1----3)-fucosyltransferase was 6.5. The enzyme was activated by Mn2+ or Mg2+ ions and was inhibited by EDTA. The apparent Km for this enzyme using N-acetyl-2-O-methyllactosamine was 20.4 mM and Vmax was 5.6 pmol/h/ml serum.

Lysosomal-enzyme targeting: the phosphorylation of synthetic d-mannosyl saccharides by UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosaminephosphotransferase from rat-liver microsomes and fibroblasts

Phosphorylation of the D-mannose residues of lysosomal enzymes is essential for the uptake and intracellular transport of these enzymes to lysosomes. The GlcNAc-P-transferase which is involved in the phosphorylation reaction seems to recognize a signal, probably a protein conformation, common to many lysosomal enzymes. To evaluate the role of the carbohydrate portion of the enzyme in these phosphorylation reactions, the acceptor specificity of GlcNAc-P-transferase from rat-liver microsomes and fibroblasts was examined with the aid of synthetic D-mannosyl disaccharides and derivatives that are closely related to the high-mannose type of oligosaccharides. Four methyl D-mannobiosides were synthesized, and their structures were established by 13C-n.m.r. spectroscopy. Of all the D-mannosyl saccharides tested, alpha-D-Man-(1----2)-alpha-D-Man-(1----OMe) was found to be the best acceptor, thereby suggesting that oligosaccharide structure may also have a role to play in recognition by this enzyme.

N-acetyl-β-d-glucosaminyltransferases related to the synthesis of mucin-type glycoproteins in human ovarian tissue

The presence of N-acetyl-beta-D-glucosaminyltransferases in microsome preparations from human ovarian tissues was investigated with UDP-GlcNAc and several synthetic oligosaccharides as acceptors. The products were identified by paper chromatography and the linkage of the 2-acetamido-2-deoxy-beta-D-glucopyranosyl group incorporated into oligosaccharides was determined by exoglycosidase digestions, 1H-n.m.r. spectroscopy, and methylation analysis. These results showed that ovarian microsome preparations contain both beta-(1----3)- and beta-(1----6)-N-acetyl-D-glucosaminyltransferase activities which might be involved in the synthesis of mucin-type glycoproteins. Substrate competition tests suggested that both UDP-GlcNAc:-Bn glycoside of beta-D-GlcpNAc-(1----6)-alpha-D-GalpNAc [GlcNAc to GalNAc] and -Bn glycoside of beta-D-Galp-(1----3)-[beta-D-GlcNAc-(1----6)]-alpha-D-GalpNAc [GlcNAc to Gal] beta-(1----3)-N-acetyl-D-glucosaminyltransferase activities reside in a single enzyme species.

α-L-fucosidase from aspergillus niger: Demonstration of a novel α-L-(1 → 6)-fucosidase acting on glycopeptides

Abstract An α-L-fucosidase which hydrolyzes fucose from α-(1 → 6)-linkage to N -acetylglucosamine was found in Aspergillus niger . The enzyme was purified by affinity chromatography with bovine IgG glycopeptide-Sepharose 4B. The enzyme preparation released fucose from bovine IgG glycopeptide and fucosylated asialoagalactofetuin, but failed to cleave 1 → 2, 1 → 3 or 1 → 4 linkages of α-L-fucosides.

Elevated serum α(1 → 3)-l-fucosyltransferase activity with synthetic low molecular weight acceptor in human ovarian cancer

Serum α(1 → 3)-l-fucosyltransferase activity was measured in 29 ovarian cancer patients with active disease, 26 ovarian cancer pateints with no clinical evidence of disease and 23 healthy females. N-Acetyl-2′-O-methyllactosamine was used as the acceptor for the enzyme. The level of the enzyme activity was significantly (P < 0.05) higher in the serum of patients with known tumor when compared to healthy controls and patients with no clinical evidence of disease.

Use of benzyl 2-acetamido-2-deoxy-3-O-(2-O-methyl-β-d-galactosyl)-β-d-glucopyranoside [2′-O-methyllacto-N-biosel βBn] as a specific acceptor for GDP-fucose: N-acetylglucosaminide α(1→4)-l-fucosyltransferase

Abstract A synthetic substrate, benzyl 2-acetamido-2-deoxy-3-O-(2-O-methyl-β- d -galactopyranosyl)-β- d -glucopyranoside, was demonstrated to be a specific acceptor for the Lewis blood group-specified α(1 → 4)- l -fucosyltransferase from human saliva and stomach mucosa. The fucosyl linkage of the product resulting from the use of this substrate isolated by paper chromatography was characterized by hydrolysis with specific α(1 → 3)/(1 → 4)- l -fucosidase. The product can be separated by adsorption onto the reverse-phase cartridge and recovered by one-step elution with methanol. The enzymatic properties of α(1 → 4)- l -fucosyltransferase from saliva and stomach mucosa have also been examined using this substrate.

Elevated activities of blood group Le gene dependent α(1→3)-L-fucosyltransferase in human saliva of Lewis negative patients with epithelial ovarian cancer

Levels of alpha(1----3)-L-fucosyltransferase activities were measured in salivas of patients with ovarian cancer. GDP-L-Fuc:GlcNAc alpha(1----3)-fucosyltransferase was found elevated in patients known to have epithelial ovarian cancer, irrespective of their ABH and Lewis blood group phenotypes. GDP-L-Fuc: Glc alpha(1----3)-fucosyltransferase was also elevated in both Lewis positive and negative patients, although the enzyme activity was very low or absent in Lewis negative healthy controls.

Synthesis of methyl 6-(ammonium 2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate)-α-D-mannopyranoside and use of this compound for the determination of N-acetylglucosamine-1-phosphotransferase

Abstract Methyl 6-(ammonium 2-acetamido-2-deoxy-α- d -glucopyranosyl phosphate)-α- d -mannopyranoside was synthesized and identified by 1H-n.m.r. and 13C-n.m.r. data, acid hydrolysis, and elemental analysis. It was utilized for the determination of UDP-N-acetylglucosamine-1-phosphotransferase in an assay procedure that employed methyl α- d -mannopyranoside as an acceptor. The assay product was identified and characterized by thin-layer chromatography with the title reference compound. The present technique does not require [32P]UDP-N-acetyl-glucosamine, but effectively uses commercially available UDP[14C]GlcNAc.

Serum β-(1→4)-galactosyltransferase activity with synthetic low molecular weight acceptor in human ovarian cancer

A modified procedure was developed for the determination of UDP-galactose: 2-acetamido-2-deoxy-glucopyranoside β-(1→4)-galactosyltransferase (GT) in huamn serum which employed the synthetic substrates p-nitrophenyl 6-0-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-β-d-galactopyranoside and p-nitrophenyl 6-0-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-α-d-mannopyranoside as receptors. THe enzyme products were identified by thin layer chromatography with authentic reference compounds, and the galactosyl linkage was characterized by hydrolysis with β-d-galactosidase from jack beans. The diagnostic value of this GT for ovarian cancer was tested by measuring the serum enzyme activity in 28 ovarian cancer patients with disease, 20 ovarian cancer patients with no clinical evidence of disease, and 22 healthy females. Although the level of the enzyme activity was significantly higher (P < 0.002) in the serum of patients with active disease when compared to healthy controls, an appreciable overlap of enzyme activity was found between them. Also, no correlation was found between enzyme activity and tumor size. Differences in methodology and selection of patients makes it difficult to compare results from other reports. However, based on our improved assay procedure, we suggest caution should be exercised in evaluating the merits of GT as a diagnostic marker for ovarian cancer.