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The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas.

ABSTRACT: The metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. Specialized carbohydrates modified with sialyl Lewis x (sLex) antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. The activity of the enzyme core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT1) in leukocytes greatly increases their ability to bind to endothelial selectins. C2GnT1 is essential for the synthesis of core 2-branched O-linked carbohydrates terminated with sLex (C2-O-sLex). Our goal was to determine the expression profiles of C2-O-sLex in the malignant progression and metastasis of colorectal adenocarcinomas. The well characterized CHO-131 monoclonal antibody (mAb) specifically recognizes C2-O-sLex present in human leukocytes and carcinoma cells. Using CHO-131 mAb, we investigated whether C2-O-sLex was present in 113 human primary colorectal adenocarcinomas, 10 colorectal adenomas, 46 metastatic liver tumors, 28 normal colorectal tissues, and 5 normal liver tissues by immunohistochemistry. We also examined mRNA levels of the enzyme core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT1) in 20 well, 15 moderately, and 2 poorly differentiated colorectal adenocarcinomas, and in 5 normal colorectal tissues by using quantitative real-time polymerase chain reactions (RT-PCR).We observed high reactivity with CHO-131 mAb in approximately 70% of colorectal carcinomas and 87% of metastatic liver tumors but a lack of reactivity in colorectal adenomas and normal colonic and liver tissues. Positive reactivity with CHO-131 mAb was very prominent in neoplastic colorectal glands of well to moderately differentiated adenocarcinomas. The most intense staining with CHO-131 mAb was observed at the advancing edge of tumors with the deepest invasive components.Finally, we analyzed C2GnT1 mRNA levels in 37 colorectal adenocarcinomas and 5 normal colorectal tissues by RT-PCR. Significantly, we observed a greater than 15-fold increase in C2GnT1 mRNA levels in colorectal adenocarcinomas compared to normal colorectal tissues.C2-O-sLex, detected by the CHO-131 mAb, is a tumor associated antigen whose expression is highly upregulated in colorectal adenocarcinomas and metastatic liver tumors compared to normal tissues. C2-O-sLex is a potentially useful early predictor of metastasis.

SUBMITTER: St Hill CA

PROVIDER: S-EPMC2662873 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas.

St Hill Catherine A CA Farooqui Mariya M Mitcheltree Gregory G Gulbahce H Evin HE Jessurun Jose J Cao Qing Q Walcheck Bruce B

BMC cancer 20090306

<h4>Background</h4>The metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. Specialized carbohydrates modified with sialyl Lewis x (sLex) antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. The activity of the enzyme core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT1) in leukocytes greatly increases their ability to bind to endothelial selectins. C2GnT1 is ess ...[more]

PMID: 19267921

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Project description:The survival of commensal bacteria in the human gut partially depends on their ability to metabolize host-derived molecules. The use of the glycosidic moiety of N-glycoproteins by bacteria has been reported, but the role of N-glycopeptides or glycoamino acids as the substrates for bacterial growth has not been evaluated. We have identified in Lactobacillus casei strain BL23 a gene cluster (alf-2) involved in the catabolism of the glycoamino acid fucosyl-α-1,6-N-GlcNAc-Asn (6'FN-Asn), a constituent of the core-fucosylated structures of mammalian N-glycoproteins. The cluster consists of the genes alfHC, encoding a major facilitator superfamily (MFS) permease and the α-l-fucosidase AlfC, and the divergently oriented asdA (aspartate 4-decarboxylase), alfR2 (transcriptional regulator), pepV (peptidase), asnA2 (glycosyl-asparaginase), and sugK (sugar kinase) genes. Knockout mutants showed that alfH, alfC, asdA, asnA2, and sugK are necessary for efficient 6'FN-Asn utilization. The alf-2 genes are induced by 6'FN-Asn, but not by its glycan moiety, via the AlfR2 regulator. The constitutive expression of alf-2 genes in an alfR2 strain allowed the metabolism of a variety of 6'-fucosyl-glycans. However, GlcNAc-Asn did not support growth in this mutant background, indicating that the presence of a 6'-fucose moiety is crucial for substrate transport via AlfH. Within bacteria, 6'FN-Asn is defucosylated by AlfC, generating GlcNAc-Asn. This glycoamino acid is processed by the glycosylasparaginase AsnA2. GlcNAc-Asn hydrolysis generates aspartate and GlcNAc, which is used as a fermentable source by L.casei These data establish the existence in a commensal bacterial species of an exclusive metabolic pathway likely to scavenge human milk and mucosal fucosylated N-glycopeptides in the gastrointestinal tract.IMPORTANCE The gastrointestinal tract accommodates more than 1014 microorganisms that have an enormous impact on human health. The mechanisms enabling commensal bacteria and administered probiotics to colonize the gut remain largely unknown. The ability to utilize host-derived carbon and energy resources available at the mucosal surfaces may provide these bacteria with a competitive advantage in the gut. Here, we have identified in the commensal species Lactobacillus casei a novel metabolic pathway for the utilization of the glycoamino acid fucosyl-α-1,6-N-GlcNAc-Asn, which is present in the core-fucosylated N-glycoproteins from mammalians. These results give insight into the molecular interactions between the host and commensal/probiotic bacteria and may help to devise new strategies to restore gut microbiota homeostasis in diseases associated with dysbiotic microbiota.

Project description:Among mucus-secreting cells, the gastric gland mucous cells, Brunner's glands, accessory glands of pancreaticobiliary tract, and pancreatic ducts exhibiting gastric metaplasia are unique in that they express class III mucin identified by paradoxical Con A staining composed of periodate oxidation, sodium borohydride reduction, Con A, and horseradish peroxidase reaction. Recently it was shown that these mucous cells secrete glycoproteins having GlcNAcalpha1-->4Galbeta-->R at nonreducing terminals of the carbohydrate moieties. Herein we describe the expression cloning of a cDNA encoding a human alpha1,4-N-acetylglucosaminyltransferase (alpha4GnT), a key enzyme for the formation of GlcNAcalpha1-->4Galbeta1-->R. COS-1 cells were thus cotransfected with a stomach cDNA library and a leukosialin cDNA. Transfected COS-1 cells were screened by using monoclonal antibodies specific for GlcNAcalpha1-->4Galbeta-->R and enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA clone that directs the expression of GlcNAcalpha1-->4Galbeta-->R. The deduced amino acid sequence predicts a type II membrane protein with 340 amino acids, showing no significant similarity with any other proteins. The alpha4GnT gene is located at chromosome 3p14.3, and its transcripts are expressed in the stomach and pancreas. An in vitro GlcNAc transferase assay by using a soluble alpha4GnT revealed that alpha1,4-linked GlcNAc residues are transferred most efficiently to core 2 branched O-glycans (Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAc), forming GlcNAcalpha1-->4Galbeta1-->4GlcNAcbeta1-->6(GlcNAca lpha1-->4Galbeta1- ->3)GalNAc. Transfection of alpha4GnT cDNA into gastric adenocarcinoma AGS cells produced class III mucin, indicating that alpha4GnT is responsible for the formation of class III Con A reactivity. These results indicate that the alpha4GnT is a glycosyltransferase that forms alpha1,4-linked GlcNAc residues, preferentially in O-glycans.

Dataset Information

The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas.

Publications

The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas.

Similar Datasets

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