LC-MS analysis of pig intestine sulfatides: interaction with Escherichia coli STb enterotoxin and characterization of molecular species present.
STb, a 48-amino acid thermostable enterotoxin is produced by enterotoxigenic Escherichia coli strains and is responsible for diarrheal diseases in many animals, including man. Our laboratory recently identified a family of molecules, from a lipid extract of porcine intestinal epithelial cells, that could bind to STb. These molecules were identified as sulfatides as they reacted with a monoclonal antibody raised against this family of molecules. However, as the epitope recognized by this monoclonal antibody was the galactose 3-sulfate, a doubt could remain as to the exact nature of the identified receptors. The goal of this study was thus to confirm the chemical nature of the STb-binding molecule as sulfatides or as distinctive molecules comprising a sulfated galactosyl residue. Using a thin-layer chromatography-overlay method we confirmed using antibodies to STb that STb recognizes the commercial sulfatides and a band migrating at the same level from the intestinal tissue lipid extract obtained from an 8-week-old piglet. The compounds recovered from the silica gel plates were analyzed by mass spectrometry in electrospray negative-ionization mode. The most abundant ions observed had m/z values of 779, 795, 879 and 907. For commercial bovine brain sulfatides the ions 795, 879 and 907 have been attributed to hydroxylated sulfatides with a saturated fatty acid chain containing 16, 22 and 24 carbons, while the 779 ion contained a saturated fatty acid chain of 16 carbons. The general profile of the ions observed was similar to the already described commercial bovine brain sulfatides.
Beausoleil
Hans-Erick
HE
Groupe de recherche sur les maladies infectieuses du porc (GREMIP), Département de pathologie et microbiologie, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada.
Lépine
François
F
Dubreuil
J Daniel
JD
D000818:Animals; D001427:Bacterial Toxins / Q000032:analysis* / Q000378:metabolism; D002518:Ceramides / Q000032:analysis; D002853:Chromatography, Liquid; D002855:Chromatography, Thin Layer; D004768:Enterotoxins / Q000032:analysis* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D007583:Jejunum / Q000382:microbiology*; D013058:Mass Spectrometry; D011485:Protein Binding; D013433:Sulfoglycosphingolipids / Q000032:analysis* / Q000378:metabolism; D013552:Swine
D001427:Bacterial Toxins; D002518:Ceramides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D013433:Sulfoglycosphingolipids; C034151:heat stable toxin (E coli)