Biochemical and enzymatic characterization of blood group ABH and related histo-blood group glycosphingolipids in the epithelial cells of porcine small intestine

Non-acid glycosphingolipids were isolated from small intestinal epithelial cells of a single blood group A pig. One very predominant blood group compound was obtained chemically pure upon HPLC fractionation. It was characterized by mass spectrometry and 1H NMR spectroscopy to be the type 1 chain blood group A hexaglycosylceramide. Support for the presence of minute amounts of additional A glycolipids was obtained by mass spectrometry and immunostaining of TLC plates with anti-A antibodies specific for A type 2 chain, A type 3 and 4 chain, and the ALeb determinant. Among precursor chains, globoside (type 4) and lactotetraosylceramide (type 1) were immunologically identified, whereas no neolactotetraosylceramide (type 2) and gangliotetraosylceramide reactivities were detected. We addressed the question whether the predominant expression of type 1 chain based A glycolipids reflects a restricted glycolipid precursor chain specificity of the α1–2 fucosyl and/or the α1–3 N-acetylgalactosaminyltransferases, or if the biosynthesis of the precursor chains themselves is regulated. All precursor core saccharides. lacto- (type 1), neolacto- (type 2), and gangliotetraosylceramide as well as globopentaosylceramide (type 4), could serve as acceptors for fucose in vitro when a crude microsomal fraction obtained from mechanically released, porcine intestinal epithelial cells was used as an enzyme source. Under the same conditions an N-acetylgalactosamine residue could be transferred to the blood group H structures based on these core saccharide chains. Lactotriaosylceramide, but not gangliotriaosylceramide, could serve as an acceptor for UDP galactose. When the product was digested with f β-galactosidase (EC 3.2.1.23) from S.pneumoniae, under conditions where it specifically cleaves Galβ1–4 residues, approximately 40% of the radioactivity was cleaved off, indicating that a substantial amount of neolactotetraosyl-ceramide was made in vitro, as opposed to the predominance of lactotetraosylceramide-based structures found in vivo.