title: Structures of the glycosyl-phosphatidylinositol anchors of porcine and human renal membrane dipeptidase. Comprehensive structural studies on the porcine anchor and interspecies comparison of the glycan core structures.
abstract: The glycan core structures of the glycosyl-phosphatidylinositol (GPI) anchors on porcine and human renal membrane dipeptidase (EC 3.4.13.19) were determined following deamination and reduction by a combination of liquid chromatography, exoglycosidase digestions, and methylation analysis. The glycan core was found to exhibit microheterogeneity with three structures observed for the porcine GPI anchor: Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN (29% of the total population), Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN (33%), and Man alpha 1-2Man alpha 1-6(Gal beta 1-3GalNAc beta 1-4)Man alpha 1-4GlcN (38%). The same glycan core structures were also found in the human anchor but in slightly different proportions (25, 52, and 17%, respectively). Additionally, a small amount (6%) of the second structure with an extra mannose alpha (1-2)-linked to the non-reducing terminal mannose was also observed in the human membrane dipeptidase GPI anchor. A small proportion (maximally 9%) of the porcine GPI anchor structures was found to contain sialic acid, probably linked to the GalNAc residue. The porcine GPI anchor was found to contain 2.5 mol of ethanolamine/mol of anchor. Negative-ion electrospray-mass spectrometry revealed the presence of exclusively diacyl-phosphatidylinositol (predominantly distearoyl-phosphatidylinositol with a minor amount of stearoyl-palmitoyl-phosphatidylinositol) in the porcine membrane dipeptidase anchor. Porcine membrane dipeptidase was digested with trypsin and the C-terminal peptide attached to the GPI anchor isolated by removal of the other tryptic peptides on anhydrotrypsin-Sepharose. The sequence of this peptide was determined as Thr-Asn-Tyr-Gly-Tyr-Ser, thereby identifying the site of attachment of the GPI anchor as Ser368. This work represents a comprehensive study of the GPI anchor structure of porcine membrane dipeptidase and the first interspecies comparison of mammalian GPI anchor structures on the same protein.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Leeds, United Kingdom.
authors.forename: I A
authors.initials: IA
authors.lastname: Brewis
authors.forename: M A
authors.initials: MA
authors.lastname: Ferguson
authors.forename: A
authors.initials: A
authors.lastname: Mehlert
authors.forename: A J
authors.initials: AJ
authors.lastname: Turner
authors.forename: N M
authors.initials: NM
authors.lastname: Hooper
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002853:Chromatography, Liquid; D002855:Chromatography, Thin Layer; D004150:Dipeptidases / Q000737:chemistry* / Q000302:isolation & purification; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013552:Swine
chemical_list: D017261:Glycosylphosphatidylinositols; D009844:Oligosaccharides; D011134:Polysaccharides; D004150:Dipeptidases