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 (*)
- Ian A. Brewis(§),
- Michael A. J. Ferguson(¶),
- Angela Mehlert,
- Anthony J. Turner and
- Nigel M. Hooper(**)
- From the Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT and the
- Department of Biochemistry, University of Dundee, Dundee DD1 4HN, United Kingdom
- ** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom. Tel.: 44-113-233-3163; Fax: 44-113-233-3167.
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: Manα1-2Manα1-6Manα1-4GlcN (29% of the total population), Manα1-2Manα1-6(GalNAcβ1-4)Manα1-4GlcN
(33%), and Manα1-2Manα1-6(Galβ1-3GalNAcβ1-4)Manα1-4GlcN (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 α(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 Ser
. 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.
Footnotes
-
↵¶ Howard Hughes International Research Scholar.
-
↵* This work was supported by The Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- GPI
-
glycosyl-phosphatidylinositol
- AHM
-
2,5-anhydromannitol
- AHT
-
anhydrotrypsin
- Du
-
Dionex units
- ES-MS
-
electrospray-mass spectrometry
- EtNP
-
ethanolamine phosphate
- GC-MS
-
gas chromatography-mass spectrometry
- Gu
-
glucose units
- HPLC
-
high performance liquid chromatography
- HPTLC
-
high performance thin layer chromatography
- HVE
-
high voltage electrophoresis
- MDP
-
membrane dipeptidase
- NANA
-
N-acetylneuraminic acid
- NCAM
-
neural cell adhesion molecule
- PARP
-
procyclic acidic repetitive protein
- PI
-
phosphatidylinositol
- PI-PLC
-
phosphatidylinositol-specific phospholipase C
- PMAA
-
partially methylated alditol acetate
- TMS
-
trimethylsilyl
- TPCK
-
L-1-tosylamido-2-phenylethylchloromethyl ketone
- VSG
-
variant surface glycoprotein
- cpm
-
counts/minute.
-
↵2N. M. Hooper and M. A. J. Ferguson, unpublished data.
-
- Received June 20, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
