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.
Highlights
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␣1–2Man␣1– 6Man␣1– 4GlcN (29% of the total population), Man␣1– 2Man␣1– 6(GalNAc1– 4)Man␣1– 4GlcN (33%), and Man␣1– 2Man␣1– 6(Gal1–3GalNAc1– 4)Man␣1– 4GlcN (38%)
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
Summary
Materials—Pig kidneys were obtained from ASDA Farm Stores, Lofthousegate, West Yorkshire, U.K., and post-mortem human kidneys were from Leeds General Infirmary or St. The sample was carefully adjusted to pH 5.0 with 5% (v/v) acetic acid, and an aliquot (equivalent to 1.0 nmol) was freeze-dried and redissolved in 50 mM sodium acetate-HCl, 20 mM CaCl2, pH 5.0 (equilibration buffer, 0.1 ml final volume) This was applied to an anhydrotrypsin (AHT)-Sepharose column prepared as described in Ishii et al (1983) (10 mg AHT, 3.5-ml bed volume, 1-ml void volume and pre-equilibrated in equilibration buffer (100 ml)). The run-through fraction was freeze-dried and redissolved in 0.1 ml of water This sample was subjected to reverse-phase HPLC using a Bondapak C18 steel column (3.9 ϫ 300 mm) with a UV (214 nm) detector at a flow rate of 2.0 ml/min and a 30-min gradient of 0 –70% (v/v) acetonitrile in 0.08% H3PO4 at pH 2.5 followed by 5 min elution at the final conditions. The fractions corresponding to the major peaks were pooled, freeze-dried, and subjected to solid-phase peptide sequencing as described in Findlay et al (1989) and Hooper et al (1990)
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