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.

TY - JOUR

T1 - Structures of the glycosyl-phosphatidylinositol anchors of porcine and human renal membrane dipeptidase.

T2 - Comprehensive structural studies on the porcine anchor and interspecies comparison of the glycan core structures.

AU - Brewis, Ian A.

AU - Ferguson, Michael A. J.

AU - Mehlert, Angela

AU - Turner, Anthony J.

AU - Hooper, Nigel M.

PY - 1995/9/29

Y1 - 1995/9/29

N2 - 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: Mana1-2Mana1-6Mana1-4GlcN (29% of the total population), Mana1-2Mana1-6(GalNAcß1-4)Mana1-4GlcN (33%), and Mana1-2Mana1-6(Galß1-3GalNAcß1-4)Mana1-4GlcN (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 a(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.

AB - 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: Mana1-2Mana1-6Mana1-4GlcN (29% of the total population), Mana1-2Mana1-6(GalNAcß1-4)Mana1-4GlcN (33%), and Mana1-2Mana1-6(Galß1-3GalNAcß1-4)Mana1-4GlcN (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 a(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.

U2 - 10.1074/jbc.270.39.22946

DO - 10.1074/jbc.270.39.22946

M3 - Article

C2 - 7559431

SN - 0021-9258

VL - 270

SP - 22946

EP - 22956

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 39

ER -