N-linked glycosylation of macrophage-derived PAF-AH is a major determinant of enzyme association with plasma HDL.
Human plasma PAF-AH (platelet-activating factor-acetylhydrolase) is a Ca(2)+-independent phospholipase A2 of hematopoietic origin associated with LDL and HDL; it degrades PAF and oxidizes phospholipids. We show that human macrophages synthesize PAF-AH as a premedial Golgi precursor containing high mannose N-linked glycans. Secreted PAF-AH possesses a molecular mass of approximately 55 kDa and contains mature N-linked glycans. Secreted PAF-AH activity (90 +/- 4% of the total) bound to a wheat germ lectin column and could be eluted with N-acetylglucosamine, whereas digestion with N-acetylneuraminidase II completely abolished enzyme absorption. Tunicamycin significantly reduced cell-associated PAF-AH activity and inhibited enzyme secretion; but it did not alter the ratio of secreted to cell-associated enzyme (1.8 at 6 h and 3.1 at 24 h), suggesting that glycosylation is not essential for PAF-AH secretion. Digestion of cell-associated PAF-AH or secreted PAF-AH with peptide N-glycosidase F affected neither catalytic activity nor its resistance to proteolysis with trypsin or proteinase K; in addition, it did not affect PAF-AH association with LDL, but significantly increased its association with HDL. We suggest that macrophage-derived PAF-AH contains heterogeneous asparagine-conjugated sugar chain(s) involving sialic acid, which hinders its association with HDL but does not influence the secretion, catalytic activity, or resistance of PAF-AH to proteases.
Tselepis
A D
AD
Laboratory of Biochemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece.
Karabina
S A
SA
Stengel
D
D
Piédagnel
R
R
Chapman
M J
MJ
Ninio
E
E
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000470:Alkaloids / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D002241:Carbohydrates / Q000032:analysis; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D019286:Endopeptidase K / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood* / Q000378:metabolism*; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D014357:Trypsin / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D000470:Alkaloids; D002241:Carbohydrates; D008075:Lipoproteins, HDL; C065629:kifunensine; D014415:Tunicamycin; D020126:Brefeldin A; D010741:Phospholipases A; D054467:Phospholipases A2; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D014357:Trypsin; D019286:Endopeptidase K