title: The C-terminus of lipoprotein lipase is essential for biological function but contains no domain for glycosylphosphatidylinositol anchoring.
abstract: In this study we present evidence that the C-terminus of lipoprotein lipase contains no glycosylphosphatidylinositol addition signal and is therefore not a glycosylphosphatidylinositol-anchored protein. Furthermore, we present additional evidence that the C-terminus of lipoprotein lipase is essential for biological function. Flow cytometric analysis and enzyme-activity monitoring experiments revealed no pool of lipoprotein lipase releasable by phosphatidylinositol-specific phospholipase present on the membrane of COS cells transfected with the human lipoprotein lipase gene while, in contrast, a heparin-releasable pool could be demonstrated. [14C]Ethanolamine, a constituent of the glycosylphosphatidylinositol anchor, was not incorporated into lipoprotein lipase during metabolic labeling. C-terminal deletion mutants were constructed and expressed in COS cells to investigate the presence of glycosylphosphatidylinositol addition signal on the C-terminus of human lipoprotein lipase (LPL). The specific activities of the mutants M442 [des-(Leu443-Gly448)-LPL] and M437 [des-(Cys438-Gly448)-LPL] were 78% and 59%, respectively, less than the wild type, while the M432 mutant [des-(Ala433-Gly449)-LPL] was catalytically inactive. Determination of the stability of the mutants revealed a decreased stability of the M437, compared with wild-type, whereas M442 showed the same stability. Flow cytometric analysis showed sustained membrane expression for all mutants including the inactive M432 mutant. These results suggest that the C-terminus of lipoprotein lipase is essential for maintaining intact catalytic activity but is not involved in any posttranslational proteolytic processing, including cleavage of a glycosylphosphatidylinositol addition signal. We therefore conclude that membrane-binding of the lipase is not mediated by such anchoring.
authors.affiliation: Center for Hemostasis, Thrombosis, Atherosclerosis and Inflammation Research, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: T
authors.initials: T
authors.lastname: Bruin
authors.forename: N B
authors.initials: NB
authors.lastname: Groot
authors.forename: J
authors.initials: J
authors.lastname: Jansen
authors.forename: J J
authors.initials: JJ
authors.lastname: Kastelein
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D017353:Gene Deletion; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D008071:Lipoprotein Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D014176:Protein Biosynthesis; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D017261:Glycosylphosphatidylinositols; D008071:Lipoprotein Lipase