Role of N-linked glycosylation in the secretion and activity of endothelial lipase

Abstract

Human endothelial lipase (EL), a member of the triglyceride lipase gene family, has five potential N-linked glycosylation sites, two of which are conserved in both lipoprotein lipase and hepatic lipase. Reduction in molecular mass of EL after treatment with glycosidases and after treatment of EL-expressing cells with the glycosylation inhibitor tunicamycin demonstrated that EL is a glycosylated protein. Each putative glycosylation site was examined by site-directed mutagenesis of the asparagine (Asn). Mutation of Asn-60 markedly reduced secretion and slightly increased specific activity. Mutation of Asn-116 did not influence secretion but increased specific activity. In both cases, this resulted from decreased apparent Km and increased apparent Vmax. Mutation of Asn-373 did not influence secretion but significantly reduced specific activity, as a result of a decrease in apparent Vmax. Mutation of Asn-471 resulted in no reduction in secretion or specific activity. Mutation of Asn-449 resulted in no change in secretion, activity, or molecular mass, indicating that the site is not utilized. The ability of mutants secreted at normal levels to mediate bridging between LDL and cell surfaces was examined.The Asn-373 mutant demonstrated a 3-fold decrease in bridging compared with wild-type EL, whereas Asn-116 and Asn-471 were similar to wild-type EL. Human endothelial lipase (EL), a member of the triglyceride lipase gene family, has five potential N-linked glycosylation sites, two of which are conserved in both lipoprotein lipase and hepatic lipase. Reduction in molecular mass of EL after treatment with glycosidases and after treatment of EL-expressing cells with the glycosylation inhibitor tunicamycin demonstrated that EL is a glycosylated protein. Each putative glycosylation site was examined by site-directed mutagenesis of the asparagine (Asn). Mutation of Asn-60 markedly reduced secretion and slightly increased specific activity. Mutation of Asn-116 did not influence secretion but increased specific activity. In both cases, this resulted from decreased apparent Km and increased apparent Vmax. Mutation of Asn-373 did not influence secretion but significantly reduced specific activity, as a result of a decrease in apparent Vmax. Mutation of Asn-471 resulted in no reduction in secretion or specific activity. Mutation of Asn-449 resulted in no change in secretion, activity, or molecular mass, indicating that the site is not utilized. The ability of mutants secreted at normal levels to mediate bridging between LDL and cell surfaces was examined. The Asn-373 mutant demonstrated a 3-fold decrease in bridging compared with wild-type EL, whereas Asn-116 and Asn-471 were similar to wild-type EL. Endothelial lipase (EL) has been identified as a member of the triglyceride lipase gene family that includes pancreatic lipase, LPL, and HL (1Rader D.J. Jaye M. Endothelial lipase: a new member of the triglyceride lipase gene family.Curr. Opin. Lipidol. 2000; 11: 141-147Crossref PubMed Scopus (87) Google Scholar, 2Goldberg I.J. Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis.J. Lipid Res. 1996; 37: 693-707Abstract Full Text PDF PubMed Google Scholar, 3Santamarina-Fojo S. Haudenschild C. Amar M. The role of hepatic lipase in lipoprotein metabolism and atherosclerosis.Curr. Opin. Lipidol. 1998; 9: 211-219Crossref PubMed Scopus (211) Google Scholar, 4Wong H. Schotz M.C. The lipase gene family.J. Lipid Res. 2002; 43: 993-999Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Similar to LPL and HL, EL functions in the plasma compartment, and our initial report showed that overexpression of EL in mice profoundly alters plasma lipoprotein levels (5Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. A novel endothelial-derived lipase that modulates HDL metabolism.Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (426) Google Scholar). More recent studies have indicated that HDL is the preferred substrate of EL (6McCoy M.G. Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase.J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar, 7Jin W. Millar J.S. Broedl U. Glick J.M. Rader D.J. Inhibition of endothelial lipase causes increased HDL cholesterol levels in vivo.J. Clin. Invest. 2003; 111: 357-362Crossref PubMed Scopus (202) Google Scholar, 8Ma K. Cilingiroglu M. Otvos J.D. Ballantyne C.M. Marian A.J. Chan L. Endothelial lipase is a major genetic determinant for high-density lipoprotein concentration, structure, and metabolism.Proc. Natl. Acad. Sci. USA. 2003; 100: 2748-2753Crossref PubMed Scopus (205) Google Scholar). In contrast to LPL and HL, EL is synthesized in endothelial cells. Initial studies indicated that EL has significant phospholipase activity (5Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. A novel endothelial-derived lipase that modulates HDL metabolism.Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (426) Google Scholar, 9Hirata K. Diechek H.L. Cioffi J.A. Choi S.Y. Leeper N.J. Quintana L. Kronmal G.S. Cooper A.D. Quertermous T. Cloning of a unique lipase from endothelial cells extends the lipase gene family.J. Biol. Chem. 1999; 274: 14170-14175Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar); more recent studies have shown that it also has triglyceride lipase activity (6McCoy M.G. Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase.J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar). Many features of the gene family are conserved in EL, including the three catalytic residues, the cysteine residues that form intramolecular disulfide bonds (10Murthy V. Julien P. Gagne C. Molecular pathobiology of the human lipoprotein lipase gene.Pharmacol. Ther. 1996; 70: 101-135Crossref PubMed Scopus (160) Google Scholar), the lid covering the catalytic pocket (11Winkler F.K. D'Arcy A. Hunziker W. Structure of human pancreatic lipase.Nature. 1990; 343: 771-774Crossref PubMed Scopus (1044) Google Scholar), and the heparin binding and lipid binding regions (12Sendak R.A. Melford K. Kao A. Bensadoun A. Identification of the epitope of a monoclonal antibody that inhibits heparin binding of lipoprotein lipase: new evidence for a carboxyl-terminal heparin-binding domain.J. Lipid Res. 1998; 39: 633-646Abstract Full Text Full Text PDF PubMed Google Scholar). Conservation of structure also exists among the N-linked glycosylation sites, with two of the five potential glycosylation sites in human EL conserved in both human LPL and human HL (Fig. 1)(13Hide W.A. Chan L. Li W.H. Structure and evolution of the lipase superfamily.J. Lipid Res. 1992; 33: 167-178Abstract Full Text PDF PubMed Google Scholar, 14Lookene A. Bengtsson-Olivecrona G. Chymotryptic cleavage of lipoprotein lipase. Identification of cleavage sites and functional studies of the truncated molecule.Eur. J. Biochem. 1993; 213: 185-194Crossref PubMed Scopus (44) Google Scholar). HL has two additional glycosylation sites that are not present in LPL; EL has three additional potential glycosylation sites that are not present in LPL and are different from those in HL. Prevention of N-linked glycosylation has a direct effect on the secretion and activity of both LPL and HL. Tunicamycin treatment of LPL- and HL-expressing cells resulted in intracellular accumulation of an inactive protein of reduced size (15Olivecrona T. Chernick S.S. Bengtsson-Olivecrona G. Garrison M. Scow R.O. Synthesis and secretion of lipoprotein lipase in 3T3-L1 adipocytes. Demonstration of inactive forms of lipase in cells.J. Biol. Chem. 1987; 262: 10748-10759Abstract Full Text PDF PubMed Google Scholar, 16Busca R. Pujana M.A. Pognonec P. Auwerx J. Deeb S.S. Reina M. Vilaro S. Absence of N-glycosylation at asparagine 43 in human lipoprotein lipase accumulation in the and alters this Lipid Res. Full Text PDF PubMed Google Scholar, A.J. H. and apparent of human hepatic lipase in the J. 1999; PubMed Google Scholar). mutagenesis was to mutant of putative site in LPL and HL, which were for on secretion and activity. In both LPL and HL, of the conserved glycosylation sites on the Mutation of the conserved site in LPL, secretion and activity R. Pujana M.A. Pognonec P. Auwerx J. Deeb S.S. Reina M. Vilaro S. Absence of N-glycosylation at asparagine 43 in human lipoprotein lipase accumulation in the and alters this Lipid Res. Full Text PDF PubMed Google Scholar, Chan L. In and mutagenesis of the human lipoprotein lipase N-linked glycosylation site asparagine 43 is for both activity and Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar). Mutation of the site in HL, resulted in a decrease in secretion G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar, G. H. Schotz M.C. H. of N-linked glycosylation on hepatic lipase Lipid Res. Full Text PDF PubMed Google Scholar, J. H. Chan L. role of N-linked glycosylation in human hepatic lipase: is for both activity and Lipid Res. 1993; Full Text PDF PubMed Google and a reduced specific activity of the that was secreted G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar, G. H. Schotz M.C. H. of N-linked glycosylation on hepatic lipase Lipid Res. Full Text PDF PubMed Google Scholar). Mutation of the conserved site in LPL, no effect on secretion or activity G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar), whereas of the site in HL, resulted in a decrease in secretion but no effect on the specific activity of the secreted G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar, G. H. Schotz M.C. H. of N-linked glycosylation on hepatic lipase Lipid Res. Full Text PDF PubMed Google Scholar). Mutation of the two sites in HL, and no effect on secretion or specific activity G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar). In this that human EL is a glycosylated that of the five potential N-linked glycosylation sites are and that of glycosylation at the N-linked glycosylation sites has different on the secretion and specific activity of the In both LPL and HL have been shown to have the ability to the binding of to cell a bridging effect S. Haudenschild C. Amar M. The role of hepatic lipase in lipoprotein metabolism and atherosclerosis.Curr. Opin. Lipidol. 1998; 9: 211-219Crossref PubMed Scopus (211) Google Scholar, U. on the role of plasma lipase in lipoprotein and 1996; Full Text PDF PubMed Scopus Google Scholar, K.J. The family of of in Clin. Invest. 100: PubMed Scopus Google Scholar, K.J. Opin. Lipidol. PubMed Scopus Google Scholar), the role of N-linked glycosylation on the ability of human EL to mediate lipoprotein and cells were in with and at and cells were for with EL, whereas cells were for of wild-type and mutant EL with (5Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. A novel endothelial-derived lipase that modulates HDL metabolism.Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (426) Google Scholar), cells were to on were with in of at a of of of heparin was was at or after and at cells were to on and were to the after on was and with of and after an additional heparin was to the on a was and at was to were on and as (5Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. A novel endothelial-derived lipase that modulates HDL metabolism.Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (426) Google a antibody to a in the of the EL protein as a an as a and and were to EL in from cells with EL. were for at and were by to and at for on the were by cells were with as with the the with the was with of heparin with and were at and both cells and were after The cells were in of and and at for at intracellular protein and were and at from the and were by of lipase activity, were three with and at the of were in of and for triglyceride lipase and phospholipase activity protein was a protein The site-directed mutagenesis was to the asparagine at the five putative glycosylation sites identified in EL to an or a of the five and of wild-type EL in the were with of the and were at for and at for for and for of The for mutagenesis of are as and were by of lipase and phospholipase were in as (6McCoy M.G. Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase.J. Lipid Res. 2002; 43: 921-929Abstract Full Text Full Text PDF PubMed Google Scholar). were an with A of the triglyceride lipase was to apparent Km and for the mutant The were and the of initial and to the of substrate were for by of the wild-type and mutant in cells were in were for substrate of the of and the in the of the substrate were or two of the mutant with the of wild-type EL. Km and were and of the were a also with shown in the apparent Km and for the wild-type between but the of to Km was for wild-type and mutant forms of for Km and are EL, endothelial lipase. in a new for Km and are EL, endothelial lipase. human EL as W. Sun G.S. Marchadier D. Glick J.M. Rader D.J. Endothelial cells triglyceride lipase and phospholipase in to as a result of endothelial Res. 2003; PubMed Scopus Google was in the was no with human LPL or human HL. The was a protein and was a the was to in and to a The was at binding sites in those as as an of were with in for at The were with and with EL has not been to a of human EL from cells was in to a of EL in the are as to this EL as the were in in and in to the The was at for The were with and with to in in was The was for at three as of a in in was to The was for The was five with and with of were in of substrate and of was The substrate was to and was to for The was with of and the was at on an A cell binding was to the of EL and the N-linked glycosylation mutants to mediate bridging between the cell and plasma in cells on were with wild-type EL or glycosylation mutants binding at after were to of with and LDL in the or of heparin were at for and were The of binding of was by the in the of heparin from those in the of heparin binding of cells were at for in heparin to protein mass and phospholipase activity secreted by the cells. were from cells with the EL protein and to as in The effect of of human EL was by (Fig. and resulted in a reduction in size of the wild-type EL EL is with the effect by an effect by and the effect by H. of the human EL resulted in a that was the result of cleavage and was with the antibody to an A reduction in size of the was in the and to a in the but no was in the with N-linked at the to the asparagine to which are and to N-linked with reduced the form to which is the size of EL. with which residues, resulted in a more decrease in size that was for both the and which has for residues of the of N-linked resulted in a decrease in the form but no decrease in the The of effect of on the form that the at the glycosylation sites the form and the tunicamycin treatment to the of of N-linked glycosylation on EL secretion and activity. were from cells with EL in the and of tunicamycin protein as a cell and were by (Fig. cells EL, a protein of reduced mass was present in the cell and of the cells compared with cells. A protein of is in size with the EL protein by the EL protein was in of cells but present in the of cells. were for both triglyceride lipase (Fig. phospholipase (Fig. activity. from the of secretion, the that both triglyceride lipase and phospholipase were in from cells. were also for both triglyceride lipase and phospholipase activity. The activity in both was in the cells compared with cells wild-type EL not studies that of N-linked glycosylation as a result of tunicamycin treatment the secretion of EL and to the intracellular of an inactive protein of reduced studies of tunicamycin treatment of LPL- and HL-expressing cells similar G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar, A.J. D. H. lipase gene is in a truncated Lipid Res. Full Text PDF PubMed Google Scholar, M. S. of N-linked and in human hepatic lipase Lipid Res. 1999; Full Text Full Text PDF PubMed Google Scholar). that EL, LPL and HL, N-linked glycosylation for activity and of tunicamycin treatment on triglyceride and phospholipase activity of cells EL. cells with EL were with tunicamycin as in were and of from the were in for triglyceride lipase and phospholipase activity as in EL-expressing cells were present as a shown are mutant were at putative N-linked glycosylation site to glycosylation of the EL protein. mutants for in an site is and for the of of N-linked The asparagine of the was to or site-directed mutagenesis and in cells in the of were and to by (Fig. which indicated that of the mutant resulted in a reduction in molecular mass from the compared with wild-type EL, the the and mutant The reduction in molecular mass is with the of N-linked glycosylation at the sites, indicating that of the five potential N-linked glycosylation sites in EL are utilized. Mutation of two of the sites, Asn-60 and also resulted in a reduction in molecular mass of the whereas of the two sites, Asn-373 and did not the size of the that the site of cleavage in EL between and N-linked glycosylation and have on the and secretion of N-linked glycosylation on and Opin. Chem. Biol. 1999; PubMed Scopus Google Scholar), it was of to the of the mutant that were secreted the The (Fig. of the that of Asn-60 or resulted in an decrease in secretion compared with wild-type EL. N-linked glycosylation site is conserved among human LPL, HL, and EL (Fig. Mutation of LPL secretion of the R. Pujana M.A. Pognonec P. Auwerx J. Deeb S.S. Reina M. Vilaro S. Absence of N-glycosylation at asparagine 43 in human lipoprotein lipase accumulation in the and alters this Lipid Res. Full Text PDF PubMed Google Scholar, Chan L. In and mutagenesis of the human lipoprotein lipase N-linked glycosylation site asparagine 43 is for both activity and Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar), and of HL resulted in a reduction in secretion G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar, G. H. Schotz M.C. H. of N-linked glycosylation on hepatic lipase Lipid Res. Full Text PDF PubMed Google Scholar). our with the of EL at this site are with for LPL and HL, but more similar to HL, in that secretion was not as it was for The EL at and Asn-471 no on secretion compared with wild-type EL. Asn-373 is conserved in LPL and HL (Fig. and as with EL, of the LPL also no effect on secretion Chan L. In and mutagenesis of the human lipoprotein lipase N-linked glycosylation site asparagine 43 is for both activity and Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar, G. H. Schotz M.C. H. of N-linked glycosylation on hepatic lipase Lipid Res. Full Text PDF PubMed Google Scholar). Mutation of the HL resulted in a decrease in secretion G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar, G. H. Schotz M.C. H. of N-linked glycosylation on hepatic lipase Lipid Res. Full Text PDF PubMed Google Scholar). secretion of HL, but not LPL or EL, is on N-linked glycosylation at this N-linked glycosylation also influence protein structure and of N-linked glycosylation on and Opin. Chem. Biol. 1999; PubMed Scopus Google Scholar), both of which have on activity. In the that examined both the asparagine and were for triglyceride lipase and phospholipase for EL mass by and specific were in specific activity among the mutants not the was on not was no evidence that the mutants from the was in which five of the mutants and wild-type EL were on from this new were and for triglyceride lipase and phospholipase as as for EL mass by and the specific were (Fig. the Asn-60 mutant reduced secretion of was no reduction in lipase specific activity (Fig. The increased specific activity was not significant in this in the this mutant increased activity. secretion was the Asn-60 mutant that was secreted lipase specific activity that was similar to or slightly that wild-type EL. is in contrast to the in HL at which resulted in a reduction in the specific activity of the secreted to of wild-type HL G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar, G. H. Schotz M.C. H. of N-linked glycosylation on hepatic lipase Lipid Res. Full Text PDF PubMed Google Scholar, J. H. Chan L. role of N-linked glycosylation in human hepatic lipase: is for both activity and Lipid Res. 1993; Full Text PDF PubMed Google Scholar). The Asn-373 secreted of significantly reduced triglyceride and phospholipase specific activity compared with wild-type EL (Fig. N-linked glycosylation site is also conserved in LPL and HL (Fig. In contrast to EL, of LPL R. Pujana M.A. Pognonec P. Auwerx J. Deeb S.S. Reina M. Vilaro S. Absence of N-glycosylation at asparagine 43 in human lipoprotein lipase accumulation in the and alters this Lipid Res. Full Text PDF PubMed Google Scholar, A.J. H. and apparent of human hepatic lipase in the J. 1999; PubMed Google HL G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar, G. H. Schotz M.C. H. of N-linked glycosylation on hepatic lipase Lipid Res. Full Text PDF PubMed Google resulted in a reduction in specific activity. N-linked glycosylation at this site is for normal specific activity of EL but not for LPL or HL. was not in the three potential N-linked glycosylation sites in EL, Asn-116 of Asn-449 of and Asn-471 of sites are unique to EL and are not present in LPL or HL (Fig. Mutation of Asn-449 did not lipase specific activity (Fig. this is not as this site not to as an N-linked glycosylation as Asn-471 is of this site did triglyceride lipase specific activity but did not significantly phospholipase specific activity (Fig. demonstrated that of Asn-116 to increased lipase specific the phospholipase specific activity was significantly that in wild-type EL for the triglyceride lipase activity, it was not significantly different In of that compared wild-type EL with the mutant EL, the increased activity of the mutant form was from site-directed mutagenesis of the two N-linked glycosylation sites in HL (Fig. indicated both sites are for at site the of the have an effect on lipase specific activity G. G. Lipoprotein lipase and hepatic lipase: the role of glycosylation in the of a functional Lipid Res. Full Text PDF PubMed Google Scholar, G. H. Schotz M.C. H. of N-linked glycosylation on hepatic lipase Lipid Res. Full Text PDF PubMed Google Scholar). more the of the mutant were as in from three are in In of the of wild-type EL were to for in of the substrate the for The Km and The of are by the binding of the to the the binding of the site to a triglyceride in the and of an R. of PubMed Scopus Google Scholar). The triglyceride a of the triglyceride present in the of the the Km the A of the the K. J.S. R. role of N-linked glycosylation in J. 1993; PubMed Scopus Google Scholar), is the between A of the for wild-type EL in the three the that is among different substrate of the among of Km and a wild-type EL in the Km are with and the of to Km are the is the of in the and to between In (Fig. the for was significantly different from that for wild-type EL with wild-type EL, a Km and similar and the of to Km was for the Km was the was and the increased that the of glycosylation at or increased the activity of the In (Fig. wild-type EL and the mutant form of the site that not to similar and as and the were not significantly the for wild-type EL and were significantly different a markedly a slightly and a compared with wild-type EL. In (Fig. showed in and to wild-type EL. a of the that were not significantly In contrast to LPL and HL, in which the N-linked glycosylation sites no change in activity or decreased activity, the site in EL to a in activity, to by an increased of the for substrate and an in the Vmax. The a more but effect on activity, a in an of similar was in a on K. J.S. R. role of N-linked glycosylation in J. 1993; PubMed Scopus Google Scholar), an substrate is also In that of to resulted in a in with the in the to an in Vmax. LPL and HL have been shown to have an effect on bridging of with the role of N-linked glycosylation in the bridging of has not been the of the of N-linked glycosylation on the ability of EL to between and cells were with wild-type EL and the three mutant EL that glycosylation and were secreted and of the EL at similar of was in both the and of a of heparin a that the of with but not with the LDL were in and binding was to cell protein (Fig. the cells that were not EL cells with the a binding of indicating that was no binding in cells in the of EL. The and mutants were similar to wild-type EL in ability to mediate binding of the mutant a 3-fold decrease in binding of by the cells compared with wild-type EL. is that of three the mutant has reduced lipase specific activity. of glycosylation at this site result in an that is in ability to or and this result in reduced specific activity. to the between specific activity and bridging activity with N-linked In that human EL is a glycosylated protein and that secretion of the is the of N-linked mutagenesis of N-linked glycosylation sites in EL demonstrated that of the five putative sites are utilized. Mutation of the conserved glycosylation site Asn-60 similar to of the sites in LPL and HL in that secretion was LPL and HL, the activity of the was Mutation of a unique glycosylation site in EL, no effect on secretion but markedly increased activity. Mutation of the conserved glycosylation site Asn-373 was markedly different compared with of the sites in LPL and HL, in that it significantly reduced the lipase specific activity. Mutation of Asn-373 was the glycosylation mutant to have an effect on bridging of EL to and indicating a potential role for N-linked glycosylation in this was by of from the and an from the and a in