Proprotein Covertases Are Responsible for Proteolysis and Inactivation of Endothelial Lipase

Plasma lipoprotein metabolism is tightly regulated by several members of the triglyceride lipase family, including endothelial lipase (EL) and lipoprotein lipase (LPL). Our previous work suggested that EL is proteolytically processed. In this report, we have used a combination of epitope tagging, mutagenesis, and N-terminal sequencing to determine the precise location of the cleavage site within EL. The cleavage occurs immediately after the sequence RNKR, a known recognition sequence for the proprotein convertase (PC) family. We demonstrate that some PCs, but not all, can proteolytically cleave EL at this site and thereby directly regulate EL enzymatic activity through modulating EL cleavage. Furthermore, specific knockdown of individual PCs proves that PCs are the proteases that cleave EL in human endothelial cells. Interestingly, a homologous site in LPL is also cleaved by PCs. This action is unusual for PCs, which are traditionally known as activators of pro-proteins, and highlights a potential role of PCs in lipid metabolism through their proteolytic processing of lipases. Plasma lipoprotein metabolism is tightly regulated by several members of the triglyceride lipase family, including endothelial lipase (EL) and lipoprotein lipase (LPL). Our previous work suggested that EL is proteolytically processed. In this report, we have used a combination of epitope tagging, mutagenesis, and N-terminal sequencing to determine the precise location of the cleavage site within EL. The cleavage occurs immediately after the sequence RNKR, a known recognition sequence for the proprotein convertase (PC) family. We demonstrate that some PCs, but not all, can proteolytically cleave EL at this site and thereby directly regulate EL enzymatic activity through modulating EL cleavage. Furthermore, specific knockdown of individual PCs proves that PCs are the proteases that cleave EL in human endothelial cells. Interestingly, a homologous site in LPL is also cleaved by PCs. This action is unusual for PCs, which are traditionally known as activators of pro-proteins, and highlights a potential role of PCs in lipid metabolism through their proteolytic processing of lipases. Site-specific proteolysis is crucial in regulating many fundamental biological pathways, including the sequential initiation of activation of blood coagulation factors and activation of caspases and digestive enzymes (1Salvesen G.S. Abrams J.M. Oncogene. 2004; 23: 2774-2784Crossref PubMed Scopus (212) Google Scholar, 2Kageyama T. Cell Mol. Life Sci. 2002; 59: 288-306Crossref PubMed Scopus (210) Google Scholar, 3Bazan J.F. Nature. 1996; 380: 21-23Crossref PubMed Scopus (14) Google Scholar). The proprotein convertase (PC) 5The abbreviations used are:PCproprotein convertaseSKI-1/S1PSubtilisin-kexin isozyme-1/Site-1 proteaseNARC-1neural apoptosis-regulated convertase 1ELendothelial lipaseLPLlipoprotein lipaseHLhepatic lipaseHEKhuman embryonic kidneyDPCdecanoyl-Arg-Val-Lys-Arg-chloromethyl ketoneKDELLys-Asp-Glu-Leuα1-PDXα1-antitrypsin PortlandHUVEChuman umbilical vein artery endothelial cellsBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. family is composed of at least nine members and belongs to the subtilisin superfamily of serine endoproteases (4Seidah N.G. Prat A. Essays Biochem. 2002; 38: 79-94Crossref PubMed Scopus (187) Google Scholar). The basic amino acid-specific PCs catalyze the proteolytic maturation of a strikingly diverse collection of substrates at paired basic amino acid processing sites to generate biologically active molecules in the secretory pathway (5Seidah N.G. Chretien M. Curr. Opin. Biotechnol. 1997; 8: 602-607Crossref PubMed Scopus (241) Google Scholar). Some PC substrates are proteins involved in lipid metabolism. Furin, the first discovered and best characterized PC, was shown to cleave the low density lipoprotein receptorrelated protein (6Willnow T.E. Moehring J.M. Inocencio N.M. Moehring T.J. Herz J. Biochem. J. 1996; 313: 71-76Crossref PubMed Scopus (108) Google Scholar), and a mutation of the processing site of chicken low density lipoprotein receptor-related protein impaired efficient exit from the endoplasmic reticulum (7Ko K.W. McLeod R.S. Avramoglu R.K. Nimpf J. FitzGerald D.J. Vukmirica J. Yao Z. J. Biol. Chem. 1998; 273: 27779-27785Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Subtilisin-kexin isozyme-1/Site-1 protease (SKI-1/S1P), an early Golgi-localized PC (8Seidah N.G. Chretien M. Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (690) Google Scholar), cleaves membrane-bound sterol regulatory element-binding proteins and releases the active subunit of sterol regulatory element-binding proteins to maintain cholesterol homeostasis (9Horton J.D. Goldstein J.L. Brown M.S. Cold Spring Harbor Symp. Quant. Biol. 2002; 67: 491-498Crossref PubMed Scopus (157) Google Scholar). proprotein convertase Subtilisin-kexin isozyme-1/Site-1 protease neural apoptosis-regulated convertase 1 endothelial lipase lipoprotein lipase hepatic lipase human embryonic kidney decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone Lys-Asp-Glu-Leu α1-antitrypsin Portland human umbilical vein artery endothelial cells 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. PCs were considered to be redundant because a large body of in vitro work had shown that several PCs could cleave the same substrate. Recently, lessons from the studies of neural apoptosis-regulated convertase 1 (NARC-1), a newly discovered PC (10Cohen J. Pertsemlidis A. Kotowski I.K. Graham R. Garcia C.K. Hobbs H.H. Nat. Genet. 2005; 37: 161-165Crossref PubMed Scopus (1076) Google Scholar, 11Abifadel M. Varret M. Rabes J.P. Allard D. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. Derre A. Villeger L. Farnier M. Beucler I. Bruckert E. Chambaz J. Chanu B. Lecerf J.M. Luc G. Moulin P. Weissenbach J. Prat A. Krempf M. Junien C. Seidah N.G. Boileau C. Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2187) Google Scholar), reinforced the importance of individual PCs in regulating plasma lipid metabolism. However, the substrate of NARC-1 is not yet identified. Endothelial lipase (EL) is a recently described modulator of lipoprotein metabolism that belongs to the triglyceride lipase family, which also includes lipoprotein lipase (LPL) and hepatic lipase (HL) (12Wong H. Schotz M.C. J. Lipid Res. 2002; 43: 993-999Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Like LPL and HL, the EL protein is secreted and binds to heparan sulfate proteoglycans on the endothelial cell surface, where it interacts with lipoproteins (13Choi S.Y. Hirata K. Ishida T. Quertermous T. Cooper A.D. J. Lipid Res. 2002; 43: 1763-1769Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 14Jaye M. Krawiec J. Curr. Opin. Lipidol. 2004; 15: 183-189Crossref PubMed Scopus (42) Google Scholar, 15Rader D.J. Jaye M. Curr. Opin. Lipidol. 2000; 11: 141-147Crossref PubMed Scopus (87) Google Scholar). Both overexpression and loss of function studies have shown that EL is important in the regulation of HDL cholesterol levels (16Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (426) Google Scholar, 17Jin W. Millar J.S. Broedl U. Glick J.M. Rader D.J. J. Clin. Investig. 2003; 111: 357-362Crossref PubMed Scopus (202) Google Scholar, 18Ma K. Cilingiroglu M. Otvos J.D. Ballantyne C.M. Marian A.J. Chan L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2748-2753Crossref PubMed Scopus (204) Google Scholar, 19Ishida T. Choi S. Kundu R.K. Hirata K. Rubin E.M. Cooper A.D. Quertermous T. J. Clin. Investig. 2003; 111: 347-355Crossref PubMed Scopus (272) Google Scholar). It may also have a role in apoB-containing lipoprotein metabolism (20Broedl U.C. Maugeais C. Millar J.S. Jin W. Moore R.E. Fuki I.V. Marchadier D. Glick J.M. Rader D.J. Circ. Res. 2004; 94: 1554-1561Crossref PubMed Scopus (69) Google Scholar). EL protein was detected in endothelial cells, smooth muscle cells, and macrophages in human atherosclerotic lesions (21Azumi H. Hirata K. Ishida T. Kojima Y. Rikitake Y. Takeuchi S. Inoue N. Kawashima S. Hayashi Y. Itoh H. Quertermous T. Yokoyama M. Cardiovasc. Res. 2003; 58: 647-654Crossref PubMed Scopus (57) Google Scholar). Unlike LPL and HL, EL is up-regulated in response to cytokines, shear stress, and cyclic stretch (22Hirata K. Ishida T. Matsushita H. Tsao P.S. Quertermous T. Biochem. Biophys. Res. Commun. 2000; 272: 90-93Crossref PubMed Scopus (83) Google Scholar, 23Jin W. Sun G.S. Marchadier D. Octtaviani E. Glick J.M. Rader D.J. Circ. Res. 2003; 92: 644-650Crossref PubMed Scopus (108) Google Scholar). Furthermore, EL deficiency reduced the development of atherosclerosis in apoE–/– mice (24Ishida T. Choi S.Y. Kundu R.K. Spin J. Yamashita T. Hirata K.I. Kojima Y. Yokoyama M. Cooper A.D. Quertermous T. J. Biol. Chem. 2004; 279: 45085-45092Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Interestingly, two major forms of the EL protein, the full-length and a smaller form, are detected in conditioned media from endothelial cells by an an N-terminal (16Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (426) Google Scholar). of EL in vitro and in in of the same two proteins C. Broedl U.C. Marchadier D. W. S. Glick J.M. Rader D.J. 2003; PubMed Scopus Google Scholar). We that the smaller of EL is the of a specific proteolytic cleavage of the full-length In this report, we the site of this the specific sequence demonstrate that specific PCs are for the cleavage of that cleavage reduced EL and that LPL is also cleaved by PCs. and from from the of human embryonic kidney cell from from from from for and from and and from was from Life and was from Endothelial cells were from ketone was from was by of Cell of cells were in at in and with cells were on on a cells were with 1 of the in 1 of after 1 of and with was after the cells were to in the of of The media were at The media with are to as conditioned for the cells were in of with protease The was at for and the was cell were used for cells, and cell were for and were as described W. Sun G.S. Marchadier D. Octtaviani E. Glick J.M. Rader D.J. Circ. Res. 2003; 92: 644-650Crossref PubMed Scopus (108) Google Scholar). The used were a human EL and of with was as (16Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (426) Google Scholar). and the form, the cleaved N-terminal of and the cleaved of PC of EL and LPL in cells EL LPL were with for of the the and cells were for in the of Cell media were to with the and the were on and as (8Seidah N.G. Chretien M. Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (690) Google Scholar, S. A. Wickham L. M. J.S. A. C. S. Chretien M. Seidah N.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, N. A. Chretien M. Seidah N.G. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, N.G. Benjannet S. Wickham L. J. S. A. Prat A. Chretien M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar). of was the for were to the previous M. Seidah N. Chretien M. J. 1997; PubMed Scopus Google Scholar). of and of were by of cells to the were by of cells in and were cells were by with and and in the of of The were shown as for for for lipoprotein were as described I.V. N. Jin W. Marchadier Millar J.S. Glick J.M. Rader D.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Both triglyceride lipase and were as described Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. J. Lipid Res. 2002; 43: Full Text Full Text PDF PubMed Google Scholar). EL media from of human endothelial cells were with and proteins to the were to an that the N-terminal epitope of EL of the endothelial cells EL as by the of an of EL. In the media also a protein by this same The of forms of EL the endothelial cells, that the processing of EL is We that the protein was from full-length EL by proteolytic cleavage. the cleavage of we full-length EL with a a of human EL in cells, the EL detected full-length the EL as as N-terminal that were in to EL and to that in endothelial cell media This that EL is proteolytically cleaved in cell endothelial cells. The detected the full-length as as a smaller and the detected the full-length as as a This that the the cleavage In by we that the on EL processing not the of cleavage of cells human were in a with EL was from media at a that and of EL W. Sun G.S. Marchadier D. Octtaviani E. Glick J.M. Rader D.J. Circ. Res. 2003; 92: 644-650Crossref PubMed Scopus (108) Google Scholar). EL protein was from the media in the of not not Both in the and of the full-length in the with the smaller The of forms of EL as a function of the that the processing of EL is of EL a PC the site of we a of that EL is cleaved amino and not the full-length EL and the were cells. of the EL proteins as as full-length EL were detected in the media with the EL The and were detected by but the and were not detected with the same the loss of the epitope through cleavage and This the cleavage in the the and of EL. In to the processing of full-length of forms of EL were cleaved the importance of the of EL in regulating this the precise cleavage we full-length EL a in cells by proteins a epitope from cell a acid proteins by and to The of was by N-terminal sequencing of this protein that the cleavage of EL occurs immediately after the of an sequence determine the amino acid that are important for cleavage of we a of in which was for the amino the two reduced EL and the of the to at EL but of for at cleavage. In two EL cleavage and the that at the to the cleavage site and at the are the two in the for proteolytic we the importance of the that may for the of a (8Seidah N.G. Chretien M. Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (690) Google and the cleavage in the is of EL the cleavage is by shown in of of within the sequence not of the EL protein the but reduced EL cleavage that an sequence is also for EL cleavage. of EL Both and cell was used to determine the location of the proteolytic cleavage which with of of the endoplasmic reticulum to the S. Y. J. Cell Sci. 92: Google Scholar), of full-length of EL the but not in of the in the cell that proteolytic with the endoplasmic reticulum not in processing EL. which with Full Text PDF PubMed Scopus Google Scholar), and which the of the endoplasmic reticulum with the early J. A. Full Text PDF PubMed Scopus Google Scholar), also not in of the N-terminal demonstrate that proteolytic processing of the EL is to to the In which with C.M. D. K. J. Cell Biol. PubMed Scopus Google Scholar), in of the N-terminal in the cell that processing of EL can in the We could not an cleavage of EL in which be the of not to the proteolytic processing of the endoplasmic reticulum sequence Lys-Asp-Glu-Leu J. PubMed Scopus Google was at the of EL. the had of EL the media not but had EL in the cell of EL that not the In the cells the N-terminal was in the cell In N-terminal was in cells This that cleavage occurs to the early and is with the that proteolytic processing of EL is a we the cell with at for full-length EL is from the conditioned that the secreted of EL is proteolysis can the full-length was at in the of for to In the of cells, full-length EL was proteolytically cleaved not In the of cells, the full-length was to the with are to at the cell surface, the that cleaves EL. for EL the sequence is a PC recognition site and PCs are known to be active after the we used PC to their on the proteolytic processing of EL. is a that binds to the substrate site of PCs and their activity W. A. E. P. PubMed Scopus Google Scholar). determine the cleavage of cells with a were with for in a of cleavage of of the full-length EL. In EL cleavage was not by serine protease including a protease from not The of and are of PC activity by to PCs, and as as and N. A. Chretien M. Seidah N.G. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J.S. A. Benjannet S. E. Chretien M. Seidah N.G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). We the of and to the processing of EL. in overexpression of in a of EL cleavage. PC was by the of α1-antitrypsin to the sequence for PCs cleavage and to as a K. L. G. Y. A.J. G. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus (241) Google Scholar). We the of to the processing of EL. The processing of EL was cells were with but of EL cleavage the of also reduced the processing of EL in as with the sequence is the site of a to this site the processing of EL. We the of two on the processing of EL after in which the and as a a an EL cleavage was reduced by had on cleavage are not to the cells, this that processing occurs at the cell in the Some PCs the to determine which of the PC family cleave we in the cell of the PCs and were to generate from full-length EL but not the (5Seidah N.G. Chretien M. Curr. Opin. Biotechnol. 1997; 8: 602-607Crossref PubMed Scopus (241) Google Scholar), The cleavage of EL were and to N-terminal protein EL sequence immediately PCs cleaved EL at the same site where EL is cleaved in cells. that loss of the of in efficient processing and are at levels not We cleavage by the recognition sequence of EL. this we EL and of EL and with in cells. of cleavage of reduced cleavage and cleavage by PCs EL was as by an which may through a cleavage PCs are The of PCs was by in human endothelial cells, human endothelial cells, and human endothelial cells. at least two were for members of the PC family. Furin, and were in cells, was in human endothelial cells and human endothelial cells, was in human endothelial cells, human endothelial cells, and human endothelial cells, and was not in of endothelial cells the PCs that are to cleave EL are in in endothelial cells. determine the of individual PCs in processing EL in endothelial cells, we several The of in their was by in cells not we and the of EL in the conditioned shown in cleavage of EL was but not all, in cells with and and in the processing of EL in EL by PCs the of cleavage on EL enzymatic which is in endothelial cells G. E. W. Chretien M. Seidah N.G. R. Cell Res. 279: PubMed Scopus Google Scholar, M. M. Seidah N.G. C. E. 1996; PubMed Scopus Google Scholar), cleaved EL in a and reduced lipase lipase and in a In the cleavage of EL and EL lipase and that the of and on EL lipase are on an we EL and a of EL with in cells. the cleavage of EL and EL lipase cleaved full-length EL and reduced but had on the cleavage enzymatic activity of the cleavage of EL at the site by PCs enzymatic to the we that the specific activity of this was EL not LPL by PCs at a is of the triglyceride lipase family to EL. In the the and of human we a sequence at which is also known LPL proteins in LPL is proteolytically we LPL with and in cells. LPL protein was detected by the which the of shown in in the LPL as a and a at in a of that full-length LPL is proteolytically with the processing of a in with the with the had on LPL LPL was with of the LPL protein was to LPL is with of the LPL as a the of cleavage of LPL the that the cleavage of LPL is at the we to LPL the same we used for EL. We the of LPL the acid and The cleavage of LPL is after the in the sequence we the of PCs to cleave LPL in a cell were to used for EL. in cells with the two with an of and to the full-length and of with of the PCs that and and to a could but not the PCs of the triglyceride lipase family, HL, be cleaved by PCs. J. M. and D. J. Our that the EL protein is proteolytically cleaved at a specific site after and that this cleavage is by several members of the PC family. are with early that a of EL was detected in W. Millar J.S. Broedl U. Glick J.M. Rader D.J. J. Clin. Investig. 2003; 111: 357-362Crossref PubMed Scopus (202) Google Scholar). In to many PC proteolytic cleavage is not a for EL cleavage of EL by PCs in a active a unusual function for PCs. We that EL is cleaved at the in the two of EL. The for efficient cleavage are and The of the recognition sequence is also This sequence is in the and The specific location of this sequence is because a sequence is also at several sites within the EL protein, but is not We that EL is a substrate of PCs. However, EL cleavage is not by and which are the PCs known to be involved in lipid metabolism and the metabolism of apoB-containing Furin, and not several are to cleave EL but not the site Furthermore, several specific of PCs EL cleavage. We that several PCs that cleave EL are with EL in endothelial cells, with previous that and are by endothelial cells G. E. W. Chretien M. Seidah N.G. R. Cell Res. 279: PubMed Scopus Google Scholar, M. M. Seidah N.G. C. E. 1996; PubMed Scopus Google Scholar). that PCs are the proteases in endothelial cells that cleave EL. the of PCs is in a of endothelial cells, it is that several PCs, including and regulate EL cleavage in a and Our that EL cleavage occurs in the and at the cell It is that a of EL is cleaved after of protein with of processing of EL. was used to cell the EL was in the full-length of the in the of cells to was processing the same was in the of cells. with the the EL cleavage sequence EL that EL is cleaved by PCs at least in on the cell The role of and in the processing of their substrates on the cell is a protein, known role on the plasma was activation of K. Biochem. J. 1997; PubMed Scopus Google Scholar). However, was shown to cleave and the protein of a of the I. T.J. S. G.S. C. 2004; PubMed Scopus (24) Google at the cell In was recently shown to be cleaved and by at the cell G. Boileau G. M. J. Cell Sci. 2003; PubMed Scopus Google Scholar). and are secretory proteins and have shown to in the through to heparan sulfate proteoglycans A. K. E. T. S. K. K. Y. Biophys. 2003; PubMed Scopus Google Scholar). was shown to cleave neural on the of endothelial cells I. B. Seidah N.G. R. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). we that the may a for the of PCs and EL. on a to the of the triglyceride lipase for which a A. W. Nature. PubMed Scopus Google Scholar), full-length EL protein is composed of an N-terminal and a by a of EL at the be to generate two smaller with We not the cleavage of EL have biological for is cleaved by PCs, and the cleavage G. G. A. R. G. Biol. PubMed Scopus Google Scholar). The N-terminal of EL with the of EL after cleavage not the of of the two of and the cleavage of EL a role in this cleavage of EL some some EL activity that full-length EL may be the EL protein is in endothelial cells as shown in of EL molecules with cleavage of EL may generate several that may have We not a lipase activity of the It is that this have on protein thereby the activity in to EL cleavage. We are amino acid at the regulation of LPL activity for S. J.M. Biophys. PubMed Scopus Google Scholar), but the of this regulation are In the the and of human we a sequence at which is also known LPL proteins in we potential of that LPL is also proteolytically cleaved by PCs. by several that LPL protein was two major but this was to be an J.M. J. Clin. Investig. PubMed Scopus (24) Google Scholar). In this we that LPL cleavage is not because of but a specific proteolytic by PCs. The processing of LPL some with EL cleavage. Both of at a PC recognition sequence within the of the and the cleavage is to PC However, are also in several individual PCs have to cleave EL the of cleavage is for the of cleavage is the cleavage of LPL by PCs is efficient of EL. the of cleavage of LPL EL and LPL are cleaved by members of the PC family at a the of the cleavage may In studies demonstrate that PCs are in the cleavage of EL at a sequence and the potential importance of this processing in regulating EL Furthermore, PCs are to cleave LPL as of lipase activity by PCs may be a In this of processing of EL and LPL may a potential for regulating lipase activity and of lipid metabolism. This work a the PC family and lipid of the processing of and may the development of to and We and Millar for We are to and for with

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