The Mutation Gly142→ Glu in Human Lipoprotein Lipase Produces a Missorted Protein That Is Diverted to Lysosomes

While the molecular characterization of lipoprotein lipase (LPL) activation is progressing, the intracellular processing, transport, and secretion signals of LPL are still poorly known. The aim of this paper is to study the involvement of glycine 142 in LPL secretion and to elucidate the intracellular destination of the altered protein that remains inside the cell. We mutated the human LPL cDNA by site-directed mutagenesis in order to produce the G142E hLPL in which the glycine 142 was replaced by a glutamic acid. The wild type human LPL (WT hLPL) and the mutant G142E hLPL were expressed by transient transfection in COS1 cells. Using Western blot assays we identified a single band that had the same molecular weight for both proteins. However, Western blots of culture media did not reveal any specific band for the mutant protein, and ELISA experiments showed that the extracellular mass of the mutant LPL was only 25% of the WT protein, indicating defective secretion of the altered enzyme. Heparin increased LPL secretion in the case of the WT hLPL but did not have any stimulatory effect when acting on G142E hLPL-transfected cells. However, heparin-Sepharose chromatography revealed that both proteins presented the same heparin affinity. Metabolic labeling and radioimmunoprecipitation studies showed that both the WT and the mutant hLPL intracellular levels decreased upon chase time. Furthermore, leupeptin had a greater effect on the intracellular level of the mutant enzyme, thus indicating its higher intracellular degradation. Immunofluorescent studies using confocal microscopy indicated high colocalization of the LPL labeling and the Lamp1 lysosomal labeling in G142E hLPL-expressing cells. This result was confirmed using immunoelectron microscopy, which in addition showed gold labeling in Golgi stacks. This finding, together with experiments performed with endoglycosidase H digestion of immunoprecipitated radiolabeled LPL, indicated that the mutant enzyme entered the Golgi compartment. The results reported in this paper show that the G142E hLPL is not efficiently secreted to the extracellular medium, but it is missorted to lysosomes for intracellular degradation. This finding suggests that lysosomal missorting might be a mechanism of cell quality control of secreted LPL. While the molecular characterization of lipoprotein lipase (LPL) activation is progressing, the intracellular processing, transport, and secretion signals of LPL are still poorly known. The aim of this paper is to study the involvement of glycine 142 in LPL secretion and to elucidate the intracellular destination of the altered protein that remains inside the cell. We mutated the human LPL cDNA by site-directed mutagenesis in order to produce the G142E hLPL in which the glycine 142 was replaced by a glutamic acid. The wild type human LPL (WT hLPL) and the mutant G142E hLPL were expressed by transient transfection in COS1 cells. Using Western blot assays we identified a single band that had the same molecular weight for both proteins. However, Western blots of culture media did not reveal any specific band for the mutant protein, and ELISA experiments showed that the extracellular mass of the mutant LPL was only 25% of the WT protein, indicating defective secretion of the altered enzyme. Heparin increased LPL secretion in the case of the WT hLPL but did not have any stimulatory effect when acting on G142E hLPL-transfected cells. However, heparin-Sepharose chromatography revealed that both proteins presented the same heparin affinity. Metabolic labeling and radioimmunoprecipitation studies showed that both the WT and the mutant hLPL intracellular levels decreased upon chase time. Furthermore, leupeptin had a greater effect on the intracellular level of the mutant enzyme, thus indicating its higher intracellular degradation. Immunofluorescent studies using confocal microscopy indicated high colocalization of the LPL labeling and the Lamp1 lysosomal labeling in G142E hLPL-expressing cells. This result was confirmed using immunoelectron microscopy, which in addition showed gold labeling in Golgi stacks. This finding, together with experiments performed with endoglycosidase H digestion of immunoprecipitated radiolabeled LPL, indicated that the mutant enzyme entered the Golgi compartment. The results reported in this paper show that the G142E hLPL is not efficiently secreted to the extracellular medium, but it is missorted to lysosomes for intracellular degradation. This finding suggests that lysosomal missorting might be a mechanism of cell quality control of secreted LPL. INTRODUCTIONLipoprotein lipase (LPL) 1The abbreviations used are: LPLlipoprotein lipasehLPLhuman LPLPBSphosphate-buffered salinerERrough endoplasmic reticulumWTwild typeELISAenzyme-linked immunosorbent assayTRITCtetrarhodamine isothiocyanate. is the major enzyme responsible for the hydrolysis of triglyceride-rich lipoproteins in plasma(1.Olivecrona T. Bengtsson-Olivecrona G. Borensztajn J. Lipoprotein Lipase. Evener Publishers, Inc., Chicago1987: 15-77Google Scholar). Genetic deficiency of LPL causes type I hyperlipoproteinemia syndrome, which is characterized by a significant increase of chylomicron levels in plasma and a marked increase in plasma triglyceride levels(2.Brunzell J.D. Scriver C.K. Beaudet A.L. Sly W.S. Valle D. Metabolic Basis of Inherited Disease. McGraw-Hill Book Co., New York1983: 1165-1180Google Scholar, 3.Eckel R.H. N. Engl. J. Med. 1989; 320: 1060-1068Google Scholar).Functional LPL is a homodimeric glycoprotein with a subunit of 448 amino acids(4.Iverius P. Östlund-Lindqvist A.M. J. Biol. Chem. 1976; 251: 7791-7795Google Scholar). LPL is synthesized in parenchymal cells of tissues such as adipose tissue, heart, skeletal muscle, brain, and ovary(5.Camps L. Reina M. Llobera M. Vilaró S. Olivecrona T. Am. J. Physiol. 1990; 258: 673-681Google Scholar, 6.Braun J.E.A. Severson D.L. Biochem. J. 1992; 284: 337-347Google Scholar). After synthesis the enzyme is secreted and bound to heparan sulfate proteoglycans on the luminal surface of the capillary endothelium(7.Jackson R.L. Baker H.N. Gillian E.B. Gotto A.M. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 1942-1945Google Scholar). At this site, LPL is rate-limiting for the hydrolysis and removal of triglycerides associated with chylomicrons and very low density lipoproteins(8.Olivecrona T. Bengtsson-Olivecrona G. Curr. Opin. Lipidol. 1993; 4: 187-196Google Scholar). The monoglycerides and fatty acids liberated by the LPL reaction are further processed for tissue storage or oxidation. An important part of LPL regulation is a tissue-specific event that is associated to post-translational modifications of the enzyme(9.Semenkovich C.F. Wims M. Noe L. Etienne J. Chan L. J. Biol. Chem. 1989; 264: 9030-9038Google Scholar, 10.Doolittle M.H. Ben-Zeev O. Elovson J. Martin D. Kirchgessner T.G. J. Biol. Chem. 1990; 265: 4570-4577Google Scholar, 11.Cisar L.A. Hoogewerf A.J. Cupp M. Rapport C.A. Bensadoun A. J. Biol. Chem. 1989; 264: 1767-1774Google Scholar). This modifications might be essential for the expression of LPL catalytic activity. Among these, asparagine-linked glycosylation (12.Semenkovich C.F. Luo C.C. Nakanishi M.K. Chen S-H. Smith L.C. Chan L. J. Biol. Chem. 1990; 265: 5429-5433Google Scholar) and dimerization of the protein (13.Osborne J.C. Bengtsson-Olivecrona G. Lee N.S. Biochemistry. 1985; 24: 5606-5611Google Scholar) have been suggested as interrelated processes that confer catalytic activity to LPL(14.Ailhaud G. Clin. Biochem. 1990; 23: 343-347Google Scholar).LPL secretion might also be a crucial regulatory point in the physiological action of the enzyme. Most secretory proteins such as LPL share a common biosynthetic origin in the rough endoplasmic reticulum (rER), from which they are transported to the Golgi complex. In the trans-Golgi network proteins destined to the regulated secretory pathway are sorted from those to be constitutively secreted or sent to lysosomes for intracellular degradation. Secretory pathways involve vesicular transfer to the plasma membrane followed by the secretory event itself and exocytic discharge of vesicle contents(15.Halban P.A. Irminger C.M. Biochem. J. 1990; 299: 1-18Google Scholar). Genetic analysis of LPL deficiency, site-directed mutagenesis, and cellular expression of altered LPL cDNAs in heterologous systems have revealed that some missense changes lead to impaired or altered LPL secretion(16.Santamarina-Fojo S. Curr. Opin. Lipidol. 1992; 3: 86Google Scholar), suggesting that both LPL secretion and enzyme activity are very sensitive to single amino acid exchanges. However, to date, the only single residue studied in some detail with a clear role in LPL secretion is Asn43. Replacement of Asn43 by Ala completely abolishes LPL enzyme activity, leading to the production of an inactive LPL, which accumulates inside the rER(12.Semenkovich C.F. Luo C.C. Nakanishi M.K. Chen S-H. Smith L.C. Chan L. J. Biol. Chem. 1990; 265: 5429-5433Google Scholar, 17.Buscà R. Pujana M.A. Pognonec P. Auwerx J. Deeb S. Reina M. Vilaró S. J. Lipid. Res. 1995; 36: 939-951Google Scholar).By searching for mutations affecting the LPL gene in Type I hypertriglyceridemic patients, Ameis et al.(18.Ameis D. Kobayashi J. Davis R.C. Ben-zeev O. Malloy M.J. Kane J.P. Lee G. Wong H. Havel R.J. Schotz M.C. J. Clin. Invest. 1991; 87: 1165-1170Google Scholar) found that the substitution of a G for an A at nucleotide position 680 of human LPL cDNA, which produced a replacement of glycine 142 by a glutamic acid in the mature LPL protein, led to an inactive enzyme that was not efficiently secreted. The aim of the present study was to elucidate the intracellular destination of the nonsecreted LPL protein to gain further insight into the the mechanism of LPL secretion. We produced, by site-directed mutagenesis, a mutant hLPL carrying the substitution of a glutamic acid for glycine 142 (G142E hLPL), and we transfected COS1 cells with this construction and the wild type one (WT hLPL). The results obtained demonstrated that G142E hLPL presented reduced secretion compared with the WT hLPL, that heparin had no evident effect on the secretion of this mutant protein, and that after transport through the Golgi complex, lysosomes were the final degradation site of this altered, nonfunctional LPL. The findings reported suggest that the missorting of the mutant hLPL to lysosomes could act as a quality control mechanism of the secretory process of LPL.DISCUSSIONOur results suggest that secretion of the G142E hLPL is defective and heparin-insensitive and that the protein is missorted and diverted to lysosomes for degradation.Several studies have indicated that most LPL regulation occurs post-translationally. N-linked glycosylation at Asn43 of hLPL is essential in the development of the enzyme activity(12.Semenkovich C.F. Luo C.C. Nakanishi M.K. Chen S-H. Smith L.C. Chan L. J. Biol. Chem. 1990; 265: 5429-5433Google Scholar, 32.Masuno H. Schultz C.J. Park J-W. Blanchette-Mackie E.J. Mateo C. Scow R.O. Biochem. J. 1991; 277: 801-809Google Scholar), and the absence of N-glycosylation at this residue leads to impaired LPL secretion and rER accumulation of the mutant protein(12.Semenkovich C.F. Luo C.C. Nakanishi M.K. Chen S-H. Smith L.C. Chan L. J. Biol. Chem. 1990; 265: 5429-5433Google Scholar, 17.Buscà R. Pujana M.A. Pognonec P. Auwerx J. Deeb S. Reina M. Vilaró S. J. Lipid. Res. 1995; 36: 939-951Google Scholar). However, the model that emerges from the present results (obtained by cellular expression of mutant G142E hLPL) is clearly different, although both mutant proteins are retained inside the cell. In contrast to N43A hLPL, the present results suggest that G142E hLPL leaves the endoplasmic reticulum, as has been shown by immunofluorescence and electron microscopy; reaches the Golgi complex, as indicated by immunogold labeling, confocal analysis of double immunolocalizations and endoglycosidase H sensitivity of the mutant enzyme; and, instead of being secreted to the extracellular medium, is diverted to lysosomes for degradation, as demonstrated using leupeptin treatment, immunofluorescence, and immunogold detection studies. Thus, although the mutation G142E in hLPL leads to impaired secretion, as happens with the N43A hLPL, the intracellular fate of these mutants is different. The molecular and cellular basis of the different intracellular behavior of this mutant G142E hLPL protein is not clear at present.Glycoproteins processed by the trans-Golgi network can be sorted and sent to (i) lysosomes, (ii) constitutive secretory vesicles, (iii) regulated secretory vesicles and (iv) constitutive-like secretory vesicles(15.Halban P.A. Irminger C.M. Biochem. J. 1990; 299: 1-18Google Scholar). Studies performed to date indicate that LPL secretion is a complex event (see (6.Braun J.E.A. Severson D.L. Biochem. J. 1992; 284: 337-347Google Scholar) for a complete review). Pulse-chase experiments have revealed that in adipocytes approximately 80% of the newly synthesized LPL is degraded(33.Cupp M. Bensadoun A. Melford K. J. Biol. Chem. 1987; 262: 6383-6388Google Scholar, 34.Vannier C. Ailhaud G. J. Biol. Chem. 1989; 264: 13206-13216Google Scholar). The main intracellular site for LPL degradation seems to be lysosomes, since leupeptin subtantially reduces the rate of LPL degradation(34.Vannier C. Ailhaud G. J. Biol. Chem. 1989; 264: 13206-13216Google Scholar). It has also been suggested that some LPL degradation may also occur in the rER(35.Ben-Zeev O. Doolittle M.H. Davis R.C. Elovson J. Schotz M.C. J. Biol. Chem. 1992; 267: 6219-6227Google Scholar). This result appears to be controverted by other authors who report no rER LPL degradation in Brefeldin A-treated Chinese hamster ovary cells(36.Pradines-Figuères A. Vannier C. Ailhaud G. J. Lipid Res. 1990; 31: 1467-1476Google Scholar). To explain the high degree of lysosomal LPL degradation observed, two different models have been proposed: Ailhaud (14.Ailhaud G. Clin. Biochem. 1990; 23: 343-347Google Scholar) proposed that active LPL homodimer is sorted either directly from the trans-Golgi to the lysosomes or the constitutive secretory pathway or to the regulated pathway, where the exocytosis of LPL from intracellular secretory vesicles might be accelerated by heparin by an unknown mechanism. It has been suggested that the control of LPL efflux from the Golgi compartment represents the main post-translational regulation of LPL secretion(27.Vannier C. Deslex S. Pradines-Figuères A. Ailhaud G. J. Biol. Chem. 1989; 264: 13199-13205Google Scholar, 37.Winkler F.K. D'Arcy A. Hunziker W. Nature. 1990; 343: 771-774Google Scholar). In contrast, Cisar et al. (11.Cisar L.A. Hoogewerf A.J. Cupp M. Rapport C.A. Bensadoun A. J. Biol. Chem. 1989; 264: 1767-1774Google Scholar) proposed another model of LPL secretion in which newly synthesized enzyme would be transported to the cell surface, where it would bind to heparan sulfate proteoglycan receptors; LPL would then be either released to the extracellular medium or internalized via the receptor and either degraded in the lysosomes or recycled back to the cell surface. In our experimental system cells expressing the WT hLPL showed some degree of intracellular degradation (mostly in the lysosomes), since leupeptin treatment decreased the intracellular disappearance of the protein. Furthermore, secretion of WT hLPL to the extracellular medium was highly sensitive to heparin, indicating that although COS1 cells normally do not express LPL, they at least process the protein similarly to normal LPL-synthesizing cells. On the other hand, cells transfected with G142E hLPL and treated with leupeptin showed a 3-fold increase in LPL intracellular level. The mutant G142E hLPL had normal heparin binding capacity, but its secretion was not stimulated to the level of the WT hLPL upon heparin treatment, indicating that the intracellular compartment where G142E hLPL is retained might be slightly sensitive or insensitive to heparin experiments performed by transfected cells with LPL indicated that at least part of the G142E hLPL the plasma membrane of the expressing cells. to was in WT hLPL expressing this mutant LPL was not sensitive to heparin suggesting that the membrane that mutant LPL might not be a heparan sulfate The of our that the mutant G142E hLPL a different intracellular pathway from that of the WT hLPL, and we suggest that both pathways at the level of the trans-Golgi The that some of the G142E hLPL intracellular degradation and is in culture medium be at active have been in the present LPL to its with H. A. C. J. Biol. Chem. Scholar). that confer the to the the the binding site, the the or surface the active site, and the active or catalytic site J. P. Biol. 4: Scholar). a crucial role in LPL and they are of or which are clearly in the of the enzyme. of the seems to the residue reported in this However, the amino acid glycine 142 is highly in the LPL are known. In paper Ameis et al. D. Kobayashi J. Davis R.C. Ben-zeev O. Malloy M.J. Kane J.P. Lee G. Wong H. Havel R.J. Schotz M.C. J. Clin. Invest. 1991; 87: 1165-1170Google Scholar) suggested that the for this high might from its to the catalytic of the enzyme Thus, the mutation which a glutamic acid for a glycine the of LPL D. Kobayashi J. Davis R.C. Ben-zeev O. Malloy M.J. Kane J.P. Lee G. Wong H. Havel R.J. Schotz M.C. J. Clin. Invest. 1991; 87: 1165-1170Google Scholar) and this other missense mutations in the enzyme lead to the of inactive proteins that are normally secreted from the expressing the results reported in this paper suggest that the residue could have an the secretion of the this the to be the molecular that the mutant G142E hLPL to lysosomes for degradation. would be that the replacement of a glycine with a glutamic acid at position 142 of LPL produced a effect on the of the its secretion. This appears since proteins carrying major changes are retained the rER by such as and N. P. S. J. Biol. Chem. 1989; 264: Scholar). This is the case of the N43A mutant R. Pujana M.A. Pognonec P. Auwerx J. Deeb S. Reina M. Vilaró S. J. Lipid. Res. 1995; 36: 939-951Google Scholar). is that this single amino acid could a specific missorting that could lead the protein to the lysosomal compartment. as we this is not the since no has been found the amino acid by the mutation and any specific we can the that the mutant G142E hLPL had a different as a of the amino acid which could confer for lysosomal to the mutant LPL through the The of the of lysosomal is reported this mechanism appears and to the plasma this could an of some of the G142E hLPL is at this site and is secreted. further studies be to elucidate the molecular that in the missorting of G142E hLPL to The finding that the G142E hLPL is missorted to lysosomes and degraded suggests that this mechanism might an intracellular protein quality control system that would the of the cell. INTRODUCTIONLipoprotein lipase (LPL) 1The abbreviations used are: LPLlipoprotein lipasehLPLhuman LPLPBSphosphate-buffered salinerERrough endoplasmic reticulumWTwild typeELISAenzyme-linked immunosorbent assayTRITCtetrarhodamine isothiocyanate. is the major enzyme responsible for the hydrolysis of triglyceride-rich lipoproteins in plasma(1.Olivecrona T. Bengtsson-Olivecrona G. Borensztajn J. Lipoprotein Lipase. Evener Publishers, Inc., Chicago1987: 15-77Google Scholar). Genetic deficiency of LPL causes type I hyperlipoproteinemia syndrome, which is characterized by a significant increase of chylomicron levels in plasma and a marked increase in plasma triglyceride levels(2.Brunzell J.D. Scriver C.K. Beaudet A.L. Sly W.S. Valle D. Metabolic Basis of Inherited Disease. McGraw-Hill Book Co., New York1983: 1165-1180Google Scholar, 3.Eckel R.H. N. Engl. J. Med. 1989; 320: 1060-1068Google Scholar).Functional LPL is a homodimeric glycoprotein with a subunit of 448 amino acids(4.Iverius P. Östlund-Lindqvist A.M. J. Biol. Chem. 1976; 251: 7791-7795Google Scholar). LPL is synthesized in parenchymal cells of tissues such as adipose tissue, heart, skeletal muscle, brain, and ovary(5.Camps L. Reina M. Llobera M. Vilaró S. Olivecrona T. Am. J. Physiol. 1990; 258: 673-681Google Scholar, 6.Braun J.E.A. Severson D.L. Biochem. J. 1992; 284: 337-347Google Scholar). After synthesis the enzyme is secreted and bound to heparan sulfate proteoglycans on the luminal surface of the capillary endothelium(7.Jackson R.L. Baker H.N. Gillian E.B. Gotto A.M. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 1942-1945Google Scholar). At this site, LPL is rate-limiting for the hydrolysis and removal of triglycerides associated with chylomicrons and very low density lipoproteins(8.Olivecrona T. Bengtsson-Olivecrona G. Curr. Opin. Lipidol. 1993; 4: 187-196Google Scholar). The monoglycerides and fatty acids liberated by the LPL reaction are further processed for tissue storage or oxidation. An important part of LPL regulation is a tissue-specific event that is associated to post-translational modifications of the enzyme(9.Semenkovich C.F. Wims M. Noe L. Etienne J. Chan L. J. Biol. Chem. 1989; 264: 9030-9038Google Scholar, 10.Doolittle M.H. Ben-Zeev O. Elovson J. Martin D. Kirchgessner T.G. J. Biol. Chem. 1990; 265: 4570-4577Google Scholar, 11.Cisar L.A. Hoogewerf A.J. Cupp M. Rapport C.A. Bensadoun A. J. Biol. Chem. 1989; 264: 1767-1774Google Scholar). This modifications might be essential for the expression of LPL catalytic activity. Among these, asparagine-linked glycosylation (12.Semenkovich C.F. Luo C.C. Nakanishi M.K. Chen S-H. Smith L.C. Chan L. J. Biol. Chem. 1990; 265: 5429-5433Google Scholar) and dimerization of the protein (13.Osborne J.C. Bengtsson-Olivecrona G. Lee N.S. Biochemistry. 1985; 24: 5606-5611Google Scholar) have been suggested as interrelated processes that confer catalytic activity to LPL(14.Ailhaud G. Clin. Biochem. 1990; 23: 343-347Google Scholar).LPL secretion might also be a crucial regulatory point in the physiological action of the enzyme. Most secretory proteins such as LPL share a common biosynthetic origin in the rough endoplasmic reticulum (rER), from which they are transported to the Golgi complex. In the trans-Golgi network proteins destined to the regulated secretory pathway are sorted from those to be constitutively secreted or sent to lysosomes for intracellular degradation. Secretory pathways involve vesicular transfer to the plasma membrane followed by the secretory event itself and exocytic discharge of vesicle contents(15.Halban P.A. Irminger C.M. Biochem. J. 1990; 299: 1-18Google Scholar). Genetic analysis of LPL deficiency, site-directed mutagenesis, and cellular expression of altered LPL cDNAs in heterologous systems have revealed that some missense changes lead to impaired or altered LPL secretion(16.Santamarina-Fojo S. Curr. Opin. Lipidol. 1992; 3: 86Google Scholar), suggesting that both LPL secretion and enzyme activity are very sensitive to single amino acid exchanges. However, to date, the only single residue studied in some detail with a clear role in LPL secretion is Asn43. Replacement of Asn43 by Ala completely abolishes LPL enzyme activity, leading to the production of an inactive LPL, which accumulates inside the rER(12.Semenkovich C.F. Luo C.C. Nakanishi M.K. Chen S-H. Smith L.C. Chan L. J. Biol. Chem. 1990; 265: 5429-5433Google Scholar, 17.Buscà R. Pujana M.A. Pognonec P. Auwerx J. Deeb S. Reina M. Vilaró S. J. Lipid. Res. 1995; 36: 939-951Google Scholar).By searching for mutations affecting the LPL gene in Type I hypertriglyceridemic patients, Ameis et al.(18.Ameis D. Kobayashi J. Davis R.C. Ben-zeev O. Malloy M.J. Kane J.P. Lee G. Wong H. Havel R.J. Schotz M.C. J. Clin. Invest. 1991; 87: 1165-1170Google Scholar) found that the substitution of a G for an A at nucleotide position 680 of human LPL cDNA, which produced a replacement of glycine 142 by a glutamic acid in the mature LPL protein, led to an inactive enzyme that was not efficiently secreted. The aim of the present study was to elucidate the intracellular destination of the nonsecreted LPL protein to gain further insight into the the mechanism of LPL secretion. We produced, by site-directed mutagenesis, a mutant hLPL carrying the substitution of a glutamic acid for glycine 142 (G142E hLPL), and we transfected COS1 cells with this construction and the wild type one (WT hLPL). The results obtained demonstrated that G142E hLPL presented reduced secretion compared with the WT hLPL, that heparin had no evident effect on the secretion of this mutant protein, and that after transport through the Golgi complex, lysosomes were the final degradation site of this altered, nonfunctional LPL. The findings reported suggest that the missorting of the mutant hLPL to lysosomes could act as a quality control mechanism of the secretory process of LPL.