Macrophage Metalloelastase, MMP-12, Cleaves Human Apolipoprotein(a) in the Linker Region between Kringles IV-4 and IV-5

In this study we found that macrophage metalloelastase, MMP-12 cleaves, in vitro, apolipoprotein(a) (apo(a)) in the Asn3518-Val3519 bond located in the linker region between kringles IV-4 and IV-5, a bond immediately upstream of the Ile3520-Leu3521 bond, shown previously to be the site of action by neutrophil elastase (NE). We have also shown that human apo(a) injected into the tail vein of control mice undergoes degradation as reflected by the appearance of immunoreactive fragments in the plasma and in the urine of these animals. To define whether either or both of these enzymes may be responsible for the in vivo apo(a) cleavage, we injected intravenously MMP-12−/−, NE−/− mice and litter mates, all of the same strain, with either lipoprotein(a) (Lp(a)), full-length free apo(a), or its N-terminal fragment, F1, obtained by the in vitro cleavage of apo(a) by NE. In the plasma of Lp(a)/apo(a)-injected mice, F1 was detected in control and NE−/− mice but was virtually absent in the MMP-12−/− mice. Moreover, fragments of the F1 type were present in the urine of the animals except for the MMP-12−/− mice. These fragments were significantly smaller in size than those observed in the plasma. All of the animals injected with F1 exhibited small sized fragments in their urine. These observations provide evidence that, in the mouse strain used, MMP-12 plays an important role in the generation of F1 from injected human Lp(a)/apo(a) and that this fragment undergoes further cleavage during renal transit via a mechanism that is neither NE- nor MMP-12-dependent. Thus, factors influencing the expression of MMP-12 may have a modulating action on the biology of Lp(a). In this study we found that macrophage metalloelastase, MMP-12 cleaves, in vitro, apolipoprotein(a) (apo(a)) in the Asn3518-Val3519 bond located in the linker region between kringles IV-4 and IV-5, a bond immediately upstream of the Ile3520-Leu3521 bond, shown previously to be the site of action by neutrophil elastase (NE). We have also shown that human apo(a) injected into the tail vein of control mice undergoes degradation as reflected by the appearance of immunoreactive fragments in the plasma and in the urine of these animals. To define whether either or both of these enzymes may be responsible for the in vivo apo(a) cleavage, we injected intravenously MMP-12−/−, NE−/− mice and litter mates, all of the same strain, with either lipoprotein(a) (Lp(a)), full-length free apo(a), or its N-terminal fragment, F1, obtained by the in vitro cleavage of apo(a) by NE. In the plasma of Lp(a)/apo(a)-injected mice, F1 was detected in control and NE−/− mice but was virtually absent in the MMP-12−/− mice. Moreover, fragments of the F1 type were present in the urine of the animals except for the MMP-12−/− mice. These fragments were significantly smaller in size than those observed in the plasma. All of the animals injected with F1 exhibited small sized fragments in their urine. These observations provide evidence that, in the mouse strain used, MMP-12 plays an important role in the generation of F1 from injected human Lp(a)/apo(a) and that this fragment undergoes further cleavage during renal transit via a mechanism that is neither NE- nor MMP-12-dependent. Thus, factors influencing the expression of MMP-12 may have a modulating action on the biology of Lp(a). apolipoprotein(a) lipoprotein(a) protein moiety of LDL neutrophil elastase macrophage metalloelastase matrix metalloproteinase N- and C-terminal domains of apo(a), respectively enzyme-linked immunosorbent assay low density lipoprotein polyacrylamide gel electrophoresis Following the original observation by Oida et al.(1Oida K. Takai H. Maeda H. Takahashi S. Shimada A. Suzuki J. Tamai T. Nakai T. Miyabo S. Clin. Chem. 1992; 38: 2244-2248Crossref PubMed Scopus (75) Google Scholar), several reports have demonstrated the presence of immunoreactive apo(a)1 in human urine. Particularly significant were the studies by Mooseret al. (2Mooser V. Seabra M.C. Abedin M. Landschulz K.T. Marcovina S. Hobbs H.H. J. Clin. Invest. 1996; 97: 858-864Crossref PubMed Scopus (82) Google Scholar, 3Mooser V. Marcovina S.M. White A.L. Hobbs H.H. J. Clin. Invest. 1996; 98: 2414-2424Crossref PubMed Scopus (86) Google Scholar) who by combining chemical and immunochemical techniques provided evidence that urinary apo(a) was represented by fragments derived from its N-terminal region. When, following purification, these urinary fragments were injected intravenously into mice, they were excreted in the urine without an apparent change in size. The same authors also reported the presence in the plasma of apo(a) fragments unattached to apoB100. Upon their intravenous injection into mice, these fragments were rapidly excreted in the urine as smaller sized components (3Mooser V. Marcovina S.M. White A.L. Hobbs H.H. J. Clin. Invest. 1996; 98: 2414-2424Crossref PubMed Scopus (86) Google Scholar). Taken together, the results were interpreted to indicate that the apo(a) fragments in human plasma are derived from Lp(a)/apo(a) and are in turn the source of the apo(a) fragments in the urine. Unanswered in those studies was the underlying cause of the apo(a) fragmentation as well as the site or sites for its occurrence. We reported previously that limited in vitroproteolysis of apo(a), by either pancreatic or leukocyte elastase, is attended by the cleavage at the Ile3520-Leu3521bond located in the linker domain between kringles IV-4 and IV-5 (4Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar,5Edelstein C. Italia J.I. Scanu A.M. J. Biol. Chem. 1997; 272: 11079-11087Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). This cleavage generates two main fragments, one representing the N-terminal domain, F1, and the other the C-terminal domain, F2. When these two fragments were separately injected intravenously into mice, F1 but not F2 was rapidly excreted in the urine (4Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar). This observation prompted us to examine whether the cleavage site on apo(a) was limited to serine proteases such as pancreatic and leukocyte or neutrophil elastase (NE) or could also apply to other inflammatory enzymes. Among them we considered matrix matalloproteinases (MMPs), which are metallo-dependent enzymes involved in the homeostasis of the extracellular matrix and also shown to play an important role in the atherosclerotic process (6Galis Z.S. Sukhova G.K. Lark M.W. P. L. J. Clin. Invest. 1994; 94: 2493-2503Crossref PubMed Scopus (2205) Google Scholar, 7Li Z. Li L. Zielke H.R. Cheng L. Xiao R. Crow M.T. Stetler-Stevenson W.G. Froehlick J. Lakatta E.G. Am. J. Pathol. 1996; 148: 121-128PubMed Google Scholar, 8Shah P.K. Falk E. Badimon J.J. Fernandez-Ortiz A. Maihac A. Villareal-Levy G. Jallon J.T. Regnstron J. Fuster V. Circulation. 1995; 92: 1565-1569PubMed Google Scholar, 9Galis Z.S. Sukhova G.K. Kranzhofer R. Clark S. Libby P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 402-406Crossref PubMed Scopus (519) Google Scholar, 10Henney A.M. Wakeley P.R. Davies M.J. Foster K. Hembry R. Murphy G. Humphries S.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8154-8158Crossref PubMed Scopus (535) Google Scholar, 11Halpert I. Sire U.I. Roby J.D. Potter-perigo S. Wight T.N. Shapiro S.D. Welgus H.G. Wickline S.A. Parks W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9748-9755Crossref PubMed Scopus (322) Google Scholar, 12Thompson R.W. Holmes D.R. Mertens R.A. Liao S. Botney M.D. Mecham R.P. Welgus H.G. Parks W.C. J. Clin. Invest. 1995; 93: 9748-9753Google Scholar, 13Nikkari S.T. Hoyhtya M. Isola J. Nikkari T. Am. J. Pathol. 1996; 148: 1427-1433Google Scholar). In this work, we directed our attention to macrophage metalloelastase, also referred to as MMP-12, which was first identified as an elastolytic metalloproteinase secreted by inflammatory macrophages (14Banda M.J. Werb Z. Biochem. J. 1981; 193: 560-569Crossref Scopus (168) Google Scholar) and structurally defined (15Shapiro S.D. Griffin G.L. Gilbert D.J. Jenkins N.A. Copeland N.G. Welgus H.G. Senior R.M. Ley T.J. J. Biol. Chem. 1992; 267: 4664-4671Abstract Full Text PDF PubMed Google Scholar, 16Shapiro S.D. Kobayashi D.K. Ley T.J. J. Biol. Chem. 1993; 268: 23824-23829Abstract Full Text PDF PubMed Google Scholar). This enzyme has a broad substrate specificity for matrix macromolecules such as fibronectin, laminin, vitronectin, and proteoglycans (17Chandler S. Cossin J. Lury J. Wells G. Biochem. Biophys. Res. Commun. 1996; 228: 421-429Crossref PubMed Scopus (197) Google Scholar). In the current study, we now show that MMP-12 cleaves apo(a) in vitro and also provide evidence that, in the mouse strain used, it is involved in the generation of F1 in vivo. Human leukocyte elastase (EC 3.4.21.37) was purchased from Sigma. Antiserum to purified preparations of apo(a), Lp(a), and LDL were raised in the rabbit and affinity purified as described previously (18Fless G.M. Snyder M.L. Scanu A.M. J. Lipid Res. 1989; 30: 651-662Abstract Full Text PDF PubMed Google Scholar). Anti-Lp(a) were shown to be devoid of immunoreactivity to LDL and plasminogen; anti-LDL was unreactive to apo(a). Mice deficient in NE (NE−/−) or MMP-12 (MMP-12−/−) in a pure 129/Svj background were generated by targeted disruption of either the NE or MMP-12 gene in RW4 embryonic stem cells as described previously (19Belaaouaj A. McCarthy R. Baumann M. Gao Z. Ley T.J. Abraham S.N. Shapiro S.D. Nat. Med. 1998; 4: 615-618Crossref PubMed Scopus (546) Google Scholar, 20Shipley J.M. Wesselschmidt R.L. Kobayashi D.K. Ley T.J. Shapiro S.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3942-3946Crossref PubMed Scopus (412) Google Scholar). All mice were housed in individual cages under normal light. The evening before the experiment, the animals were given a 10% sucrose solution to drinkad libitum in place of water. The following morning the mice were anesthetized with Metafane prior to the injection into the tail vein of 25–250 μg of either Lp(a), apo(a), or fragment F1 in a volume of 100 μl. The mice were then placed in metabolic cages, given access to standard lab chow and 10% sucrose solution to drink. Blood samples were withdrawn from the orbital vein into heparinized hematocrit tubes at the specified time points and immediately iced. Urine was collected at 0–3, 3–5, and 5–24 h. ELISA quantitation, sensitive to < 0.0015 mg/dl of apo(a), was performed on the urine samples to determine the levels of apo(a)-reacting material. These results indicated to what degree the sample was to be concentrated for electrophoretic detection. The latter was estimated to be > 0.03 mg/dl. The urine was concentrated in Amicon Centriprep filters. The subjects used for the preparation of Lp(a) and LDL were healthy donors with plasma Lp(a) protein levels in the range of 15–43 mg/dl determined as described previously (4Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar). Plasmapheresis was performed in the Blood Bank of the University of Chicago. All of the subjects used in the study gave a written informed consent. The steps for Lp(a) isolation were carried out immediately after blood drawing using the procedure outlined below. We used an additional five subjects for studying the apo(a) fragments in their plasma and urine. These were also healthy subjects with a known phenotype and genotype. Their plasma Lp(a) protein levels varied between 1 and 12 mg/dl, and the apo(a) size isoforms varied between 350 and 550 kDa. In each subject, 4-h urine samples were collected, centrifuged for 15 min at 3000 and either used or immediately at they were concentrated in All of the subjects used in the study gave a written informed consent. To lipoprotein the plasma obtained by was with and 1 Lp(a) were by and as described previously G.M. Snyder M.L. M.T. R. 1994; PubMed Scopus Google Scholar). The of the was by on and of and The LDL preparations used in this study were at density of by as described previously PubMed Scopus Google Scholar). To lipoprotein the plasma obtained by was with and 1 was performed on apo(a) samples by by using C. M. Scanu A.M. 1995; PubMed Scopus Google Scholar). The of the individual apo(a) was with apo(a) isoforms of known G.M. Snyder M.L. M.T. R. 1994; PubMed Scopus Google Scholar). apo(a) were from blood cells and by gel and the were with an as described C. E. T. Hobbs H.H. J. Clin. Invest. 1991; PubMed Scopus Google Scholar). was from Lp(a) as described previously C. M. Scanu A.M. 1995; PubMed Scopus Google Scholar). Lp(a), 1 was with at a of to a of 100 was then and the was at for 1 under an volume of sucrose in the same was and the placed into a and in a at for h. the LDL free of apo(a), and Lp(a). The free apo(a) in pure The latter was in the sucrose solution at These fragments were the of the limited of apo(a) by purified human leukocyte elastase as described previously C. Italia J.I. Scanu A.M. J. Biol. Chem. 1997; 272: 11079-11087Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). the enzyme 1 was in 100 of the enzyme was μg of Lp(a) or LDL protein or 15 μg of apo(a) at for The was with for min at the fragments were purified by affinity (4Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar). and was performed on a for at at The samples were by at for min in sample which of and with or without after the were placed that were previously with a was performed on a at for min at the were in and by with or The were and with the were with the to the of the were under as outlined the were as described in the The were in with in and with with and with was performed on the which was then to degradation on an using by the All were carried out in the of the and at the University of Lp(a) and LDL protein were by a ELISA as described previously (18Fless G.M. Snyder M.L. Scanu A.M. J. Lipid Res. 1989; 30: 651-662Abstract Full Text PDF PubMed Google Scholar) except that was used as the and to as the the ELISA of apo(a), to was used as the an was for apo(a) in the sucrose were performed by the MMP-12 was and purified as described previously (15Shapiro S.D. Griffin G.L. Gilbert D.J. Jenkins N.A. Copeland N.G. Welgus H.G. Senior R.M. Ley T.J. J. Biol. Chem. 1992; 267: 4664-4671Abstract Full Text PDF PubMed Google Scholar). The enzyme was with human Lp(a)/apo(a) at of protein to enzyme and at in of samples were was to the and was performed under and The of MMP-12 to Lp(a) was at of apo(a) to enzyme using and of the of Lp(a) after an at under with or The of of apo(a) and as a of enzyme to in the with an in as the enzyme other of were also present one of which exhibited a to of the same with that a of the of in the Lp(a) with to the apo(a) in the the were two that at an apparent of and The of these to fragments F1 and F2 previously reported (4Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar). The to F1 with a on than under of the of the on the of the of the with 1 that the gel was the same as observed in the gel 1 When apo(a) was with MMP-12, the was the same as shown in 1 for Lp(a) under The results that apo(a) in Lp(a) was at a site two fragments, F1 and the latter to of apoB100. The of F1 and F2 obtained by MMP-12 of Lp(a) and apo(a) are shown in these with those of apo(a), using the we located the cleavage site at the Asn3518-Val3519 bond in the linker region between kringles IV-4 and These fragments to the N- and C-terminal domain of apo(a) kringles to IV-4 and IV-5 and the region was also by MMP-12, it was of to determine whether the fragments on the of the Lp(a) or lipoprotein each of of to F2. To this Lp(a) was on polyacrylamide that were with and Lp(a) exhibited two one of which also with The with the in to In LDL was with MMP-12, the was the same as for LDL and Taken together, we interpreted these results to show that MMP-12 both apo(a) and components of the of apo(a) fragment F1 and a in which the C-terminal region of apo(a), fragment is to of and the fragments on the of the the N-terminal fragment of apo(a), F1, is We on of plasma samples from mice after intravenous injection of either Lp(a), apo(a), or The electrophoretic of the plasma samples after injection of apo(a) into NE−/− and control mice to full-length apo(a) and fragments one in the of F1, that a fragmentation process in vivo. In the plasma sample of MMP-12−/− mice exhibited a to full-length apo(a) with of low but with the of F1 The electrophoretic under of the plasma from mice injected with Lp(a) not were with those shown in to The of plasma after injection of F1 was all of the mice and the injected All the a at and other of in the range of kDa. The electrophoretic of the plasma of all the mice at each time after the of these the F1 with time not We the urine of mice, collected after injection of either Lp(a), apo(a) or F1, by of The urinary of apo(a) after injection into control mice were and and a broad of those at and kDa. In the urine from MMP-12−/− mice was devoid of after the urine than that of either the NE−/− or control mice. In the electrophoretic of the urine from all of the mice injected with F1, fragments that were smaller in size than the injected and to those observed in NE−/− and control mice injected with full-length apo(a) and To out to the of fragments, the collected urine samples from injected mice were at and with collected urine on of In fragments were observed the urine of a control mouse was with Lp(a) or its These results provide evidence that F1 is the of apo(a) targeted for renal and that in our mouse this fragment is generated by the action of MMP-12 on apo(a). All subjects exhibited results in of electrophoretic plasma sample is shown in In to apo(a), were at other one of which to the of In of the of apo(a) fragments was with those of and NE−/− mice injected with apo(a) or Lp(a) and to all mice injected with fragment studies have shown that, in vitro, MMP-12 cleaves human apo(a) at the Asn3518-Val3519 bond located in the linker between kringles IV-4 and This bond is immediately upstream of the Ile3520-Leu3521bond that we previously to be the site of cleavage by either pancreatic or leukocyte elastase (4Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar, C. Italia J.I. Scanu A.M. J. Biol. Chem. 1997; 272: 11079-11087Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The for the substrate specificity by the and MMP-12 is is that the linker between kringles IV-4 and IV-5, to the other of apo(a), is as on the of G.M. Scanu A.M. R.M. PubMed Scopus Google Scholar). is of that the of the bond sites by NE and MMP-12, results in the generation of two main fragments of a size. of the of our current studies was the observation that Lp(a)/apo(a)-injected MMP-12−/−, to the NE−/− mice, virtually fragments in their plasma and in their urine. the MMP-12−/− mice were to in the urine intravenously injected These observations with the that MMP-12 was to in vitro, apo(a), that in the this enzyme was responsible for the generation of F1 from injected human apo(a). The results also that the has a site for free F1 but not it is a of a full-length apo(a). The for the of F1 by the is size and degree of by of F1 a process as by et al. (3Mooser V. Marcovina S.M. White A.L. Hobbs H.H. J. Clin. Invest. 1996; 98: 2414-2424Crossref PubMed Scopus (86) Google Scholar). mechanism is a our that Lp(a)/apo(a) is not by the in the of apo(a) has J.T. D.K. J. Clin. Invest. 1997; PubMed Scopus Google Scholar). on the current it be of to determine whether F1 is a for this is important to that, in all of the animals injected with either Lp(a)/apo(a) or F1, the urinary fragments, were in size and smaller than those in the that in our mouse strain neither NE nor MMP-12 was involved in the degradation by the by additional In this and have reported in the renal R. Murphy G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (44) Google Scholar, G. Murphy G. J. Biol. 1997; PubMed Scopus Google Scholar). that was not in the current studies was the of the first of apo(a) fragmentation The plasma to be an source on the by et al. (2Mooser V. Seabra M.C. Abedin M. Landschulz K.T. Marcovina S. Hobbs H.H. J. Clin. Invest. 1996; 97: 858-864Crossref PubMed Scopus (82) Google Scholar, 3Mooser V. Marcovina S.M. White A.L. Hobbs H.H. J. Clin. Invest. 1996; 98: 2414-2424Crossref PubMed Scopus (86) Google Scholar) and our (4Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar, C. Italia J.I. Scanu A.M. J. Biol. Chem. 1997; 272: 11079-11087Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) that neither mouse nor human plasma has the to in vitro, Moreover, MMP-12 in the plasma be by of at under is that the apo(a) fragments are generated at sites by the action of secreted by macrophages by of F1 and F2 are generated in vitro by the action of both NE C. Italia J.I. Scanu A.M. J. Biol. Chem. 1997; 272: 11079-11087Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and MMP-12 on apo(a), one has to F1 was present in the plasma of the NE−/− and control mice injected intravenously with apo(a). This may be of the that F2 but not F1 to matrix macromolecules such as and (4Edelstein C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar) and as by our on the O. C. L. Scanu A.M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the other F1, for which has identified A.M. C. J. Lipid Res. 1997; 38: Full Text PDF PubMed Google Scholar), at sites be free to and to the blood by a The current studies the as to whether the results on the may apply to is of to that mouse MMP-12 has a degree of with its human S.D. Kobayashi D.K. Ley T.J. J. Biol. Chem. 1993; 268: 23824-23829Abstract Full Text PDF PubMed Google Scholar). Moreover, the apo(a) fragments that are present in the plasma of human subjects are of a size as those found in the plasma of mice injected with human apo(a) (2Mooser V. Seabra M.C. Abedin M. Landschulz K.T. Marcovina S. Hobbs H.H. J. Clin. Invest. 1996; 97: 858-864Crossref PubMed Scopus (82) Google Scholar, 3Mooser V. Marcovina S.M. White A.L. Hobbs H.H. J. Clin. Invest. 1996; 98: 2414-2424Crossref PubMed Scopus (86) Google Scholar, C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar) and also as shown by the current results in In the urine of human subjects has shown previously (2Mooser V. Seabra M.C. Abedin M. Landschulz K.T. Marcovina S. Hobbs H.H. J. Clin. Invest. 1996; 97: 858-864Crossref PubMed Scopus (82) Google Scholar, 3Mooser V. Marcovina S.M. White A.L. Hobbs H.H. J. Clin. Invest. 1996; 98: 2414-2424Crossref PubMed Scopus (86) Google Scholar, C. Italia J.A. Klezovitch O. Scanu A.M. J. Lipid Res. 1996; 37: 1786-1801Abstract Full Text PDF PubMed Google Scholar) and in this study to apo(a) immunoreactive fragments which are smaller in size than those in the that the human is involved in the of degradation of apo(a). these is to that MMP-12, or other are involved in the fragmentation of apo(a) in human In our results have shown that MMP-12 cleaves apo(a) in the linker region between kringles IV-4 and IV-5 and also provide evidence from mouse that such a cleavage may for the generation in the plasma of F1, which is by the The that the generation of F1 may be by the of MMP-12, that the of this enzyme may play an important role in the of Lp(a). We for in the intravenous of apo(a) and into NE and mice and their litter