Cubbing in proapolipoprotein maturation

Disruption of the murine procollagen C-proteinase enhancer 2 gene causes accumulation of pro-apoA-I and increased HDL levelsJournal of Lipid ResearchVol. 52Issue 11PreviewGiven the increased prevalence of cardiovascular disease in the world, the search for genetic variations that impact risk factors associated with the development of this disease continues. Multiple genetic association studies demonstrate that procollagen C-proteinase enhancer 2 (PCPE2) modulates HDL levels. Recent studies revealed an unexpected role for this protein in the proteolytic processing of pro-apolipoprotein (apo) A-I by enhancing the cleavage of the hexapeptide extension present at the N-terminus of apoA-I. Full-Text PDF Open Access In this issue of the Journal of Lipid Research, Francone and colleagues have described an interesting HDL phenotype in a mouse deficient in procollagen proteinase enhancer 2 (PCPE2). Present in the plasma of the mice are preβ HDL containing as its major, if not only, apolipoprotein pro-apoA-I, accompanied by the accumulation of an enlarged HDL also containing predominantly pro-apoA-I. This highlights a potentially new actor in the regulation of HDL homeostasis. Recent genetic association studies have suggested that PCPE2 variation may be weakly associated with HDL levels (1Hinds DA. Seymour AB. Durham LK. Banerjee P. Milos PG. Cox DR. Thompson JF. Frazer KA. Application of pooled genotyping to scan candidate regions for association with HDL cholesterol levels.Hum. Genomics. 2004; 1: 421-434Crossref PubMed Scopus (54) Google Scholar). The phenotype described in the Francone paper, while of great interest, is not readily understandable in all of its details. In addition, this study raises some fascinating evolutionary and physiological questions: a) How and where is the apoA-I proprotein converted to the mature protein? What is the biochemical mechanism for this conversion? b) What is the functional role of the secretion of apoA-I as a proprotein? c) Does the proprotein differ from the mature protein in its function and regulation of cholesterol homeostasis? d) What is the mechanism for the accumulation of an enlarged HDL containing pro-apoA-I? How does this HDL compare in function to HDL in which mature apoA-I is the predominant apoprotein? ApoA-I is the major protein of HDL. It is an amphipathic, lipid binding protein containing 243 amino acids. Its primary translation product includes an 18 amino acid signal peptide that directs the primary translation product into the endoplasmic reticulum and is removed cotranslationally followed by a hexapeptide propeptide that is removed extracellularly to yield the mature apoprotein. Other secreted proteins, including several other apoproteins, are also secreted with a prosequence. The precise function of the prosequence is not always clear. The suggestion is that the prosequence often allows the mature protein to assume the “correct” conformation in the appropriate biological environment. Several examples of this mechanism are well known, e.g., pro-insulin and procollagen. Procollagen has additional sequences at both the N- and C-termini. They are cleaved by different proteinases prior to fibrillogenesis to form the mature collagen with the cleavage of the C-terminal prosequence being especially critical (2Canty EG. Kadler KE. Procollagen trafficking, processing and fibrillogenesis.J. Cell Sci. 2005; 118: 1341-1353Crossref PubMed Scopus (543) Google Scholar). It would be unproductive for fibrillogenesis to occur while the protein is being synthesized as the collagen fibers are major extracellular matrix components. The cleavage of the C-terminal procollagen sequence is accomplished by the tolloid metalloproteinase bone morphogenetic protein-1 (BMP-1) and enhanced by PCPE1, a close relative of PCPE2, with which it shares 43% amino acid homology (3Hopkins DR. Keles S. Greenspan DS. The bone morphogenetic protein 1/Tolloid-like metalloproteinases.Matrix Biol. 2007; 26: 508-523Crossref PubMed Scopus (201) Google Scholar–4Steiglitz BM. Keene DR. Greenspan DS. PCOLCE2 encodes a functional procollagen C-proteinase enhancer (PCPE2) that is a collagen-binding protein differing in distribution of expression and post-translational modification from the previously described PCPE1.J. Biol. Chem. 2002; 277: 49820-49830Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Both of BMP-1 and the PCPE proteins are CUB domain containing proteins (C = complement components C1r/C1s; U = sea urchin protein Uegf; B = BMP-1Q3: Please confirm that (2–4) in “…homology (2–4).” are reference citations or amend.). The CUB domains facilitate protein-protein interactions, in this case BMP-1 and PCPE1 to allow for the productive cleavage of the C-terminal prosequence from procollagen. The cleavage of pro-apoA-I is by a very similar process involving BMP-1 (5Chau P. Fielding PE. Fielding CJ. Bone morphogenetic protein-1 (BMP-1) cleaves human proapolipoprotein A1 and regulates its activation for lipid binding.Biochemistry. 2007; 46: 8445-8450Crossref PubMed Scopus (40) Google Scholar) and PCPE2 forming a complex with the proapoprotein, again via the CUB domains of the first two components (6Zhu J. Gardner J. Pullinger CR. Kane JP. Thompson JF. Francone OL. Regulation of apoAI processing by procollagen C-proteinase enhancer-2 and bone morphogenetic protein-1.J. Lipid Res. 2009; 50: 1330-1339Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Binding studies suggest that the order of addition of these components to the complex may favor the association of pro-apoA-I with BMP-1 followed by the addition of PCPE2. Indeed, quite limited but detectable cleavage of pro-apoA-I may occur in the absence of PCPE2, which, as its name suggests, serves as an enhancer for the cleavage. It is not clear from the studies of Francone and colleagues whether the plasma of the PCPE2 knockout animals contains any mature apoA-I, as the authors did not have the reagents to make such a quantitative assessment. This might be accomplished using an anti-peptide antibody that selectively recognizes the proprotein as has been described previously (7Barkia A. Martin C. Puchois P. Gesquiere JC. Cachera C. Tartar A. Fruchart JC. Enzyme-linked immunosorbent assay for human proapolipoprotein A-I using specific antibodies against synthetic peptide.J. Lipid Res. 1988; 29: 77-84Abstract Full Text PDF PubMed Google Scholar). In procollagen, BMP-1 and PCPE1 cleave Ala-Asp or Gly-Asp bonds (8Li SW. Sieron AL. Fertala A. Hojima Y. Arnold WV. Prockop DJ. The C-proteinase that processes procollagens to fibrillar collagens is identical to the protein previously identified as bone morphogenic protein-1.Proc. Natl. Acad. Sci. USA. 1996; 93: 5127-5130Crossref PubMed Scopus (201) Google Scholar). In the case of pro-apoA-I, BMP-1 in concert with PCPE2 removes the N-terminal hexapeptide WHVWQQ from the N-terminal aspartic acid residue of the mature mouse proprotein. The human prosequence is RHFWQQ, again upstream of the N-terminal aspartic acid residue. The proprotein sequence was first alluded to by Gordon and colleagues (9Gordon JI. Smith DP. Andy R. Alpers DH. Schonfeld G. Strauss AW. The primary translation product of rat intestinal apolipoprotein A-I mRNA is an unusual preproprotein.J. Biol. Chem. 1982; 257: 971-978Abstract Full Text PDF PubMed Google Scholar) almost 30 years ago when they were studying apolipoprotein mRNA in rat intestine. The sequence of the human proprotein hexapeptide was determined from lymph (10Brewer Jr., HB. Fairwell T. Kay L. Meng M. Ronan R. Law S. Light JA. Human plasma proapoA-I: isolation and amino-terminal sequence.Biochem. Biophys. Res. Commun. 1983; 113: 626-632Crossref PubMed Scopus (30) Google Scholar) or from the primary translation product in HepG2 cells a year later (11Zannis VI. Karathanasis SK. Keutmann HT. Goldberger G. Breslow JL. Intracellular and extracellular processing of human apolipoprotein A-I: secreted apolipoprotein A-I isoprotein 2 is a propeptide.Proc. Natl. Acad. Sci. USA. 1983; 80: 2574-2578Crossref PubMed Scopus (79) Google Scholar). The Gln dipeptide at the end of the prosequence is characteristic of the apoA-I prosequence found across fish, birds, and mammals (12Bashtovyy D. Jones MK. Anantharamaiah GM. Segrest JP. Sequence conservation of apolipoprotein A-I affords novel insights into HDL structure-function.J. Lipid Res. 2011; 52: 435-450Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). This pathway is distinct from that employed for the processing of proneurohormones like insulin, which involves endoproteolytic cleavage between dibasic residues Arg-Arg or Lys-Arg (13Steiner DF. Smeekens SP. Ohagi S. Chan SJ. The new enzymology of precursor processing endoproteases.J. Biol. Chem. 1992; 267: 23435-23438Abstract Full Text PDF PubMed Google Scholar). The cleavage of the prohormones occurs within the secretory pathway. In contrast, the cleavage of the pro-apoA-I is an extracellular event either in the plasma or tissue fluid though a particular micro-environment is not excluded. All three components of the complex involved in the conversion of the proprotein to the mature apoA-I, pro-apoA-I, BMP-1, and PCPE2, are secreted proteins, but precisely where they associate is not clear. Pro-apoA-I is secreted from the intestine and liver. BMP-1, the procollagen C-terminal proteinase, likely has a similar tissue distribution as the procollagen N-terminal proteinase with expression mostly in tissues rich in collagen, like the tendon, skin, bone, and aorta and more limited expression in the kidney and liver (14Colige A. Li SW. Sieron AL. Nusgens BV. Prockop DJ. Lapière CM. cDNA cloning and expression of bovine procollagen I N-proteinase: a new member of the superfamily of zinc-metalloproteinases with binding sites for cells and other matrix components.Proc. Natl. Acad. Sci. USA. 1997; 94: 2374-2379Crossref PubMed Scopus (157) Google Scholar). PCPE2 has a more limited expression than PCPE1 and is most abundantly expressed in the heart, the intestine, and to a lesser extent in the kidney, with very little expression in the liver. It is a heparin binding protein and is associated with the cell surface (4Steiglitz BM. Keene DR. Greenspan DS. PCOLCE2 encodes a functional procollagen C-proteinase enhancer (PCPE2) that is a collagen-binding protein differing in distribution of expression and post-translational modification from the previously described PCPE1.J. Biol. Chem. 2002; 277: 49820-49830Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), raising the possibility that the cleavage of pro-apoA-I may occur on a cell surface. PCPE2 is also found on HDL (5Chau P. Fielding PE. Fielding CJ. Bone morphogenetic protein-1 (BMP-1) cleaves human proapolipoprotein A1 and regulates its activation for lipid binding.Biochemistry. 2007; 46: 8445-8450Crossref PubMed Scopus (40) Google Scholar), suggesting that it may associate with lipid associated pro-apoA-I. At steady state, about 7% of the total plasma apoA-I is the proprotein (7Barkia A. Martin C. Puchois P. Gesquiere JC. Cachera C. Tartar A. Fruchart JC. Enzyme-linked immunosorbent assay for human proapolipoprotein A-I using specific antibodies against synthetic peptide.J. Lipid Res. 1988; 29: 77-84Abstract Full Text PDF PubMed Google Scholar), suggesting that the extracellular conversion to the mature protein is not quantitatively efficient or that it takes place in a particular micro-environment. The corollary to the hypothesis that the cleavage of pro-apoA-I occurs extracellulary is that the mature protein should not be found intracellularly in producing cells (i.e., hepatocytes and enterocytes). Although this expectation appears to hold for most of the species studied, it is not the case for chicken hepatocytes, which in culture contained 83% mature apoA-I even in the absence of serum in the culture medium (15Banerjee D. Grieninger G. Parkes JL. Mukherjee TK. Redman CM. Regulation of apo-A-I processing in cultured hepatocytes.J. Biol. Chem. 1986; 261: 9844-9849Abstract Full Text PDF PubMed Google Scholar). The molecular basis for this unexpected result remains to be clarified. Several investigators have studied the possible function of the prosequence in the secretion of pro-apoA-I. Using an in vitro transcription/translation/microsomal membrane system, it was shown that, in the absence of the prohexapeptide, there was a reduced efficiency of cotranslational translocation into the membrane vesicles (16Folz RJ. Gordon JI. The effects of deleting the propeptide from human preproapolipoprotein A-I on co-translational translocation and signal peptidase processing.J. Biol. Chem. 1987; 262: 17221-17230Abstract Full Text PDF PubMed Google Scholar). McLeod and colleagues (17McLeod RS. Robbins C. Burns A. Yao Z. Pritchard PH. Deletion of the propeptide of apolipoprotein A-I impairs exit of nascent apolipoprotein A-I from the endoplasmic reticulum.Biochem. J. 1994; 302: 641-648Crossref PubMed Scopus (15) Google Scholar) also showed that, in the absence of the prosequence, transport from the endoplasmic reticulum to the Golgi apparatus was impaired. Mutation of the Gln residues at the end of the prosequence, thus preventing its cleavage, had no impact on the ability of the mutated protein to be secreted from transfected C127 cells (18Roghani A. Zannis VI. Alterations of the glutamine residues of human apolipoprotein AI propeptide by in vitro mutagenesis. Characterization of the normal and mutant protein forms.Biochemistry. 1988; 27: 7428-7435Crossref PubMed Scopus (15) Google Scholar). It is widely thought that most of the lipidation of apoA-I to form HDL occurs extracellularly as a result of interaction with ABCA1. However, some lipidation, about 20%, occurs intracellularly, meaning that pro-apoA-I can be lipidated (19Chisholm JW. Burleson ER. Shelness GS. Parks JS. ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL.J. Lipid Res. 2002; 43: 36-44Abstract Full Text Full Text PDF PubMed Google Scholar).This has also been studied in primary hepatocytes when the newly synthesized apoA-I is initially phospholipidated in the endoplasmic reticulum and, more significantly, in the Golgi. The former lipidation appears to be ABCA1 independent whereas the latter is dependent on the transporter function (20Maric J. Kiss RS. Franklin V. Marcel YL. Intracellular lipidation of newly synthesized apolipoprotein A-I in primary murine hepatocytes.J. Biol. Chem. 2005; 280: 39942-39949Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The role of ABCA1 in the conversion of the proapolipoprotein to the mature protein is highlighted by the changing balance between the proprotein and the mature protein in Tangier disease, now known to be attributable to a deficiency of the ABCA1 transporter. In this disorder, the relative concentration of proprotein and mature protein markedly favors the former (21Zannis VI. Lees AM. Lees RS. Breslow JL. Abnormal apoprotein A-I isoprotein composition in patients with Tangier disease.J. Biol. Chem. 1982; 257: 4978-4986Abstract Full Text PDF PubMed Google Scholar), but this is not attributable to a fundamental defect in the conversion machinery in Tangier disease (22Brewer Jr., HB. Fairwell T. Meng M. Kay L. Ronan R. Human proapoA-ITangier: isolation of proapoA-ITangier and amino acid sequence of the propeptide.Biochem. Biophys. Res. Commun. 1983; 113: 934-940Crossref PubMed Scopus (16) Google Scholar, 23Gordon JI. Sims HF. Lentz SR. Edelstein C. Scanu AM. Strauss AW. Proteolytic processing of human preproapolipoprotein A-I. A proposed defect in the conversion of pro A-I to A-I in Tangier’s disease.J. Biol. Chem. 1983; 258: 4037-4044Abstract Full Text PDF PubMed Google Scholar). Even though it has been shown that the free proprotein can be converted to the mature form, it is possible that in vivo, a particular lipidated form of the proapoprotein may be the preferred substrate for its maturation. It would be worth studying whether the Tangier phenotype with respect to the relative predominance of pro-apoA-I in the very small pool of circulating apoA-I could be established in mice lacking ABCA1 function. Timmins and colleagues (24Timmins JM. Lee JY. Boudyguina E. Kluckman KD. Brunham LR. Mulya A. Gebre AK. Coutinho JM. Colvin PL. Smith TL. et al.Targeted inactivation of hepatic Abca1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism of apoA-I.J. Clin. Invest. 2005; 115: 1333-1342Crossref PubMed Scopus (423) Google Scholar) have shown that the selective knockout of hepatic ABCA1 is associated with a substantial decline in circulating HDL accompanied by hypercatabolism of apoA-I in the kidney. They did not examine the catabolism of pro-apoA-I in this model. It is not clear whether the hepatic knockout is sufficient to establish the model for pro-apoA-I catabolism or whether a global knockout would be more useful. The fate of the hexapeptide removed from the proprotein has also not been examined. It is not clear whether it is stable after separation from the protein. If it is reasonably stable, its measurement in the urine, for example, could provide information on the combined rate of apoA-I synthesis in the liver and intestine and the antibody described by Barkia (7Barkia A. Martin C. Puchois P. Gesquiere JC. Cachera C. Tartar A. Fruchart JC. Enzyme-linked immunosorbent assay for human proapolipoprotein A-I using specific antibodies against synthetic peptide.J. Lipid Res. 1988; 29: 77-84Abstract Full Text PDF PubMed Google Scholar) could be used for such a study. The differences in these two proteins remains unclear. Both are capable of binding lipid, apparently to the same extent. They both self-associate and appear to activate LCAT similarly (25McGuire KA. Davidson WS. Jonas A. High yield overexpression and characterization of human recombinant proapolipoprotein A-I.J. Lipid Res. 1996; 37: 1519-1528Abstract Full Text PDF PubMed Google Scholar). In their paper, Francone and colleagues provide a potentially important difference by suggesting that when normalized for apoA-I concentration, preβ HDL containing pro-apoA-I is defective in ABCA1-dependent cholesterol efflux. They refer to the knockout HDL as “dysfunctional,” though the precise basis for this dysfunction remains to be clarified. A potential caveat in interpreting these results is that the acceptors were probably constituted of mixtures of HDL particles that were not precisely defined. It will be necessary to undertake experiments like this with more discretely defined acceptors containing either pro-apoA-I or mature apoA-I, probably using reconstituted “HDL” particles. However, two other studies have also suggested that pro-apoA-I is not as efficient in promoting ABCA1-dependent cholesterol efflux as is the mature apoprotein (5Chau P. Fielding PE. Fielding CJ. Bone morphogenetic protein-1 (BMP-1) cleaves human proapolipoprotein A1 and regulates its activation for lipid binding.Biochemistry. 2007; 46: 8445-8450Crossref PubMed Scopus (40) Google Scholar, 26Sviridov D. Pyle LE. Fidge N. Efflux of cellular cholesterol and phospholipid to apolipoprotein A-I mutants.J. Biol. Chem. 1996; 271: 33277-33283Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The interaction of the proprotein with the ABCA1 efflux mechanism needs to be systematically investigated. Mature apoprotein in the model of Vedhachalam et al. (27Vedhachalam C. Duong PT. Nickel M. Nguyen D. Dhanasekaran P. Saito H. Rothblat GH. Lund-Katz S. Phillips MC. Mechanism of ATP-binding cassette transporter A1-mediated cellular lipid efflux to apolipoprotein A-I and formation of high density lipoprotein particles.J. Biol. Chem. 2007; 282: 25123-25130Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar) interacts with ABCA1 and its “membrane product” at two stages and which of these is affected by the hexapeptide prosequence on the acceptor would be of interest. It would also be important to complement the cell culture findings with a study of reverse cholesterol transport in vivo (28Zhang Y. Zanotti I. Reilly MP. Glick JM. Rothblat GH. Rader DJ. Overexpression of apolipoprotein A-I promotes reverse transport of cholesterol from macrophages to feces in vivo.Circulation. 2003; 108: 661-663Crossref PubMed Scopus (372) Google Scholar). In the face of the cholesterol efflux dysfunction just discussed, the accumulation of an enlarged HDL in the PCPE2-deficient mice observed by Francone et al. seems counterintuitive. The enlargement of HDL implies that ongoing LCAT activity is normal. One of the factors that influences LCAT activity in a large HDL is the apoprotein composition of the HDL. However, no change in apoprotein composition was noted in the enlarged HDL. This highlights what is becoming widely recognized; namely, that the function of HDL is more important from an atheroprotective point of view than its level in the plasma. Cholesterol efflux or reverse cholesterol transport is but one of the atheroprotective effects of HDL. In this model, it would clearly be of interest to explore some of the other anti-inflammatory properties of this HDL in contrast to normal HDL. Further studies of the function of reconstituted HDL containing predominantly pro-apoA-I are indicated. But what accounts for the increased level of this large HDL? Not studied here is HDL clearance that depends on uptake in the liver, selective cholesterol influx into sterol using cells and tissues, and the release of apoA-I from intact HDL with resulting filtration in the kidney (29Rader DJ. Molecular regulation of HDL metabolism and function: implications for novel therapies.J. Clin. Invest. 2006; 116: 3090-3100Crossref PubMed Scopus (472) Google Scholar). The capacity of the apoA-I to be filtered may be a significant determinant and this could be influenced by the size of the particle containing the protein. For example, the interaction of the HDL with lipases may influence the release of the apoprotein. The filtration of mature and pro-apoA-I is likely to be similar though this should be tested. of HDL that could influence its metabolism is the conformation of the which would to be by the and of the proteins in the HDL for example, R. A. LK. Davidson WS. A-I in from human Biol. 2011; PubMed Scopus Google Scholar). particular interest in the of the is the high interaction of apoA-I in the kidney with a CUB domain protein that has a which does not the apoA-I S. CJ. P. A. Jr., HB. WS. the for lipoprotein Natl. Acad. Sci. USA. PubMed Scopus Google Scholar, JL. C. WS. in concert with to of high density Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This in the catabolism of apoA-I. the interaction of pro-apoA-I with is of study. Its interaction with the proprotein may a role in the balance between the proprotein and the mature protein in Tangier The of an N-terminal extension of apoA-I is not to this protein the the of in the plasma in two which differ by the absence or of the N-terminal hexapeptide G. of the bovine to human and on and evidence for proteolytic J. PubMed Scopus Google Scholar, AL. RJ. RS. of the human and A in the 1987; 26: PubMed Scopus Google Scholar). This hexapeptide sequence is very to that found in and in with propeptide cleavage between the glutamine in the propeptide and the N-terminal aspartic acid residue of the mature similar to apoA-I. Both of appear to be in is also secreted as a proprotein. In this the prosequence is a with dibasic amino which is removed extracellularly by a and thus is quite different in processing than that for pro-apoA-I JI. Sims HF. Edelstein C. Scanu AM. Strauss AW. Human proapolipoprotein is cleaved secretion from cells by a Biol. Chem. Full Text PDF PubMed Google Scholar, JI. Sims HF. Edelstein C. Scanu AM. Strauss AW. processing of proapolipoprotein in cell is by a to Biol. Chem. Full Text PDF PubMed Google Scholar). Although it is that at three HDL are secreted as the biological of this is from clear. HDL is different than the in that most of the lipidation takes place with most more are than might be in the of a This is the case for the by Francone and alluded to in the there is more to be In addition, if a similar model for the were an additional of would

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