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• W2020531284 abstract "Lipoproteins consist of lipids solubilized by apolipoproteins. The lipid-binding structural motifs of apolipoproteins include amphipathic α-helixes and β-sheets. Plasma apolipoprotein (apo) M lacks an external amphipathic motif but, nevertheless, is exclusively associated with lipoproteins (mainly high density lipoprotein). Uniquely, however, apoM is secreted to plasma without cleavage of its hydrophobic NH2-terminal signal peptide. To test whether the signal peptide serves as a lipoprotein anchor for apoM in plasma, we generated mice expressing a mutated apoMQ22A cDNA in the liver (apoMQ22A-Tg mice (transgenic mice)) and compared them with mice expressing wild-type human apoM (apoM-Tg mice). The substitution of the amino acid glutamine 22 with alanine in apoMQ22A results in secretion of human apoM without a signal peptide. The human apoM mRNA level in liver and the amount of human apoM protein secretion from hepatocytes were similar in apoM-Tg and apoMQ22A-Tg mice. Nevertheless, human apoM was not detectable in plasma of apoMQ22A-Tg mice, whereas it was easily measured in the apoM-Tg mice. To examine the plasma metabolism, recombinant apoM lacking the signal peptide was produced in Escherichia coli and injected into wild-type mice. The apoM without signal peptide did not associate with lipoproteins and was rapidly cleared in the kidney. Accordingly, ligation of the kidney arteries in apoMQ22A-Tg mice resulted in rapid accumulation of human apoM in plasma. The data suggest that hydrophobic signal peptide sequences, if preserved upon secretion, can anchor plasma proteins in lipoproteins. In the case of apoM, this mechanism prevents rapid loss by filtration in the kidney. Lipoproteins consist of lipids solubilized by apolipoproteins. The lipid-binding structural motifs of apolipoproteins include amphipathic α-helixes and β-sheets. Plasma apolipoprotein (apo) M lacks an external amphipathic motif but, nevertheless, is exclusively associated with lipoproteins (mainly high density lipoprotein). Uniquely, however, apoM is secreted to plasma without cleavage of its hydrophobic NH2-terminal signal peptide. To test whether the signal peptide serves as a lipoprotein anchor for apoM in plasma, we generated mice expressing a mutated apoMQ22A cDNA in the liver (apoMQ22A-Tg mice (transgenic mice)) and compared them with mice expressing wild-type human apoM (apoM-Tg mice). The substitution of the amino acid glutamine 22 with alanine in apoMQ22A results in secretion of human apoM without a signal peptide. The human apoM mRNA level in liver and the amount of human apoM protein secretion from hepatocytes were similar in apoM-Tg and apoMQ22A-Tg mice. Nevertheless, human apoM was not detectable in plasma of apoMQ22A-Tg mice, whereas it was easily measured in the apoM-Tg mice. To examine the plasma metabolism, recombinant apoM lacking the signal peptide was produced in Escherichia coli and injected into wild-type mice. The apoM without signal peptide did not associate with lipoproteins and was rapidly cleared in the kidney. Accordingly, ligation of the kidney arteries in apoMQ22A-Tg mice resulted in rapid accumulation of human apoM in plasma. The data suggest that hydrophobic signal peptide sequences, if preserved upon secretion, can anchor plasma proteins in lipoproteins. In the case of apoM, this mechanism prevents rapid loss by filtration in the kidney. Lipoproteins consist of lipids (mainly cholesterol, phospholipids, and triglycerides) solubilized by apolipoproteins. The apolipoproteins play essential roles in controlling plasma and tissue lipid homeostasis by interacting with cellular lipoprotein receptors (the low density lipoprotein receptor, the scavenger receptor class B type-I, the ATP-binding cassette transporter AI, etc.) and enzymes (e.g. lecithin-cholesterol acyltransferase, hepatic lipase, and lipoprotein lipase) and lipid transfer protein (cholesteryl ester transfer protein and phospholipid transfer protein). However, several apolipoproteins have roles beyond lipid metabolism. Indeed, a recent shotgun proteomic approach revealed that HDL 3The abbreviations used are: HDL, high density lipoprotein; HRP, haptoglobin-related protein; PON-1, paraoxonase 1; TC, tyramine cellobiose; Tg, transgenic; apo, apolipoprotein; ELISA, enzyme-linked immunosorbent assay. 3The abbreviations used are: HDL, high density lipoprotein; HRP, haptoglobin-related protein; PON-1, paraoxonase 1; TC, tyramine cellobiose; Tg, transgenic; apo, apolipoprotein; ELISA, enzyme-linked immunosorbent assay. on average contains >40 proteins, of which many affect complement activation or protease inhibition (1Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. Chea H. Knopp R.H. Brunzell J. Geary R. Chait A. Zhao X.Q. Elkon K. Marcovina S. Ridker P. Oram J.F. Heinecke J.W. J. Clin. Investig. 2007; 117: 746-756Crossref PubMed Scopus (786) Google Scholar). Also, several well known HDL apolipoproteins have roles in inflammation and host defense against microbial infections (1Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. Chea H. Knopp R.H. Brunzell J. Geary R. Chait A. Zhao X.Q. Elkon K. Marcovina S. Ridker P. Oram J.F. Heinecke J.W. J. Clin. Investig. 2007; 117: 746-756Crossref PubMed Scopus (786) Google Scholar). For instance, apoL is involved in fighting Trypanosoma infections (2Vanhamme L. Paturiaux-Hanocq F. Poelvoorde P. Nolan D.P. Lins L. Van Den A.J. Pays A. Tebabi P. Van X.H. Jacquet A. Moguilevsky N. Dieu M. Kane J.P. De B.P. Brasseur R. Pays E. Nature. 2003; 422: 83-87Crossref PubMed Scopus (362) Google Scholar), and apoJ (clusterin) has even been proposed to play a significant role in tumorigenesis (3Trougakos I.P. Gonos E.S. Int. J. Biochem. Cell Biol. 2002; 34: 1430-1448Crossref PubMed Scopus (318) Google Scholar). For apolipoproteins without any (known) effect on plasma lipid metabolism, it is conceivable that the host HDL particle mainly serves as a carrier that prevents rapid removal of its associated apolipoproteins (e.g. by filtration in the kidney) or perhaps aids in their assembly at sites of inflammation. The structural motifs conferring the lipid-binding capacity of apolipoproteins has been most intensively studied for apoB in low density lipoprotein (4Segrest J.P. Jones M.K. De L.H. Dashti N. J. Lipid Res. 2001; 42: 1346-1367Abstract Full Text Full Text PDF PubMed Google Scholar) and apoA-I in HDL (5Davidson W.S. Silva R.A. Curr. Opin. Lipidol. 2005; 16: 295-300Crossref PubMed Scopus (75) Google Scholar, 6Davidson W.S. Hazlett T. Mantulin W.W. Jonas A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13605-13610Crossref PubMed Scopus (133) Google Scholar). Both amphipathic α-helixes (7Segrest J.P. Jackson R.L. Morrisett J.D. Gotto Jr., A.M. FEBS Lett. 1974; 38: 247-258Crossref PubMed Scopus (486) Google Scholar) and β-sheets (4Segrest J.P. Jones M.K. De L.H. Dashti N. J. Lipid Res. 2001; 42: 1346-1367Abstract Full Text Full Text PDF PubMed Google Scholar) bind to the lipids. In the case of apoA-I and several other HDL apolipoproteins (e.g. apoE and the apoCs), the non-covalent binding of the amphipathic α-helixes are rather weak, allowing exchange of the apolipoproteins between the triglyceride-rich very low density lipoprotein and chylomicron particles and HDL (8Atkinson D. Small D.M. Annu. Rev. Biophys. Biophys. Chem. 1986; 15: 403-456Crossref PubMed Scopus (136) Google Scholar). Indeed, some apolipoproteins are so loosely bound to HDL (e.g. apoJ (9Christoffersen C. Nielsen L.B. Axler O. Andersson A. Johnsen A.H. Dahlback B. J. Lipid Res. 2006; 47: 1833-1843Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), apoA-IV (10Christoffersen C. Jauhiainen M. Moser M. Porse B. Ehnholm C. Boesl M. Dahlback B. Nielsen L.B. J. Biol. Chem. 2007; 283: 1839-1847Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), and lecithin-cholesterol acyltransferase (11Cheung M.C. Wolf A.C. Lum K.D. Tollefson J.H. Albers J.J. J. Lipid Res. 1986; 27: 1135-1144Abstract Full Text PDF PubMed Google Scholar) that they are often stripped from the lipoprotein particles during preparative ultracentrifugation. ApoM (188 amino acids) was discovered in 1999 by Xu and Dahlback (12Xu N. Dahlback B. J. Biol. Chem. 1999; 274: 31286-31290Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). In plasma, apoM is exclusively found in association with lipoproteins (mainly HDL). Recent studies of mice with genetically modified apoM expression showed that apoM affects plasma HDL metabolism and development of atherosclerosis (10Christoffersen C. Jauhiainen M. Moser M. Porse B. Ehnholm C. Boesl M. Dahlback B. Nielsen L.B. J. Biol. Chem. 2007; 283: 1839-1847Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 13Wolfrum C. Poy M.N. Stoffel M. Nat. Med. 2005; 11: 418-422Crossref PubMed Scopus (265) Google Scholar), but apoM also has antioxidant effects, and its physiological roles remain to be determined (10Christoffersen C. Jauhiainen M. Moser M. Porse B. Ehnholm C. Boesl M. Dahlback B. Nielsen L.B. J. Biol. Chem. 2007; 283: 1839-1847Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Analysis of the tertiary structure of apoM by in silico homology modeling has revealed that apoM belongs to the lipocalin protein superfamily (14Duan J. Dahlback B. Villoutreix B.O. FEBS Lett. 2001; 499: 127-132Crossref PubMed Scopus (84) Google Scholar). Like in other lipocalins, the structure of apoM contains eight antiparallel β-sheets forming a small lipid-binding pocket. Recent experimental data confirmed the lipocalin structure of apoM and showed that it can bind retinoic acid and retinol (15Ahnstrom J. Faber K. Axler O. Dahlback B. J. Lipid Res. 2007; 48: 1754-1762Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Human apoM can be glycosylated at amino acid Asn-135 surrounded by the glycosylation signal Asn-Glu-Thr (12Xu N. Dahlback B. J. Biol. Chem. 1999; 274: 31286-31290Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Hence, Western blotting of SDS-PAGE gels with reduced human plasma samples typically reveals both glycosylated apoM and a non-glycosylated apoM of ∼25 and ∼20 kDa (9Christoffersen C. Nielsen L.B. Axler O. Andersson A. Johnsen A.H. Dahlback B. J. Lipid Res. 2006; 47: 1833-1843Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 12Xu N. Dahlback B. J. Biol. Chem. 1999; 274: 31286-31290Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The predicted structure of apoM lacks external amphipathic motifs that would explain the lipoprotein association of apoM. NH2-sequencing of plasma apoM has shown that it is secreted to the blood with its NH2-terminal signal peptide (12Xu N. Dahlback B. J. Biol. Chem. 1999; 274: 31286-31290Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). This is unusual because intracellular cleavage of the NH2-terminal signal peptide by signal peptidases in the endoplasmic reticulum conventionally is seen as a prerequisite for cellular release of proteins to plasma. We are, however, aware of at least two other plasma proteins with preserved NH2-terminal signal peptides, i.e. paraoxonase-1 (PON-1) (16Sorenson R.C. Bisgaier C.L. Aviram M. Hsu C. Billecke S. La Du B.N. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2214-2225Crossref PubMed Scopus (282) Google Scholar) and haptoglobin-related protein (HRP) (17Raper J. Fung R. Ghiso J. Nussenzweig V. Tomlinson S. Infect. Immun. 1999; 67: 1910-1916Crossref PubMed Google Scholar). Like apoM, PON-1 and HRP are mainly found in a subfraction of HDL (although not the same HDL subfraction as that with apoM (9Christoffersen C. Nielsen L.B. Axler O. Andersson A. Johnsen A.H. Dahlback B. J. Lipid Res. 2006; 47: 1833-1843Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar)), and in vitro studies suggest that the signal peptide in PON-1 can mediate its association with lipoproteins (16Sorenson R.C. Bisgaier C.L. Aviram M. Hsu C. Billecke S. La Du B.N. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2214-2225Crossref PubMed Scopus (282) Google Scholar). Based on these findings and the highly hydrophobic nature of the signal peptide, we hypothesized that the signal peptide serves as an anchor for apoM in plasma lipoprotein particles. To test this idea and explore the impact of the signal peptide on the metabolism of plasma apoM, we generated transgenic mice that express a mutated human apoMQ22A cDNA in the liver. The cDNA encodes a mutated human apoMQ22A protein where glutamine in position 22 is replaced by alanine. The Gln-22 → Ala substitution results in secretion of apoM without its hydrophobic signal peptide (15Ahnstrom J. Faber K. Axler O. Dahlback B. J. Lipid Res. 2007; 48: 1754-1762Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The mice expressing apoMQ22A were compared with transgenic mice expressing wild-type human apoM. ApoM-Tg and apoMQ22A-Tg Mice—Transgenic (Tg) mice expressing a truncated human apoM protein without the signal peptide were generated. Site-directed mutagenesis resulting in substitution of amino acid Gln-22 with Ala in human apoM (apoMQ22A) was done previously (15Ahnstrom J. Faber K. Axler O. Dahlback B. J. Lipid Res. 2007; 48: 1754-1762Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Secretion of apoMQ22A cDNA from transfected HEK293 cells was assessed as described (15Ahnstrom J. Faber K. Axler O. Dahlback B. J. Lipid Res. 2007; 48: 1754-1762Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) by Western blotting (9Christoffersen C. Nielsen L.B. Axler O. Andersson A. Johnsen A.H. Dahlback B. J. Lipid Res. 2006; 47: 1833-1843Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and ELISA (18Axler O. Ahnstrom J. Dahlback B. J. Lipid Res. 2007; 48: 1772-1780Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). A 774-bp fragment of the apoMQ22A-encoding cDNA was amplified with Pfu DNA polymerase system (Stratagene, AH Diagnostic) and primers (pcDNA3-apoMQ22A-51:5′-gcggccgcactggcggccgttactagtggat-3′ and pcDNA3-apoMQ22A-31:5′-gcggccgcgcagtaggtgtccaccatgttcc-3′) and cloned into a PCR-Blunt II-TOPO vector (Invitrogen A/S). The ApoMQ22A encoding cDNA was subsequently cloned into a pGEMAlbSV40 vector (obtained from Ragnar Matsson, Lund University) between a murine albumin promotor/enhancer sequence and an SV40 intron/poly(A) sequence. The correctness of apoMQ22A encoding cDNA was verified by DNA sequencing (primers will be given by request). The 4446-bp albumin-apoMQ22A-SV40 fragment was purified from the plasmid by restriction digestion with Nsi and ApaI, agarose gel electrophoresis and the QIAEX II gel extraction kit (Qiagen-Nordic) and used for pronuclear microinjections into fertilized mouse oocytes (C57/Bl X CBA) at the Transgenic Core Facility, University of Lund, Sweden. Human apoMQ22A transgenic founders were identified by real-time PCR amplification (LightCycler, Roche Diagnostic) of a 175-bp fragment of human APOM from tail DNA with primers h-apoM-51 and h-apoM-31 (10Christoffersen C. Jauhiainen M. Moser M. Porse B. Ehnholm C. Boesl M. Dahlback B. Nielsen L.B. J. Biol. Chem. 2007; 283: 1839-1847Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The human apoM-transgenic mice expressing wild-type human apoM (apoM-Tg) were described recently elsewhere (as apoM-TgN mice (10Christoffersen C. Jauhiainen M. Moser M. Porse B. Ehnholm C. Boesl M. Dahlback B. Nielsen L.B. J. Biol. Chem. 2007; 283: 1839-1847Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar)). Both apoM-Tg and apoMQ22A-Tg mice were backcrossed with C57Bl/6 mice for at least five generations before the present studies. The mice were housed at the Panum Institute (University of Copenhagen) and fed standard chow (Altromin 1314, Brogaarden). Blood samples were taken from the venous plexus in the orbital cavity into Na2EDTA- or heparin-containing tubes and kept on ice. Plasma was obtained by centrifugation at 3000 rpm for 10 min at 4 °C and stored at -80 °C or -20 °C. All procedures were approved by the Animal Experiments Inspectorate, Ministry of Justice, Denmark. mRNA Quantification—RNA was isolated from frozen tissue (-80 °C) with TRIzol (Invitrogen) and examined on an RNA Nano LabChip (Agilent Technologies). First-strand cDNA synthesis and quantitative real-time PCR analysis with a LightCycler was performed as described (19Bartels E.D. Lauritsen M. Nielsen L.B. Diabetes. 2002; 51: 1233-1239Crossref PubMed Scopus (104) Google Scholar) with primers for human APOM (10Christoffersen C. Jauhiainen M. Moser M. Porse B. Ehnholm C. Boesl M. Dahlback B. Nielsen L.B. J. Biol. Chem. 2007; 283: 1839-1847Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), mouse APOM (20Faber K. Axler O. Dahlback B. Nielsen L.B. J. Lipid Res. 2004; 45: 1272-1278Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and β-actin (19Bartels E.D. Lauritsen M. Nielsen L.B. Diabetes. 2002; 51: 1233-1239Crossref PubMed Scopus (104) Google Scholar). Quantification of Human apoM—Human wild-type apoM and recombinant human apoM22–188 were measured with a sandwich ELISA employing two monoclonal antibodies against human apoM (18Axler O. Ahnstrom J. Dahlback B. J. Lipid Res. 2007; 48: 1772-1780Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The primary antibodies against human apoM used for 12% SDS-PAGE Western blotting (9Christoffersen C. Nielsen L.B. Axler O. Andersson A. Johnsen A.H. Dahlback B. J. Lipid Res. 2006; 47: 1833-1843Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) were polyclonal rabbit anti-human apoM IgG (2.5 μg/ml), a monoclonal mouse anti-human apoM antibody (M58; 1:1000 (18Axler O. Ahnstrom J. Dahlback B. J. Lipid Res. 2007; 48: 1772-1780Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar)), or a monoclonal mouse anti-human apoM antibody (from BD Biosciences, 1:10.000). Mouse apoM was detected with a polyclonal rabbit anti-mouse apoM antiserum (1:500) (10Christoffersen C. Jauhiainen M. Moser M. Porse B. Ehnholm C. Boesl M. Dahlback B. Nielsen L.B. J. Biol. Chem. 2007; 283: 1839-1847Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Primary Hepatocyte Cell Culture—Primary hepatocytes were isolated from ApoM-TgN, ApoMQ22A-Tg, and wild-type mice (21Sabol S.L. Brewer Jr., H.B. Santamarina-Fojo S. J. Lipid Res. 2005; 46: 2151-2167Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Briefly, mice were perfused with 5 ml of liver perfusion medium (Invitrogen) followed by 5 ml of digestion medium containing 4 mg/ml collagenase type I (C2624, Sigma-Aldrich). The liver was then placed in a sterile tube with 5 ml of digestion medium at 37 °C for 20 min before being placed in ice-cold L-15 cell medium (Invitrogen). Subsequently, the tissue was homogenized with a Pasteur pipette, passed through a 70-μm nylon filter, and washed with hepatocyte washing medium (Invitrogen). Centrifugation between washes was done at 50 × g for 5 min. The cells were resuspended in 5 ml of F-12/Dulbecco's modified Eagle's medium (Invitrogen) with 1% penicillin/streptomycin, 10% bovine calf serum, and 2 mm l-glutamine (G6392, Sigma-Aldrich) and grown on 6-well collagen-coated plates (152034, Nunc). The concentration of human apoM in cell culture medium was measured after 48 h. Lipoproteins (d < 1.21 g/ml) were separated from non-lipoproteins in plasma or concentrated (×4) cell culture medium by adjusting 20 μl of sample to d = 1.21 g/ml with NaBr and ultracentrifugation in a TLA-100 ultracentrifuge (Beckman) using 200-μl tubes at 15 °C for 4 h and 100,000 rpm. Plasma Turnover and Tissue Elimination of Human apoM22–188—To study the plasma turnover of apoM without the signal peptide, recombinant apoM22–188 or wild-type human apoM were injected intravenously into recipient wild-type mice, and the plasma turnover was assessed by measuring human apoM plasma concentrations with ELISA. Recombinant human apoM22–188 was produced as described (18Axler O. Ahnstrom J. Dahlback B. J. Lipid Res. 2007; 48: 1772-1780Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and protein concentration was measured with the Pierce BCA protein assay kit (Bie and Berntsen A-S) using bovine serum albumin as a standard (9Christoffersen C. Nielsen L.B. Axler O. Andersson A. Johnsen A.H. Dahlback B. J. Lipid Res. 2006; 47: 1833-1843Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). For each recipient mouse, apoM22–188 (100 μg) in 148 μl of phosphate-buffered saline was mixed with 90 μl of plasma from a wild-type mouse and injected intravenously into a wild-type mouse (n = 3); control mice (n = 3) received 148 μl of phosphate-buffered saline mixed with 90 μl of plasma from human apoM-Tg mice that overexpress human apoM 10-fold (10Christoffersen C. Jauhiainen M. Moser M. Porse B. Ehnholm C. Boesl M. Dahlback B. Nielsen L.B. J. Biol. Chem. 2007; 283: 1839-1847Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Blood samples were taken after 5, 30, 120, and 1400 min for measurement of human apoM. To assess the lipoprotein association of human apoM, lipoproteins (d < 1.21 g/ml) were separated from proteins in plasma samples taken after 30 min as described, above and the human apoM concentration was determined in the fractions by ELISA. To determine tissue uptake and degradation, we labeled apoM22–188 or albumin with 125I-TC and injected it into wild-type mice. The 125I-TC moiety is trapped inside cells upon lysosomal degradation of the labeled protein, enabling in vivo studies of degradation sites of plasma proteins (22Pittman R.C. Carew T.E. Glass C.K. Green S.R. Taylor Jr., C.A. Attie A.D. Biochem. J. 1983; 212: 791-800Crossref PubMed Scopus (222) Google Scholar). First, tyramine cellobiose (TC) (50 nmol) was labeled with 37 MBq 125I in tubes coated with 10 μg of Iodogen (Invitrogen) (23Juul K. Nielsen L.B. Munkholm K. Stender S. Nordestgaard B.G. Circulation. 1996; 94: 1698-1704Crossref PubMed Scopus (64) Google Scholar). Then, the 125I-labeled TC was activated and coupled to 100–120 μg of apoM22–188 or albumin at pH 9–10. To separate the unbound 125I and 125I-TC from 125I-TC-albumin and 125I-TC-apoM22–188, the mixture was passed over a PD-10 column (Amersham Biosciences). The labeling efficiency was 34% for apoM22–188 and 22% for albumin, and 91 and 86% of the 125I in the doses were precipitated with 15% trichloroacetic acid for apoM22–188 and albumin, respectively. The integrity of the labeled proteins was examined by 12% SDS-PAGE, and visualization of the radioactivity was done with a Fujix BAS 200 bioimaging analyzer (Fuji Photo Film). The concentration of 125I-TC-labeled proteins was determined by the BCA Pierce kit (Pierce), and 125I-TC-apoM22–188 or 125I-TC-albumin (10 μg, 3.2–5.3 × 106 cpm) was injected into a tail vein of wild-type mice (n = 2 × 3) in a total volume of 330 μl. Blood samples were taken 10 min and 6 h after injection before the mice were anesthetized and perfused with phosphate-buffered saline. Plasma samples and biopsies from liver, kidney, lung, spleen, eyes, heart, brain, gut, and gall bladder were placed in 1 ml of phosphate-buffered saline and counted in a 1470 automatic gamma counter (PerkinElmer Life Sciences) for 30 min. To examine the cellular distribution of labeled apoM22–188, 1-μm Epon sections were obtained from kidneys fixed by immersion in 4% formalin 6 h after injection of labeled protein. The tissue was postfixed in OsO4, dehydrated, and embedded in Epon 812. The Epon sections were processed for autoradiography using Ilford K2 emulsion, exposed for 1–4 weeks, and developed in Kodak D19. The sections were examined in a Leica DMR microscope equipped with a Leica DFC320 camera. Images were transferred by a Leica TFC Twain 6.1.0 program and processed using Adobe PhotoShop 8.0. Exclusion of Kidney Elimination of Plasma Proteins—To exclude kidney elimination of plasma proteins, apoMQ22A-Tg (n = 6), apoM-TgN (n = 3), and wild-type mice (n = 3), animals were anesthetized, and the kidney was dissected free as described previously (24Bro S. Moeller F. Andersen C.B. Olgaard K. Nielsen L.B. J. Am. Soc. Nephrol. 2004; 15: 1495-1503Crossref PubMed Scopus (66) Google Scholar) to enable bilateral ligation of the kidney artery. The musculofascial and skin incisions were then sutured, and the animals were given buprenorphine (0.001 mg/10 g of body, subcutaneously) for analgesia while kept anesthetized for another 4 h. Blood samples were taken before and 1 and 4 h after the kidney artery ligation. Expression of ApoMQ22A in Transgenic Mice—To examine the impact of the signal peptide sequences on the metabolism of apoM, we used a mutated human apoMQ22A cDNA driven by albumin enhancer-promoter sequences to make Tg mice. The Gln-22 → Ala substitution introduces a signal peptidase cleavage site and results in secretion of a truncated apoM without the signal peptide from HEK293 cells (Fig. 1a). Twelve founders were generated by pronuclear injection of the transgene into fertilized mouse eggs. One transgenic line with an estimated ∼32 copies of the transgene was bred for further studies and compared with Tg mice expressing wild-type human apoM (10Christoffersen C. Jauhiainen M. Moser M. Porse B. Ehnholm C. Boesl M. Dahlback B. Nielsen L.B. J. Biol. Chem. 2007; 283: 1839-1847Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The apoMQ22A-Tg mice thrived well, bred normally, and were indistinguishable from wild-type mice. The liver expression of human apoM mRNA in the apoMQ22A-Tg mice was ∼40% of that in apoM-Tg mice (Fig. 1b). However, on Western blotting of mouse plasma with a human apoM-specific polyclonal antibody, apoM was only detectable in the apoM-Tg and not in apoMQ22A-Tg mice (Fig. 1c). Of note, the polyclonal anti-human apoM antibody recognized recombinant apoM22–188 as well as wild-type human apoM (Fig. 1c). This implies that the lack of apoM in apoMQ22A-Tg mouse plasma does not reflect the lack of reactivity of the antibodies toward truncated apoM. In agreement with the Western blotting results, human apoM could not be detected in the plasma from apoMQ22A-Tg mice with a human apoM-specific sandwich ELISA when the plasma was diluted as little as 50 times. The ELISA uses two different monoclonal antibodies to human apoM and reacts well with both wild-type human apoM and recombinant apoM without the signal peptide sequences (Fig. 1e). The detection limit is ∼0.3 nm. Hence, the concentration of human apoM in apoMQ22A-Tg mice is <15 nm. In contrast, the concentration of human apoM is ∼2 μm in the human apoM-Tg mice. The absence of detectable apoMQ22A in plasma was also seen in analyses of eight additional apoMQ22A-Tg founders and thus was not dependent on the site of transgene integration in the mouse genome. Overexpression of human apoM had no discernable effect on the plasma levels of endogenous mouse apoM (Fig. 1d). Since apoMQ22A is secreted from transfected HEK293 cells, we reasoned that the absence of apoMQ22A in the apoMQ22A-Tg mouse plasma most likely did not reflect impaired secretion from the mouse liver. To test this assumption, primary hepatocytes from apoMQ22A-Tg, apoM-Tg, and wild-type mice were grown in tissue culture plates. Analysis of the medium by ELISA (Fig. 2) and Western blotting (not shown) showed that human apoM was secreted into the medium from both apoMQ22A-Tg and apoM-Tg hepatocytes. The concentration of apoMQ22A in the medium was ∼40% of that of wild-type human apoM. Hence, the relative rate of secretion of apoMQ22A and wild-type human apoM from the cultured hepatocytes was similar to the relative amount of apoMQ22A and wild-type human apoM mRNA expression in the liver. The virtual absence of apoMQ22A in plasma despite ample secretion from the liver suggested that the lack of the signal peptide might cause rapid clearance of apoMQ22A from plasma because apoMQ22A cannot associate with plasma lipoproteins. Impact of the Signal Peptide on Plasma Metabolism of apoM—To explore the impact of the signal peptide on the plasma metabolism of apoM, we produced a recombinant truncated human apoM22–188 without the signal peptide (corresponding to amino acids 22–188 of wild-type human apoM) in Escherichia coli and injected it (100 μg) into wild-type mice. ELISA measurements showed that apoM22–188 was rapidly cleared from plasma with only ∼5% of the injected dose remaining in plasma 2 h after the injection (Fig. 3a). We also examined the plasma metabolism of wild-type human apoM by injecting plasma from apoM-Tg mice into wild-type mice; the clearance of wild-type human apoM was much slower than that of apoM22–188 with ∼50 and ∼5% of the injected wild-type human apoM remaining in plasma after 2 and 24 h, respectively (Fig. 3a). To analyze the lipoprotein association of apoM22–188, lipoproteins were separated from the remaining plasma proteins by ultracentrifugation at d = 1.21 g/ml using plasma collected 30 min after injection of apoM22–188 or wild-type human apoM particles. ApoM22–188 was recovered in the protein fraction, whereas wild-type human apoM predominantly was in the lipoprotein fraction (Fig. 3b). Due to the small size (∼20 kDa) and the lack of lipoprotein association of apoM22–188, we suspected that it would be rapidly removed by filtration in the kidney. We could, however, not detect any human apoM in the urine of apoMQ22A-Tg mice by ELISA or Western blotting, suggesting that if the kidney was a primary site for removal of apoM without the signal peptide, then the filtered apoM might be taken up in the proximal tubule rather than being excreted into the urine. Indeed, we previously showed that the endocytotic receptor megalin, which is highly expressed on the luminal side of the proximal tubule epithelium, can bind and internalize apoM (26Faber K. Hvidberg V. Moestrup S.K. Dahlback B. Nielsen L.B. Mol. Endocrinol. 2006; 20: 212-218Crossref PubMed Scopus (61) Google Scholar). To explore the tissue sites for uptake and degradation of apoM22–188, we labeled apoM22–188 with 125I-TC and injected it into wild-type mice. Parallel studies were done with human albumin. 125I-TC labeling did not affect the integrity of apoM22–188 or albumin as both proteins migrated as one band with the expected size in SDS-PAGE gels (Fig. 4a). Similar to non-labeled apoM22–188, 125I-TC-apoM22–188 was rapidly removed from plasma (Fig. 4b). Six hours after injection, the main portion of the 125I-TC-apoM22–188 was recovered in the kidney, whereas 125I-TC-albumin was recovered in the plasma, liver, and kidney (Fig. 4c). To examine which cells had taken up 125I-TC-apoM22–188, sections of the recipient mouse kidneys were subjected to autoradiography. The vast proportion of 125I in the kidney was seen in proximal tubule epithelial cells, whereas only a few grains were seen in glomerular cells or distal tubules (Fig. 4d). The rapid plasma elimination and uptake by kidney proximal tubule cells of human apoM22–188 were reiterated when using 125I-TC-labeled recombinant mouse apoM without its signal peptide sequences (data not shown). The turnover studies using recombinant apoM22–188 suggested that the absence of human apoM in the plasma of apoMQ22A-Tg mice reflects rapid removal of apoMQ22A in the kidney and that human apoM would accumulate in the plasma of the apoMQ22A-Tg mice if kidney elimination were prevented. To test this idea, we excluded kidney degradation of plasma proteins by bilateral ligation of the renal arteries in anesthetized apoMQ22A-Tg, apoM-Tg, and wild-type mice and measured human apoM in plasma with ELISA. Indeed, human apoM became detectable in plasma of the apoMQ22A-Tg mice already 1 h after eliminating kidney excretion (Fig. 5), which strongly supports the conclusion that apoM can be secreted from the liver without its signal peptide but that the lack of signal peptide results in rapid elimination of the truncated plasma protein by the kidney. The concentration of human apoM in apoM-Tg mice did not increase after bilateral ligation of the kidney arteries (Fig. 5). The NH2-terminal signal peptide plays important roles in the intracellular processing of proteins. It can determine protein folding (e.g. EspP (27Szabady R.L. Peterson J.H. Skillman K.M. Bernstein H.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 221-226Crossref PubMed Scopus (109) Google Scholar)) and direct proteins into specific secretory pathways (e.g. pro-opiomelanocortin (28Cool D.R. Fenger M. Snell C.R. Loh Y.P. J. Biol. Chem. 1995; 270: 8723-8729Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar)) or cellular compartments (e.g. PB2 of the influenza virus is directed to the mitochondria (29Carr S.M. Carnero E. Garcia-Sastre A. Brownlee G.G. Fodor E. Virology. 2006; 344: 492-508Crossref PubMed Scopus (55) Google Scholar)). Also, the hydrophobic nature of the signal peptide can anchor proteins in the cell membrane. Failure of cleavage of the signal peptide of proteins that are normally secreted can result in intracellular degradation (30Kostova Z. Wolf D.H. EMBO J. 2003; 22: 2309-2317Crossref PubMed Scopus (363) Google Scholar). In the present study, however, human wild-type apoM and human apoMQ22A appeared to be equally effectively secreted from primary mouse hepatocytes. The results suggest that the signal peptide is responsible for apoM's lipoprotein association and is crucial to prevent rapid clearance of apoM from plasma. The present in vivo findings are in accord with a recent in vitro observation by Axler et al. (25Axler O. Ahnstrom J. Dahlback B. FEBS Lett. 2008; 582: 826-828Crossref PubMed Scopus (45) Google Scholar), suggesting that apoMQ22A fails to associate with HDL in the culture medium when expressed in HEK293 cells. ApoM shares its preservation of the NH2-terminal signal peptide sequences with PON-1 and HRP (16Sorenson R.C. Bisgaier C.L. Aviram M. Hsu C. Billecke S. La Du B.N. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2214-2225Crossref PubMed Scopus (282) Google Scholar, 17Raper J. Fung R. Ghiso J. Nussenzweig V. Tomlinson S. Infect. Immun. 1999; 67: 1910-1916Crossref PubMed Google Scholar). In plasma, all three proteins are mainly in the HDL fraction. The apoM-containing HDL fraction is not enriched in PON-1 and HRP (9Christoffersen C. Nielsen L.B. Axler O. Andersson A. Johnsen A.H. Dahlback B. J. Lipid Res. 2006; 47: 1833-1843Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), and HRP is mainly part of an HDL subfraction named the trypanosome lytic complex, which also contains apoA-I and apoL (17Raper J. Fung R. Ghiso J. Nussenzweig V. Tomlinson S. Infect. Immun. 1999; 67: 1910-1916Crossref PubMed Google Scholar, 31Vanhollebeke B. Nielsen M.J. Watanabe Y. Truc P. Vanhamme L. Nakajima K. Moestrup S.K. Pays E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 4118-4123Crossref PubMed Scopus (59) Google Scholar). Thus, the signal peptides do not anchor the three proteins to the same HDL subfraction. Ovalbumin (32Palmiter R.D. Gagnon J. Walsh K.A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 94-98Crossref PubMed Scopus (199) Google Scholar) and PAI-2 (33Ye R.D. Wun T.C. Sadler J.E. J. Biol. Chem. 1988; 263: 4869-4875Abstract Full Text PDF PubMed Google Scholar) are also secreted without cleavage of their signal peptides. However, in those cases, the signal peptide sequences are not at the NH2 terminus of the proteins, and the hydrophobic residues are embedded in the three-dimensional structures of the proteins (34von H.G. Liljestrom P. Mikus P. Andersson H. Ny T. J. Biol. Chem. 1991; 266: 15240-15243Abstract Full Text PDF PubMed Google Scholar). Hence, neither ovalbumin nor PAI-2 is lipoprotein-bound. In silico sequence analyses using the signalP software predict that the preservation of the signal peptides in apoM and PON-1 results from the lack of a signal peptide cleavage site and, for both proteins, single amino acid substitutions can result in removal of the signal peptide prior to secretion (15Ahnstrom J. Faber K. Axler O. Dahlback B. J. Lipid Res. 2007; 48: 1754-1762Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 16Sorenson R.C. Bisgaier C.L. Aviram M. Hsu C. Billecke S. La Du B.N. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2214-2225Crossref PubMed Scopus (282) Google Scholar). In the case of HRP, however, the signalP software predicts that the signal peptide can be cleaved, and in vitro expression in 293 cells of an HRP encoding cDNA showed that the major fraction of HRP indeed is secreted from the cultured cells without the signal peptide (35Nielsen M.J. Petersen S.V. Jacobsen C. Oxvig C. Rees D. Moller H.J. Moestrup S.K. Blood. 2006; 108: 2846-2849Crossref PubMed Scopus (82) Google Scholar). Nevertheless, plasma HRP does contain the signal peptide. The present data provide a possible explanation for the apparent discrepancy; even if only a subfraction of HRP is secreted with the signal peptide sequences, the anchoring of HRP with the signal peptide in HDL may prevent clearance from plasma, whereas HRP without the signal peptide (∼35 kDa) predictably will be rapidly removed from plasma. The physiological role of apoM remains to be elucidated. Genetic elimination or overexpression of apoM in mice affects plasma lipid metabolism (10Christoffersen C. Jauhiainen M. Moser M. Porse B. Ehnholm C. Boesl M. Dahlback B. Nielsen L.B. J. Biol. Chem. 2007; 283: 1839-1847Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 13Wolfrum C. Poy M.N. Stoffel M. Nat. Med. 2005; 11: 418-422Crossref PubMed Scopus (265) Google Scholar), and in healthy humans, the plasma apoM concentration is positively associated with total cholesterol concentration (18Axler O. Ahnstrom J. Dahlback B. J. Lipid Res. 2007; 48: 1772-1780Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Nevertheless, apoM may have roles beyond lipid metabolism; HDL carries multiple proteins with roles in inflammation and innate immune responses, and HDL has pronounced anti-inflammatory effects in animal models (1Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. Chea H. Knopp R.H. Brunzell J. Geary R. Chait A. Zhao X.Q. Elkon K. Marcovina S. Ridker P. Oram J.F. Heinecke J.W. J. Clin. Investig. 2007; 117: 746-756Crossref PubMed Scopus (786) Google Scholar, 36Barter P.J. Nicholls S. Rye K.A. Anantharamaiah G.M. Navab M. Fogelman A.M. Circ. Res. 2004; 95: 764-772Crossref PubMed Scopus (1056) Google Scholar). Thus, it is possible that apoM, only being present in ∼5% of the plasma HDL particles (9Christoffersen C. Nielsen L.B. Axler O. Andersson A. Johnsen A.H. Dahlback B. J. Lipid Res. 2006; 47: 1833-1843Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), may also have effects unrelated to lipid metabolism. For instance, apoM protects against Cu2+-induced lipid oxidation (9Christoffersen C. Nielsen L.B. Axler O. Andersson A. Johnsen A.H. Dahlback B. J. Lipid Res. 2006; 47: 1833-1843Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 10Christoffersen C. Jauhiainen M. Moser M. Porse B. Ehnholm C. Boesl M. Dahlback B. Nielsen L.B. J. Biol. Chem. 2007; 283: 1839-1847Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and binds retinol and retinoic acid in vitro (15Ahnstrom J. Faber K. Axler O. Dahlback B. J. Lipid Res. 2007; 48: 1754-1762Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The latter observation is explained by the lipocalin structure of apoM where a small hydrophobic binding pocket enables binding of small lipophilic molecules (14Duan J. Dahlback B. Villoutreix B.O. FEBS Lett. 2001; 499: 127-132Crossref PubMed Scopus (84) Google Scholar). It is interesting to note that apoM is also found in a highly conserved major histocompatibility complex class III cluster in primitive organisms such as fish, indicating conservation of the cluster for more than 450 million years (37Deakin J.E. Papenfuss A.T. Belov K. Cross J.G. Coggill P. Palmer S. Sims S. Speed T.P. Beck S. Graves J.A. BMC. Genomics. 2006; 7: 281Crossref PubMed Scopus (55) Google Scholar), further suggesting that apoM may have essential biological functions. The present data document that the signal peptide in apoM serves an important function by anchoring the apolipoprotein to lipoproteins, thus inhibiting the elimination of apoM in the kidney and maintaining stable apoM concentrations in plasma. We thank Karen Rasmussen, Maria Kristensen, Charlotte Wandel, Tina Axen, and Inger Kristoffersen for technical assistance." @default.
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• W2020531284 title "The Signal Peptide Anchors Apolipoprotein M in Plasma Lipoproteins and Prevents Rapid Clearance of Apolipoprotein M from Plasma" @default.
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• W2020531284 cites W1490634134 @default.
• W2020531284 cites W1573267684 @default.
• W2020531284 cites W1607246558 @default.
• W2020531284 cites W1878414703 @default.
• W2020531284 cites W1968045991 @default.
• W2020531284 cites W1974294182 @default.
• W2020531284 cites W1982007286 @default.
• W2020531284 cites W1999030993 @default.
• W2020531284 cites W2003558962 @default.
• W2020531284 cites W2013681281 @default.
• W2020531284 cites W2019825424 @default.
• W2020531284 cites W2021986191 @default.
• W2020531284 cites W2032392307 @default.
• W2020531284 cites W2033299688 @default.
• W2020531284 cites W2039307785 @default.
• W2020531284 cites W2040812386 @default.
• W2020531284 cites W2044728266 @default.
• W2020531284 cites W2053453886 @default.
• W2020531284 cites W2054847996 @default.
• W2020531284 cites W2063790487 @default.
• W2020531284 cites W2070382695 @default.
• W2020531284 cites W2083315579 @default.
• W2020531284 cites W2084528656 @default.
• W2020531284 cites W2102697057 @default.
• W2020531284 cites W2105790696 @default.
• W2020531284 cites W2119277085 @default.
• W2020531284 cites W2129089207 @default.
• W2020531284 cites W2131553564 @default.
• W2020531284 cites W2134969640 @default.
• W2020531284 cites W2140919506 @default.
• W2020531284 cites W2144473676 @default.
• W2020531284 cites W2147589893 @default.
• W2020531284 cites W2166536336 @default.
• W2020531284 cites W2181164354 @default.
• W2020531284 cites W2305216237 @default.
• W2020531284 cites W2309042381 @default.
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