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• W2067907816 abstract "Cathelicidins are a family of antibacterial and lipopolysaccharide-binding proteins. hCAP-18, the only human cathelicidin, is a major protein of the specific granules of human neutrophils. The plasma level of hCAP-18 is >20-fold higher than that of other specific granule proteins relative to their levels within circulating neutrophils. The aim of this study was to elucidate the background for this high plasma level of hCAP-18. Plasma was subjected to molecular sieve chromatography, and hCAP-18 was found in distinct high molecular mass fractions that coeluted with apolipoproteins A-I and B, respectively. The association of hCAP-18 with lipoproteins was validated by the cofractionation of hCAP-18 with lipoproteins using two different methods for isolation of lipoproteins from plasma. Furthermore, the level of hCAP-18 in delipidated plasma was <1% of that in normal plasma. Immunoprecipitation of very low, low, and high density lipoprotein particles with anti-apolipoprotein antibodies resulted in coprecipitation of hCAP-18. The binding of hCAP-18 to lipoproteins was mediated by the antibacterial C-terminal part of the protein. The binding of hCAP-18 to lipoproteins suggests that lipoproteins may play an important role as a reservoir of this antimicrobial protein. Cathelicidins are a family of antibacterial and lipopolysaccharide-binding proteins. hCAP-18, the only human cathelicidin, is a major protein of the specific granules of human neutrophils. The plasma level of hCAP-18 is >20-fold higher than that of other specific granule proteins relative to their levels within circulating neutrophils. The aim of this study was to elucidate the background for this high plasma level of hCAP-18. Plasma was subjected to molecular sieve chromatography, and hCAP-18 was found in distinct high molecular mass fractions that coeluted with apolipoproteins A-I and B, respectively. The association of hCAP-18 with lipoproteins was validated by the cofractionation of hCAP-18 with lipoproteins using two different methods for isolation of lipoproteins from plasma. Furthermore, the level of hCAP-18 in delipidated plasma was <1% of that in normal plasma. Immunoprecipitation of very low, low, and high density lipoprotein particles with anti-apolipoprotein antibodies resulted in coprecipitation of hCAP-18. The binding of hCAP-18 to lipoproteins was mediated by the antibacterial C-terminal part of the protein. The binding of hCAP-18 to lipoproteins suggests that lipoproteins may play an important role as a reservoir of this antimicrobial protein. hCAP-18 belongs to the cathelicidins, a group of antimicrobial peptides found in mammalian neutrophils (1Zanetti M. Gennaro R. Romeo D. FEBS Lett. 1995; 374: 1-5Crossref PubMed Scopus (615) Google Scholar). The cathelicidins share a highly conserved N-terminal prosequence that is homologous to cathelin, a protein first isolated from porcine leukocytes (2Ritonja A. Kopitar M. Jerala R. Turk V. FEBS Lett. 1989; 255: 211-214Crossref PubMed Scopus (129) Google Scholar). The active antimicrobial domains of the cathelicidins generally reside in their C termini. The antimicrobial activity is observed only when the C-terminal domain is cleaved from the holoprotein (3Scocchi M. Skerlavaj B. Romeo D. Gennaro R. Eur. J. Biochem. 1992; 209: 589-595Crossref PubMed Scopus (123) Google Scholar, 4Zanetti M. Litteri L. Griffiths G. Gennaro R. Romeo D. J. Immunol. 1991; 146: 4295-4300PubMed Google Scholar, 5Panyutich P. Shi J. Boutz P.L. Zhao C. Ganz T. Infect. Immun. 1997; 65: 978-985Crossref PubMed Google Scholar). The C termini of the cathelicidins show great variability in amino acid sequence, but they are all highly cationic and hydrophobic. The antimicrobial part of many cathelicidins, including the C terminus of hCAP-18 (also named LL-37), has been shown to bind lipopolysaccharide (6Larrick J.W. Michimasa H. Balint R.F. Lee J. Zhong J. Wright S.C. Infect. Immun. 1995; 63: 1291-1297Crossref PubMed Google Scholar). Porcine and bovine neutrophils contain a variety of cathelicidins, whereas hCAP-18 is the only cathelicidin identified in humans (6Larrick J.W. Michimasa H. Balint R.F. Lee J. Zhong J. Wright S.C. Infect. Immun. 1995; 63: 1291-1297Crossref PubMed Google Scholar, 7Agerberth B. Gunne H. Odeberg J. Kogner P. Boman H.G. Gudmundsson G.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 195-199Crossref PubMed Scopus (438) Google Scholar, 8Cowland J.B. Johnsen A.H. Borregaard N. FEBS Lett. 1995; 368: 173-176Crossref PubMed Scopus (300) Google Scholar). hCAP-18 is a major protein of the specific granules of human neutrophils (9Sørensen O. Arnljots K. Cowland J.B. Bainton D.F. Borregaard N. Blood. 1997; 90: 2796-2803Crossref PubMed Google Scholar), but is also present in squamous epithelia (10Frohm Nilsson M. Sandstedt B. Sørensen O. Weber G. Borregaard N. Ståhle-Bäckdahl M. Infect. Immun. 1999; 67: 2561-2566Crossref PubMed Google Scholar) and in keratinocytes during inflammatory skin diseases (11Frohm M. Agerberth B. Ahangari G. Ståhle-Bäckdahl M. Lidén S. Wigzell H. Gudmundsson G.H. J. Biol. Chem. 1997; 272: 15258-15263Crossref PubMed Scopus (676) Google Scholar). Transcripts for hCAP-18 have been found in lung tissue by in situhybridization (12Bals R. Wang X. Zasloff M. Wilson J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9541-9546Crossref PubMed Scopus (620) Google Scholar). We have previously shown that the relative plasma levels of specific granule proteins from neutrophils are very low compared with the levels in circulating neutrophils (<1%) (13Lollike K. Kjeldsen L. Sengeløv H. Borregaard N. Leukemia (Baltimore). 1995; 9: 159-164PubMed Google Scholar). In contrast, the concentration of hCAP-18 in plasma is ∼1.2 μg/ml, which is >20% of the amount present in circulating neutrophils (14Sørensen O. Cowland J.B. Askaa J. Borregaard N. J. Immunol. Methods. 1997; 206: 53-59Crossref PubMed Scopus (171) Google Scholar). In general, neutrophils are activated to release their granule proteins only when present outside the circulation. Thus, degranulation is unlikely to be the cause of this high plasma level of hCAP-18 since other granule proteins localized in the same granule subset would be expected to have equally high plasma levels as hCAP-18. This is not the case. Thus, a specific mechanism must exist to sequester hCAP-18 in the circulation and provide the relatively high concentration of this pro-bactericidal protein in plasma. Since hCAP-18 partitions mainly in the hydrophobic phase during Triton X-114 phase separation (8Cowland J.B. Johnsen A.H. Borregaard N. FEBS Lett. 1995; 368: 173-176Crossref PubMed Scopus (300) Google Scholar), we reasoned that hCAP-18 might partition into lipoprotein particles, possibly in the same way as the bactericidal C terminus of hCAP-18 is believed to insert into the phospholipid bacterial membrane and cause bacterial lysis. In the following, we demonstrate that hCAP-18 is found in plasma complexed with lipoproteins, and we suggest that lipoproteins may serve as a reservoir of a pro-bactericidal substance in plasma. Blood was obtained from healthy volunteers and used to prepare human plasma anticoagulated with EDTA. Specific rabbit anti-hCAP-18 antibodies were generated by immunization of rabbits with recombinant hCAP-18 (14Sørensen O. Cowland J.B. Askaa J. Borregaard N. J. Immunol. Methods. 1997; 206: 53-59Crossref PubMed Scopus (171) Google Scholar). SDS-PAGE (15Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207856) Google Scholar) and immunoblotting (16Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44969) Google Scholar) were performed according to the instructions given by the manufacturer (Bio-Rad). For immunoblotting, polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA) were blocked for 1 h with 5% skimmed milk in PBS after the transfer of proteins from 14% polyacrylamide gels. For visualization of hCAP-18, the polyvinylidene fluoride membranes were incubated overnight with protein A-purified rabbit anti-hCAP-18 antibodies. The following day, the membranes were incubated for 2 h with peroxidase-conjugated porcine antibodies to rabbit immunoglobulins (Dako, Glostrup, Denmark) and visualized by diaminobenzidine/metal concentrate and Stable Substrate Buffer (Pierce). Human neutrophils were isolated from freshly prepared buffy coats as described (17Boyum A. Scand. J. Clin. Lab. Invest. 1968; 21: 77-90Crossref PubMed Scopus (990) Google Scholar). Briefly, after sedimentation with 2% dextran T-500 (Amersham Pharmacia Biotech, Uppsala, Sweden) in isotonic NaCl, the leukocyte-rich supernatant was pelleted and resuspended in saline for subsequent centrifugation on Lymphoprep (Nycomed Pharma A/S, Oslo, Norway) at 400 ×g for 30 min for removal of lymphocytes and monocytes. The remaining erythrocytes were lysed in ice-cold water for 30 s. Tonicity was restored by the addition of 1 volume of 1.8% NaCl. The cells were washed once and resuspended in the desired buffer. All steps were carried out at 4 °C, except sedimentation in dextran, which was carried out at room temperature. Neutrophils were disrupted by nitrogen cavitation after the addition of 5 mm diisopropyl fluorophosphate (Sigma). The post-nuclear supernatant was loaded on a three-layer Percoll gradient (1.05/1.09/1.12 g/ml; Amersham Pharmacia Biotech) (18Kjeldsen L. Sengeløv H. Lollike K. Nielsen M.H. Borregaard N. Blood. 1994; 6: 1640-1649Crossref Google Scholar) and centrifuged for 30 min at 37,000 ×g. This resulted in four visible bands. Starting at the bottom, the bands were designated the α-band, which contains the azurophil granules; the β1-band, which contains the specific granules; the β2-band, which contains the gelatinase granules; and the γ-band, which contains the plasma membranes and the secretory vesicles. The β1-band containing specific granules was harvested, and the Percoll removed by ultracentrifugation. The granules were lysed in PBS containing 1% Triton X-100 (Roche Molecular Biochemicals, Heidelberg, Germany), 1 mm phenylmethylsulfonyl fluoride (Sigma), 100 kalikrein inhibitory units/ml aprotinin (Bayer, Leverkusen, Germany), 100 μg/ml leupeptin (Sigma), and 1 mm EDTA (Sigma). The lysate was centrifuged, and the supernatant was applied to an affinity chromatography column with anti-hCAP-18 antibodies immobilized on CNBr-activated Sepharose (Amersham Pharmacia Biotech) as described by the manufacturer. The column was washed extensively, and the bound protein was eluted with 0.2 m glycine HCl (pH 2.5). The purity of the eluted protein was ascertained by SDS-PAGE. Isolated neutrophils from freshly prepared buffy coats were resuspended in Krebs-Ringer phosphate solution (10 mmNaH2PO4/Na2HPO4, 130 mm NaCl, 5 mm KCl, 0.95 mmCaCl2, and 5 mm glucose) at a concentration of 3 × 108 cells/ml. Cells were preincubated at 37 °C for 5 min and then stimulated with 1 μm ionomycin (Calbiochem) for 20 min at 37 °C. The stimulation was stopped by placing the cells on ice. The cells were pelleted by centrifugation, and the supernatant containing the exocytosed material was harvested and stored at −20 °C until further use. A recombinant form of cathelin, the N-terminal part of hCAP-18, was produced using the baculovirus expression vector system. The cDNA for the cathelin part of hCAP-18 was polymerase chain reaction-amplified from a human bone marrow cDNA library (CLONTECH, Palo Alto, CA) using specific primers and was cloned into the pAcGP67(b) vector (Pharmingen, San Diego, CA). The sequence of the construct was checked by DNA sequencing. Recombinant protein was produced by Sf9 cells (Pharmingen) after cotransfection of the cells with recombinant pAcGP67(b) and BaculoGold DNA (Pharmingen). The recombinant protein was harvested from the supernatant of the infected Sf9 cells and purified by affinity chromatography as described above for native hCAP-18. SDS-PAGE and subsequent staining with Coomassie Blue of the purified protein showed a single band of the expected molecular mass. This band reacted with anti-hCAP-18 antibodies in immunoblotting. Recombinant hCAP-18 was produced and purified as described (14Sørensen O. Cowland J.B. Askaa J. Borregaard N. J. Immunol. Methods. 1997; 206: 53-59Crossref PubMed Scopus (171) Google Scholar). Fragments of hCAP-18, exocytosed from neutrophils after stimulation with ionomycin, were affinity-purified on an anti-hCAP-18 antibody column. The eluted material was subjected to anion-exchange chromatography on a MonoQ column using fast protein liquid chromatography (Amersham Pharmacia Biotech). Bound material was eluted with a 0–1 m NaCl gradient in 50 mm Tris (pH 8.0). One peak containing a 14-kDa protein was eluted at 0.2m NaCl. The sample was concentrated on a Centricon 10 microconcentrator (Amicon, Inc., Beverly, MA) and subjected to gel filtration on a Superose 12 column (Amersham Pharmacia Biotech). The protein was re-purified and desalted by reverse-phase HPLC employing a Vydac C4 column (2.1 × 150 mm) equilibrated with 10% solvent B (0.1% trifluoroacetic acid in acetonitrile) and eluted with a 1%/min gradient from solvent A (0.1% trifluoroacetic acid) to solvent B. An aliquot was analyzed by mass spectrometry (see below) using horse myoglobin as an internal standard, whereas the remainder was reduced and derivatized with iodoacetamide, as described by Matsudaira (19Matsudaira P.T. A Practical Guide to Protein and Peptide Purification for Microsequencing. Academic Press, New York1989: 20-23Google Scholar), followed by HPLC purification as described above. The derivatized cathelin was digested with endoproteinase Asp-N (Roche Molecular Biochemicals) as described by the manufacturer. The resulting fragments were separated by HPLC on a Vydac C8 column (2.1 × 150 mm) using the solvents described above. Peak fractions were collected manually. Intact cathelin and proteolytic fragments were subjected to matrix-assisted laser desorption mass spectrometry in a Biflex instrument (Bruker-Franzen) using α-cyano-4-hydroxycinnamic acid as the matrix. Sequence analysis was performed on a 494 A Procise Protein Sequencer (Perkin-Elmer). Immunodiffusion of affinity-purified hCAP-18 from plasma was carried out as described by Ouchterlony and Nilsson (20Ouchterlony Ö. Nilsson L.-A. Handbook of Experimental Immunology. 6. Blackwell Publishers, Oxford1986: 32.1-32.50Google Scholar). LDL was precipitated from plasma as described (21Finley P.R. Schifman R.B. Williams R.J. Lichti D.A. Clin. Chem. 1978; 24: 931-933Crossref PubMed Scopus (365) Google Scholar) with minor modifications. Plasma (0.5 ml) was mixed with 0.05 ml (10 g/liter) of dextran sulfate (Amersham Pharmacia Biotech) in 0.5 mMgCl2 and incubated for 10 min at room temperature, followed by centrifugation at 12,000 × g for 5 min. The supernatant was collected manually, and the pellet was resuspended in 0.5 ml of PBS. Plasma was adjusted to densities of 1.060 and 1.215 by the addition of solid KBr. The 350-μl sample was centrifuged in an Airfuge at 100,000 × g for 2.5 h as described (22Bronzert T.J. Brewer Jr., H.B. Clin. Chem. 1977; 23: 2089-2098Crossref PubMed Scopus (177) Google Scholar). Fractions of 100 μl were collected manually from the top and the bottom, respectively, for analysis of plasma proteins as described below. The density of plasma was adjusted to 1.215 by the addition of solid KBr. Samples of 400 μl were centrifuged for 28 h in an Airfuge at 100,000 × g. Delipidated plasma (200 μl) was collected from the bottom of the tube with a syringe to prevent contamination with lipoproteins from the top phase. Delipidated plasma was dialyzed against PBS to remove the KBr before further use. IgG, IgM, IgA, apolipoprotein A-I (a marker of HDL), apolipoprotein B (a marker of VLDL and LDL), and albumin were quantitated by a semiquantitative enzyme-linked immunosorbent assay. The samples were diluted in 50 mmNa2CO3/NaHCO3 buffer (pH 9.6) and incubated in 96-well flat-bottom immunoplates (Nunc, Roskilde, Denmark) overnight at 4 °C. Unspecific binding was blocked by incubation with 200 μl/well dilution buffer (0.5 m NaCl, 3 mmKCl, 8 mmNa2HPO4/KH2PO4, 1% bovine serum albumin (Sigma), and 1% Triton X-100 (pH 7.2)) for 1 h. Rabbit antibodies (Dako) against the above-mentioned antigens were diluted 2000-fold in dilution buffer and incubated for 2 h. Horseradish peroxidase-labeled goat anti-rabbit antibodies (Dako) were diluted 1000-fold in dilution buffer and incubated for 1 h. The plates were washed three times in wash buffer (0.5 m NaCl, 3 mm KCl, 8 mmNa2HPO4/KH2PO4, and 1% Triton X-100 (pH 7.2)) after each incubation using a Microwash-II (Skatron, Roskilde). The plates were washed once in substrate buffer (0.1 m sodium phosphate and 0.1 m citric acid (pH 5.0)) prior to color development and then incubated with substrate buffer containing 0.04% o-phenylenediamine (Kem-En-Tec, Copenhagen, Denmark) and 0.03% H2O2. 100 μl were added to each well at each incubation step unless otherwise stated. The color development was stopped by the addition of 100 μl of 1 m H2SO4; absorbance was measured at 492 nm in a Multiscan Plus ELISA Reader (Labsystems, Helsinki, Finland); and the concentrations are expressed as absorbance units (read at 492 nm). An arbitrary standard of diluted plasma was used in the experiments with immunoprecipitations and delipidation of plasma. hCAP-18 was measured by enzyme-linked immunosorbent assay as described previously (14Sørensen O. Cowland J.B. Askaa J. Borregaard N. J. Immunol. Methods. 1997; 206: 53-59Crossref PubMed Scopus (171) Google Scholar). Plasma was diluted with 1 volume of PBS. A 200-μl sample was applied to a Superose 12 column. Fractions of 0.5 ml were collected and analyzed for their content of hCAP-18, apolipoproteins B and A-I, and IgA. Molecular mass standards (Amersham Pharmacia Biotech) and endogenous IgA were used to estimate the molecular sizes of the plasma proteins investigated. Antibodies against apolipoproteins A-I and B, normal rabbit immunoglobulins, and hCAP-18 were immobilized on CNBr-activated Sepharose. Plasma was diluted 200-fold in PBS, and 400 μl were incubated with 40 μl of antibodies coupled to Sepharose. The Sepharose particles were pelleted by centrifugation after 4 h of incubation at room temperature. The supernatants were aspirated, and the pellets were washed three times in PBS before elution with glycine HCl (0.2 m, pH 2.5). The Sepharose beads were pelleted again, and the eluted material was aspirated and neutralized by the addition of 2 m Tris-HCl (pH 8). Protein concentrations were measured in the supernatant and pellet after immunoprecipitation. The fractions containing the highest concentration of hCAP-18 after gel filtration of plasma (fractions 17 and 23) were diluted 5-fold in PBS with 1% bovine serum albumin, and immunoprecipitation was performed as described above. When hCAP-18 isolated from the specific granules of neutrophils was applied to molecular sieve chromatography on a Superose 12 column, a major peak of hCAP-18 was found in fraction 29 (∼15–30 kDa) as expected for monomeric hCAP-18. When isolated hCAP-18 was subjected to chromatography in the presence of HSA, a minor high molecular mass peak of hCAP-18 was observed in fraction 22 (Fig.1 A). When human plasma was subjected to gel filtration, two major high molecular mass peaks of hCAP-18 were observed at fraction 17 (void volume) and fraction 22 (150 kDa), and no hCAP-18 was found at 15–30 kDa (Fig. 1 A). This indicates that the high molecular mass complexes of hCAP-18 found in plasma were not caused by self-aggregation of hCAP-18. When plasma was separated by SDS-PAGE under reducing and nonreducing conditions, followed by immunoblotting with anti-hCAP-18 antibodies, the only band observed was at the expected molecular mass of 18 kDa (14Sørensen O. Cowland J.B. Askaa J. Borregaard N. J. Immunol. Methods. 1997; 206: 53-59Crossref PubMed Scopus (171) Google Scholar). This indicates that hCAP-18 is bound noncovalently to a high molecular mass component present in plasma. To identify the nature of these high molecular mass complexes, hCAP-18 was isolated from plasma by affinity chromatography using anti-hCAP-18 antibodies. The resulting eluate contained several proteins, including hCAP-18 and apolipoproteins, as judged by immunodiffusion and SDS-PAGE (data not shown). It has previously been found that hCAP-18 from neutrophil granules is very hydrophobic and partitions into the Triton-rich (hydrophobic) phase during phase separation of neutrophil granules with Triton X-114 (8Cowland J.B. Johnsen A.H. Borregaard N. FEBS Lett. 1995; 368: 173-176Crossref PubMed Scopus (300) Google Scholar). We therefore reasoned that hCAP-18 might be associated with lipoproteins. Analysis of the fractions obtained by gel filtration of plasma showed that the low molecular mass peak of hCAP-18 co-localized with apolipoprotein A-I and that the high molecular mass peak co-localized with apolipoprotein B (Fig. 1 B). To investigate this further, lipoproteins were isolated from plasma, and their content of hCAP-18 was determined. 80% of hCAP-18 and almost 100% of apolipoprotein B, but no IgG, IgM, albumin, or apolipoprotein A-I present in plasma, coprecipitated with LDL/VLDL (by the dextran sulfate method) (Fig. 2 A). To ensure that hCAP-18 itself is not precipitated by this method, a sample of purified hCAP-18 was precipitated under the same conditions. No significant precipitation of hCAP-18 was found (Fig.2 B). Ultracentrifugation was used to further examine the association of plasma hCAP-18 with lipoproteins. When plasma was subjected to ultracentrifugation at a density of 1.060, VLDL and LDL were found in the top fraction, and HDL was found in the bottom fraction; hCAP-18 was found in both the HDL- and LDL/VLDL-enriched fractions (data not shown). When the density was increased to 1.215, >95% of hCAP-18 was found in the top fraction, together with VLDL, LDL, and HDL (Fig. 3). Common plasma proteins like IgG and albumin were more evenly distributed in the fractions at all densities examined. Thus, hCAP-18 in plasma was found to partition with lipoproteins obtained by two different separation procedures. As an additional control, plasma was delipidated through a 28-h ultracentrifugation after adjustment of the density to 1.215 with solid KBr. Less than 1% of the original concentration of hCAP-18 was found in plasma after delipidation, whereas the concentrations of the common plasma proteins like albumin, IgG, and IgM were increased (TableI). A small amount of hCAP-18 was incubated with delipidated plasma for 2 h at 37 °C. hCAP-18 in delipidated plasma was then subjected either to gel filtration or to ultracentrifugation (after adjustment of the density to 1.215). In the gel filtration experiment, exogenous hCAP-18 was found in the low molecular mass fractions (Fig. 1 C). Following ultracentrifugation of the delipidated plasma, the added hCAP-18 was present at the same concentrations in the top and bottom fractions (Table I), as expected when no formation of complexes takes place.Table IUltracentrifugation of plasma (d = 1.215)FractionhCAP-18ApoA-IApoBAlbuminIgGIgMhCAP-18 in delipidated plasmaBottom fraction0.37.21.683.168.554.249.9Middle fraction35.919.622.91329.543.525Top fraction63.873.275.53.922.325.2All concentrations are given as percent of the total amount. The volume of the samples was 400 μl. After centrifugation, 200 μl were collected from the bottom (bottom fraction) and 100 μl from the top (top fraction); the remaining 100 μl were the middle fraction. The bottom fraction represents delipidated plasma. In the last column, hCAP-18 was incubated with delipidated plasma (dialyzed against PBS), followed by adjustment of the density and ultracentrifugation. Open table in a new tab All concentrations are given as percent of the total amount. The volume of the samples was 400 μl. After centrifugation, 200 μl were collected from the bottom (bottom fraction) and 100 μl from the top (top fraction); the remaining 100 μl were the middle fraction. The bottom fraction represents delipidated plasma. In the last column, hCAP-18 was incubated with delipidated plasma (dialyzed against PBS), followed by adjustment of the density and ultracentrifugation. To further substantiate the association of hCAP-18 with lipoproteins in plasma, immunoprecipitation was performed with antibodies against hCAP-18, apolipoprotein A-I (HDL), and apolipoprotein B (VLDL and LDL). 46% of hCAP-18 was found in the apolipoprotein B precipitate (n = 4, S.D. = 10.5), and 17.3% was found in the apolipoprotein A-I precipitate (n = 4, S.D. = 12.2). It was not possible, however, to immunoprecipitate all of the lipoproteins from plasma without increasing the antibody concentration to an extent that resulted in high unspecific precipitation. No hCAP-18 was found after precipitation with preimmune rabbit immunoglobulins, whereas >95% of hCAP-18 was precipitated with anti-hCAP-18 antibodies. Immunoprecipitation of hCAP-18 present in plasma resulted in coprecipitation of 30% of LDL/VLDL (n = 4, S.D. = 15.2) and 12.4% of HDL (n = 4, S.D. = 10.8), indicating that not all lipoproteins in plasma are associated with hCAP-18. The association of hCAP-18 with apolipoproteins during gel filtration of plasma was investigated by immunoprecipitation of the fractions that contained peak concentrations of hCAP-18. No hCAP-18 was precipitated from fraction 17 (void volume), but 48.3% (n = 4, S.D. = 10.3) of hCAP-18 was precipitated with anti-apolipoprotein A-I antibodies from fraction 23 (150 kDa). 72.9% of hCAP-18 in fraction 17 was precipitated with anti-apolipoprotein B antibodies (n = 4, S.D. = 19.8), and 40.5% was precipitated in fraction 23 (n = 3, S.D. = 11.5). All of apolipoproteins A-I and B were precipitated, respectively. The results of the specific lipoprotein isolation and the immunoprecipitation indicate that 80% of hCAP-18 is bound to LDL or VLDL. The 20% of hCAP-18 in plasma that is not isolated with LDL/VLDL-specific precipitation is bound to HDL and represents the amount of hCAP-18 that was precipitated with anti-lipoprotein A-I antibodies in fractions with a molecular mass of ∼150 kDa after gel filtration of plasma. To study the nature of the binding between lipoproteins and hCAP-18, gel filtration of plasma was performed either with 3 m KSCN or at pH 4.5. The high molecular mass complexes were not dissociated by 3m KSCN (Fig. 4) or by lowering the pH to 4.5. This indicates that the binding of hCAP-18 to lipoproteins in plasma is not due to electrostatic interactions, but is caused by hydrophobic interactions. Different amounts of hCAP-18 purified from neutrophils were added to EDTA-treated plasma and incubated for 2 h at 37 °C, followed by gel filtration. The fractions were subsequently analyzed for their content of hCAP-18. More than 85% of hCAP-18 was still found in the high molecular mass complexes even when >20 times the endogenous amount of hCAP-18 was added to the plasma (Fig. 5). The antimicrobial active domain of hCAP-18 (LL-37) is cleaved off during exocytosis from human neutrophils (23Gudmundsson G.H. Agerberth B. Odeberg J. Bergman T. Olsson B. Salcedo R. Eur. J. Biochem. 1996; 238: 325-332Crossref PubMed Scopus (462) Google Scholar). Immunoblotting (with anti-hCAP-18 antibodies) of the exocytosed material revealed three bands with apparent molecular masses of 18, 14, and 4 kDa (Fig. 6, lane A). The molecular mass of hCAP-18, calculated from the amino acid sequence, is 16 kDa, 11.5 kDa for cathelin and 4.5 kDa for LL-37. To confirm that the 14-kDa band seen by immunoblotting was the cathelin part of hCAP-18, the exocytosed material from neutrophils was affinity-purified on an anti-hCAP-18 antibody column. As the different parts of hCAP-18 have different pI values (with the holoprotein and LL-37 being very cationic and the cathelin part being anionic), the eluate obtained from the antibody column was subjected to anion-exchange chromatography. The 14-kDa fragment of hCAP-18 was eluted at 0.2 m NaCl and further purified and desalted by gel filtration and reverse-phase HPLC. The N terminus of hCAP-18 (and of the cathelin part) is blocked for protein sequence analysis (8Cowland J.B. Johnsen A.H. Borregaard N. FEBS Lett. 1995; 368: 173-176Crossref PubMed Scopus (300) Google Scholar). Fragments of the 14-kDa protein were therefore generated by cleavage with endoproteinase Asp-N, separated by HPLC, and identified by mass spectrometry (TableII). Endoproteinase Asp-N was chosen to recover both an N- and a C-terminal fragment so that both termini of the protein could be identified. It was expected that several of the small very hydrophilic fragments were not retained on the HPLC column, as was also observed (Table II). All the fragments generated were from the cathelin part of hCAP-18, and the last fragment detected (DNKRFA) was consistent with the C terminus of cathelin (Fig.7). Furthermore, the molecular mass of the purified intact 14-kDa fragment was determined by mass spectrometry. The protein was heterogeneous, showing three peaks at 11,910, 12,178, and 12,462 Da, respectively, compared with 11,520 Da calculated for the cathelin part of hCAP-18 (residues 1–103 of the holoprotein) (Fig. 7). The heterogeneity of the protein and the discrepancy from the calculated value remain unexplained, but do suggest either a post-translational modification located in one or more of the non-recovered fragments or a non-consensus cleavage by Asp-N after the C-terminal alanine (Fig. 7). Thus, the data obtained by mass spectrometry and the sequences from the Asp-N-derived fragments identified the 14-kDa fragment as the cathelin part of hCAP-18. To substantiate that the 4-kDa fragments seen by immunoblotting represented the antibacterial C terminus of hCAP-1" @default.
• W2067907816 created "2016-06-24" @default.
• W2067907816 creator A5006196652 @default.
• W2067907816 creator A5025006519 @default.
• W2067907816 creator A5025400563 @default.
• W2067907816 creator A5033986551 @default.
• W2067907816 creator A5062477843 @default.
• W2067907816 date "1999-08-01" @default.
• W2067907816 modified "2023-10-18" @default.
• W2067907816 title "The Human Antibacterial Cathelicidin, hCAP-18, Is Bound to Lipoproteins in Plasma" @default.
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