Antimicrobial and Protease Inhibitory Functions of the Human Cathelicidin (hCAP18/LL-37) Prosequence
2003; Elsevier BV; Volume: 120; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2003.12132.x
ISSN1523-1747
AutoresMohamed Zaiou, Victor Nizet, Richard L. Gallo,
Tópico(s)Immune Response and Inflammation
ResumoCathelicidins are a class of small cationic peptide antibiotics that are expressed in skin and in other epithelial cells and are an active component of mammalian innate immunity. Human cathelicidin (hCAP18/LL-37) consists of a conserved prosequence called the cathelin-like domain and a C-terminal peptide named LL-37. To date, our understanding of the cathelin-like domain was very limited. To bring insight into the function of this evolutionarily conserved prosequence, we produced recombinant human cathelin-like protein and full-length hCAP18/LL-37 in Escherichia coli. As the cathelin-like protein shares homology with the cystatin family of cysteine protease inhibitors, we first analyzed the effect of the cathelin-like recombinant protein on the cysteine protease cathepsin L. We found that the cathelin-like protein inhibited protease activity. Next, we tested the cathelin-like protein for antimicrobial activity using solid phase radial diffusion and liquid phase killing assays. The cathelin-like prosequence, but not full-length hCAP18/LL-37, killed human pathogens including E. coli and methicillin-resistant Staphylococcus aureus at concentrations ranging from 16 to 32 μM. Together these findings suggest that after proteolytic cleavage the cathelin-like domain can contribute to innate host defense through inhibition of bacterial growth and limitation of cysteine-proteinase-mediated tissue damage. As these dual functions are complementary to the LL-37 peptide released from the C-terminus of full-length hCAP18/LL-37, human cathelicidin represents an elegant multifunctional effector molecule for innate immune defense of the skin. Cathelicidins are a class of small cationic peptide antibiotics that are expressed in skin and in other epithelial cells and are an active component of mammalian innate immunity. Human cathelicidin (hCAP18/LL-37) consists of a conserved prosequence called the cathelin-like domain and a C-terminal peptide named LL-37. To date, our understanding of the cathelin-like domain was very limited. To bring insight into the function of this evolutionarily conserved prosequence, we produced recombinant human cathelin-like protein and full-length hCAP18/LL-37 in Escherichia coli. As the cathelin-like protein shares homology with the cystatin family of cysteine protease inhibitors, we first analyzed the effect of the cathelin-like recombinant protein on the cysteine protease cathepsin L. We found that the cathelin-like protein inhibited protease activity. Next, we tested the cathelin-like protein for antimicrobial activity using solid phase radial diffusion and liquid phase killing assays. The cathelin-like prosequence, but not full-length hCAP18/LL-37, killed human pathogens including E. coli and methicillin-resistant Staphylococcus aureus at concentrations ranging from 16 to 32 μM. Together these findings suggest that after proteolytic cleavage the cathelin-like domain can contribute to innate host defense through inhibition of bacterial growth and limitation of cysteine-proteinase-mediated tissue damage. As these dual functions are complementary to the LL-37 peptide released from the C-terminus of full-length hCAP18/LL-37, human cathelicidin represents an elegant multifunctional effector molecule for innate immune defense of the skin. 7-amido-4-methylcoumarin antimicrobial peptide colony-forming unit methicillin-resistant Staphylococcus aureus trypticase soy broth Small, cationic antimicrobial peptides (AMPs) are naturally occurring antibiotics of the innate immune system. AMPs are widely distributed in animals and plants and are among the most ancient host defense factors (Hoffmann et al., 1999Hoffmann J.A. Kafatos F.C. Janeway C.A. Ezekowitz R.A. Phylogenetic perspectives in innate immunity.Science. 1999; 284: 1313-1318Crossref PubMed Scopus (2069) Google Scholar). Their spectrum of activity includes Gram-positive and Gram-negative bacteria as well as fungi and certain viruses (Boman, 2000Boman H.G. Innate immunity and the normal microflora.Immunol Rev. 2000; 173: 5-16Crossref PubMed Scopus (277) Google Scholar;Zasloff, 2002Zasloff M. Antimicrobial peptides of multicellular organisms.Nature. 2002; 415: 389-395Crossref PubMed Scopus (6269) Google Scholar). As resistance of pathogenic microbes to conventional antibiotics increases, researchers are exploring these endogenous antibiotics as a potential source for new therapies against a variety of infectious diseases. In humans, there are several classes of known AMPs including α-defensins, β-defensins, and cathelicidins. Cathelicidins are found in several mammalian species and accumulating evidence supports a key role for this class of AMPs in innate immune defense (for review seeGennaro and Zanetti, 2000Gennaro R. Zanetti M. Structural features and biological activities of the cathelicidin-derived antimicrobial peptides.Biopolymers. 2000; 55: 31-49Crossref PubMed Scopus (272) Google Scholar;Ramanathan et al., 2002Ramanathan B. Davis E.G. Ross C.R. Blecha F. Cathelicidins: microbicidal activity, mechanisms of action, and roles in innate immunity.Microbes Infect. 2002; 4: 361-372Crossref PubMed Scopus (206) Google Scholar;Zaiou and Gallo, 2002Zaiou M. Gallo R.L. Cathelicidins, essential gene-encoded mammalian antibiotics.J Mol Med. 2002; 80: 549-561Crossref PubMed Scopus (184) Google Scholar). Recently, development and characterization of a cathelicidin knockout mouse provided evidence that endogenous expression of an AMP protects the host against invasive bacterial infection of the skin (Nizet et al., 2001Nizet V. Ohtake T. Lauth X. et al.Innate antimicrobial peptide protects the skin from invasive bacterial infection.Nature. 2001; 414: 454-457Crossref PubMed Scopus (980) Google Scholar). Additional investigations have identified roles for cathelicidins in specific clinical disease states. Production of cathelicidins is induced in response to epithelial wounding or infectious challenge (Dorschner et al., 2001Dorschner R.A. Pestonjamasp V.K. Tamakuwala S. et al.Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus.J Invest Dermatol. 2001; 117: 91-97Crossref PubMed Scopus (461) Google Scholar;Schaller-Bals et al., 2002Schaller-Bals S. Schulze A. Bals R. Increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection.Am J Respir Crit Care Med. 2002; 165: 992-995Crossref PubMed Scopus (189) Google Scholar), or suppressed by the virulence mechanisms of certain bacterial pathogens, e.g., Shigella dysenteriae (Islam et al., 2001Islam D. Bandholtz L. Nilsson J. Wigzell H. Christensson B. Agerberth B. Gudmundsson G. Downregulation of bactericidal peptides in enteric infections: A novel immune escape mechanism with bacterial DNA as a potential regulator.Nat Med. 2001; 7: 180-185Crossref PubMed Scopus (352) Google Scholar). Cathelicidin expression is also differentially affected in certain chronic inflammatory disorders. In psoriasis, cathelicidin levels are elevated and secondary infection is rare (Frohm et al., 1997Frohm M. Agerberth B. Ahangari G. Stahle-Backdahl M. Liden S. Wigzell H. Gudmundsson G.H. The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders.J Biol Chem. 1997; 272: 15258-15263Crossref PubMed Scopus (650) Google Scholar), whereas in atopic dermatitis cathelicidin expression is deficient and bacterial or viral superinfection is common (Ong et al., 2002Ong P.Y. Ohtake T. Brandt C. et al.Endogenous antimicrobial peptides and skin infections in atopic dermatitis.N Engl J Med. 2002; 347: 1151-1160Crossref PubMed Scopus (1550) Google Scholar). Therapeutic benefits of cathelicidin have been demonstrated experimentally, including decreased bacterial colonization of skin wounds following topical administration (Cole et al., 2001Cole A.M. Shi J. Ceccarelli A. Kim Y.H. Park A. Ganz T. Inhibition of neutrophil elastase prevents cathelicidin activation and impairs clearance of bacteria from wounds.Blood. 2001; 97: 297-304Crossref PubMed Scopus (114) Google Scholar) and improved pulmonary bacterial clearance with cathelicidin overexpression through viral gene transfer (Bals et al., 1999aBals R. Weiner D.J. Meegalla R.L. Wilson J.M. Transfer of a cathelicidin peptide antibiotic gene restores bacterial killing in a cystic fibrosis xenograft model.J Clin Invest. 1999; 103: 1113-1117Crossref PubMed Scopus (177) Google Scholar,Bals et al., 1999bBals R. Weiner D.J. Moscioni A.D. Meegalla R.L. Wilson J.M. Augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide.Infect Immun. 1999; 67: 6084-6089Crossref PubMed Google Scholar). Cathelicidin proteins are composed of two distinct domains: an N-terminal “cathelin-like” or “prosequence” domain and the C-terminal domain of the mature AMP. The highly variable C-terminal domains of cathelicidins were among the earliest mammalian AMPs to show potent, rapid, and broad-spectrum killing activity (Zanetti et al., 1990Zanetti M. Litteri L. Gennaro R. Horstmann H. Romeo D. Bactenecins, defense polypeptides of bovine neutrophils, are generated from precursor molecules stored in the large granules.J Cell Biol. 1990; 111: 1363-1371Crossref PubMed Scopus (125) Google Scholar;Gennaro et al., 1998Gennaro R. Scocchi M. Merluzzi L. Zanetti M. Biological characterization of a novel mammalian antimicrobial peptide.Biochim Biophys Acta. 1998; 1425: 361-368Crossref PubMed Scopus (41) Google Scholar). In contrast, the function of the conserved prosequence domain of cathelicidins is less well understood. The term “cathelin-like” derives from the similarity of this sequence with that of cathelin, a 12 kDa protein isolated from porcine neutrophils that shares similarity with the cystatin superfamily of cysteine protease inhibitors (Ritonja et al., 1989Ritonja A. Kopitar M. Jerala R. Turk V. Primary structure of a new cysteine proteinase inhibitor from pig leucocytes.FEBS Lett. 1989; 255: 211-214Abstract Full Text PDF PubMed Scopus (128) Google Scholar;Storici et al., 1996Storici P. Tossi A. Lenarcic B. Romeo D. Purification and structural characterization of bovine cathelicidins, precursors of antimicrobial peptides.Eur J Biochem. 1996; 238: 769-776Crossref PubMed Scopus (53) Google Scholar;Scocchi et al., 1997Scocchi M. Wang S. Zanetti M. Structural organization of the bovine cathelicidin gene family and identification of a novel member.FEBS Lett. 1997; 417: 311-315Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Cathelicidins are expressed in neutrophils and myeloid bone marrow cells (Zanetti et al., 1990Zanetti M. Litteri L. Gennaro R. Horstmann H. Romeo D. Bactenecins, defense polypeptides of bovine neutrophils, are generated from precursor molecules stored in the large granules.J Cell Biol. 1990; 111: 1363-1371Crossref PubMed Scopus (125) Google Scholar;Gallo et al., 1997Gallo R.L. Kim K.J. Bernfield M. Kozak C.A. Zanetti M. Merluzzi L. Gennaro R. Identification of CRAMP, a cathelin-related antimicrobial peptide expressed in the embryonic and adult mouse.J Biol Chem. 1997; 272: 13088-13093Crossref PubMed Scopus (305) Google Scholar;Ganz and Lehrer, 1997Ganz T. Lehrer R.I. Antimicrobial peptides of leukocytes.Curr Opin Hematol. 1997; 4: 53-58Crossref PubMed Scopus (147) Google Scholar;Sorensen et al., 1997Sorensen O. Arnljots K. Cowland J.B. Bainton D.F. Borregaard N. The human antibacterial cathelicidin, hCAP-18, is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils.Blood. 1997; 90: 2796-2803Crossref PubMed Google Scholar) and at most epithelial surfaces (Frohm et al., 1997Frohm M. Agerberth B. Ahangari G. Stahle-Backdahl M. Liden S. Wigzell H. Gudmundsson G.H. The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders.J Biol Chem. 1997; 272: 15258-15263Crossref PubMed Scopus (650) Google Scholar;Harder et al., 1997Harder J. Bartels J. Christophers E. Schroder J.M. A peptide antibiotic from human skin.Nature. 1997; 387: 861Crossref PubMed Scopus (1162) Google Scholar;Bals et al., 1998Bals R. Wang X. Zasloff M. Wilson J.M. The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface.Proc Natl Acad Sci USA. 1998; 95: 9541-9546Crossref PubMed Scopus (584) Google Scholar;Dorschner et al., 2001Dorschner R.A. Pestonjamasp V.K. Tamakuwala S. et al.Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus.J Invest Dermatol. 2001; 117: 91-97Crossref PubMed Scopus (461) Google Scholar), and were the first AMPs discovered in mammalian skin due to their presence in wound fluid (Gallo et al., 1994Gallo R.L. Ono M. Povsic T. Page C. Eriksson E. Klagsbrun M. Bernfield M. Syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide from wounds.Proc Natl Acad Sci USA. 1994; 91: 11035-11039Crossref PubMed Scopus (319) Google Scholar). In the neutrophil, cathelicidins are synthesized as a full-length precursor and targeted to the secondary granules where they are stored. Upon stimulation, the full-length cathelicidin protein is proteolytically processed to unleash the microbicidal activity of the C-terminal peptide from the cathelin-like domain (Zanetti et al., 1991Zanetti M. Litteri L. Griffiths G. Gennaro R. Romeo D. Stimulus-induced maturation of probactenecins, precursors of neutrophil antimicrobial polypeptides.J Immunol. 1991; 146: 4295-4300PubMed Google Scholar;Sorensen et al., 2001Sorensen O.E. Follin P. Johnsen A.H. Calafat J. Tjabringa G.S. Hiemstra P.S. Borregaard N. Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3.Blood. 2001; 97 (3395): 3951Crossref PubMed Scopus (663) Google Scholar). Although the killing activities and biologic functions of mature cathelicidin antimicrobial peptides have been investigated extensively, a satisfactory description and evidence of the function of the conserved cathelin-like prosequence are still unavailable. The structure of this 96–104 residue protein domain is believed to be stabilized by four cysteines engaged in two disulfide bonds (Storici et al., 1996Storici P. Tossi A. Lenarcic B. Romeo D. Purification and structural characterization of bovine cathelicidins, precursors of antimicrobial peptides.Eur J Biochem. 1996; 238: 769-776Crossref PubMed Scopus (53) Google Scholar;Sanchez et al., 2002aSanchez J.F. Hoh F. Strub M.P. Aumelas A. Dumas C. Structure of the cathelicidin motif of the protegrin-3 precursor: structural insights into the activation mechanism of an antimicrobial protein.Structure. 2002; 10: 1363-1370Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar,Sanchez et al., 2002bSanchez J.F. Wojcik F. Yang Y.S. et al.Overexpression and structural study of the cathelicidin motif of the protegrin-3 precursor.Biochemistry. 2002; 41: 21-30Crossref PubMed Scopus (13) Google Scholar). These four cysteines as well as their relative positions are well conserved in all species. The strict evolutionary conservation of this domain and its similarity to cystatins, a family of proteinase inhibitors, suggests it plays a specific and independent biologic function in host defense (Gennaro and Zanetti, 2000Gennaro R. Zanetti M. Structural features and biological activities of the cathelicidin-derived antimicrobial peptides.Biopolymers. 2000; 55: 31-49Crossref PubMed Scopus (272) Google Scholar). To investigate possible function(s) of the cathelin-like domain, we selected the sole human cathelicidin, hCAP18/LL-37 (cationic antibacterial protein of 18 kDa). The C-terminal mature AMP of 37 amino acids (LL-37) of CAP18/LL-37 has been well characterized (Agerberth et al., 1995Agerberth B. Gunne H. Odeberg J. Kogner P. Boman H.G. Gudmundsson G.H. FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis.Proc Natl Acad Sci USA. 1995; 92: 195-199Crossref PubMed Scopus (417) Google Scholar). Here we report prokaryotic expression of recombinant full-length hCAP18/LL-37 and its cathelin-like prosequence. We show that the human cathelin-like domain acts as a cysteine proteinase inhibitor and discover that it exhibits antibacterial activity against pathogens including Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). This antimicrobial activity is distinct from that of the LL-37 peptide. These findings suggest that the cathelin-like domain of hCAP18/LL-37 is a distinct contributor to skin innate host defense through inhibition of both bacterial growth and cysteine-proteinase-mediated tissue damage. Expression plasmids containing the human full-length cathelicidin cDNA (hCAP18/LL-37) residues (31–170) or the cathelin-like domain residues (31–131) were constructed as fusion proteins in the pET-28a vector (Novagen, Madison, WI) using standard methods (Sambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1989Google Scholar). High-fidelity polymerase chain reaction was used to amplify the coding sequence of hCAP18/LL-37 with primers designed from the published sequence (Gudmundsson et al., 1996Gudmundsson G.H. Agerberth B. Odeberg J. Bergman T. Olsson B. Salcedo R. The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes.Eur J Biochem. 1996; 238: 325-332Crossref PubMed Scopus (435) Google Scholar): forward primer P1, 5′-TCC-GAGCTCGACGATGACGATAAGCTGCTGGGTGATTTCTTCCGG-3′, containing a SacI recognition site and enterokinase cleavage site (underlined), and reverse primer P2, 5′-CCGCTCGAGCTAGGACTCT-GTCCTGGGTACAAGATTCCG-3′. For plasmid pET-Cath, we used primer P1 (above) and reverse primer P3, 5′-CCGCTCGAGCTACTAGG-CAAATCTCTTGTTATCCTT-3′. P2 and P3 both contain stop codons and XhoI restriction site extensions. SacI and XhoI digested polymerase chain reaction amplicons were used for unidirectional ligation into pET-28 vector. The pET-hCAP18 and pET-Cath constructs were confirmed by plasmid purification and direct DNA sequencing. pET-hCAP18 or pET-Cath were transformed into protease-deficient E. coli strain BL21 (DE3). Overnight cultures of each in Luria–Bertani broth (LB) (1% bactotryptone, 0.5% yeast extract, 1% NaCl) supplemented with kanamycin (50 μg per ml) were used to inoculate 1 l LB broth and then grown at 37°C with agitation to 0.6 OD at 600 nm. Expression was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a concentration of 0.5 mM. Cells were harvested by centrifugation (6500×g) for 10 min at 4°C and then resuspended in 50 ml ice-cold sonication buffer (0.1 M Tris–HCl pH 8.0; 0.01 M Na2HPO4, 0.1 M NaCl; 0.05 M ethylenediamine tetraacetic acid (EDTA); 0.005 M β-mercaptoethanol) supplemented with the protease inhibitors 0.1% aprotinin and 2 mM phenylmethylsulfonyl fluoride. Cells were disrupted by sonication on ice and the mixture was centrifuged at 20,000×g for 30 min to separate the insoluble material. Recombinant protein solubility was assessed by comparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation of both the supernatant and the pellet fractions. The supernatant fraction containing soluble recombinant proteins was collected and guanidine–HCl and β-mercaptoethanol were added to final concentrations of 6 M and 0.1%, respectively. The pellet resulting from the centrifugation was washed with 50 mM Tris–HCl and 5 mM EDTA. Insoluble full-length hCAP18/LL-37 and cathelin-like proteins were extracted overnight at 4°C with lysis buffer supplemented with 6 M guanidine–HCl and β-mercaptoethanol with a yield of 70%–90% without degradation. The suspension was then centrifuged at 20,000×g for 30 min to remove the remaining insoluble material. The supernatants of both extractions were dialyzed against 200 mM NaCl, 200 mM L-arginine, 10 mM β-mercaptoethanol, and 50 mM Tris–HCl pH 8.0, followed by extensive dialysis against 10 mM Tris–HCl pH 7.5. The solution was centrifuged for 10 min at 15,000×g to remove any precipitate. Proteins were pooled with the soluble fractions obtained earlier. Immobilized metal affinity chromatography was employed. The supernatant was dialyzed against buffer A consisting of 0.5 M NaCl, 0.02 M Na2HPO4 pH 7.5. Proteins were loaded onto an Ni2+–NTA 5 ml His-Trap column (Pharmacia Biotech, Piscataway, NJ) previously equilibrated with buffer A to which 40 mM imidazole was added (flow rate 1 ml per min). The column was washed with 50 volumes of buffer A containing 40 mM imidazole to remove unspecific bound materials and bound proteins were eluted with 500 mM imidazole in buffer A, collecting 1 ml fractions. The elution profile was monitored by separation of samples by SDS-PAGE. Fractions containing proteins of interest were pooled and dialyzed against 0.5 M NaCl, 0.02 M Na2HPO4 pH 7.5 and the purification step was repeated at least twice. Eluted fractions were pooled and were dialyzed against enterokinase buffer (50 mM Tris–HCl pH 8.0, 1 mM CaCl2, 0.1% Tween-20). The upstream 43-residue N-terminal fusion sequence of pET containing the enterokinase recognition sequence DDDDK was cleaved from the recombinant hCAP18/LL-37 and Cath proteins by digestion with enterokinase (obtained as a 1 unit per μl solution from Invitrogen, Carlsbad, CA). The reaction was incubated at 37°C overnight with an enzyme:protein substrate ratio of 1:25. Digested proteins were dialyzed against 10 mM Tris–HCl pH 7.5 and then proteins were applied onto a Superdex-75 column (2.6×100 cm) and eluted using 10 mM phosphate buffer (pH 7.5) containing 150 mM NaCl at room temperature. Fractions containing proteins of interest were collected and dialyzed against 10 mM Tris–HCl pH 7.5. The purity of cathelin-like or hCAP18/LL-37 protein was first confirmed by SDS-PAGE followed by Coomassie blue and silver staining. For Western blot, protein was separated by SDS-PAGE and was then transferred to nitrocellulose membranes using the Bio-Rad system. Membranes were blocked for 1 h at room temperature with 0.1% low fat milk in 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.2% Tween-20, and were probed overnight at 4°C with chicken polyclonal anticathelin-like antibodies (1:15,000) to detect cathelin-like protein or with rabbit anti-LL-37 antibodies (1:6000) to detect full-length hCAP18/LL-37, followed by extensive washing. Immunoreactive materials were detected by enhanced chemiluminescence using horseradish peroxidase conjugated antichicken antibodies (1:20,000) or horseradish peroxidase conjugated antirabbit antibodies (1:5000). A MALDI-TOF mass spectrometer (Applied Biosystems, Framingham, MA) was used to analyze purified proteins and further confirm identity and purity. Protein samples were prepared for analysis by mixing in a 1:1 ratio with sinapinic acid matrix (3,5-dimethoxy-4-hydroxycinnamic acid). Calibration was performed using internal standards: bovine insulin, apomyoglobin, and thioredoxin (Core facility at the University of California, San Diego). Proteinase inhibitory activity of recombinant cathelin-like protein was assayed spectrofluorometrically by measuring its inhibitory action against human liver cathepsin L (Calbiochem, CA). For the reaction assay, cathepsin L (0.1 mU) in the assay buffer (340 nM sodium acetate, 60 mM acetic acid, 8 mM dithiothreitol, and 4 mM EDTA, pH 5.5, supplemented with 0.1% BRIJ 35) was preincubated for 2 min at 30°C with 10-6 M cathelin-like protein before adding 20 μM of substrate Z-Phe-Arg-7-amido-4-methylcoumarin (Calbiochem). When hydrolyzed by cathepsin L, this substrate releases highly fluorescent 7-amido-4-methylcoumarin (AMC). AMC intensity was determined using a spectrophotometer at 370 nm excitation and an emission wavelength of 460 nm. One unit was defined as the amount of enzyme that will hydrolyze 1.0 μmol of Z-Phe-Arg-AMC per min at 25°C, pH 5.5. E. coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 8427), Salmonella typhimurium (ATCC 13311), Proteus vulgaris (ATCC 8427), Staphylococcus epidermidis (ATCC 12228), and MRSA (ATCC 33591) isolates were maintained on trypticase soy broth (TSB) agar plates. Individual colonies were selected and cultured overnight in TSB, subcultured once at 1:50 in fresh TSB, and then grown to stationary phase for use in all experiments. The radial diffusion assay was performed as described previously (Steinberg and Lehrer, 1997Steinberg D.A. Lehrer R.I. Designer assays for antimicrobial peptides. Disputing the ‘one-size- fits-all’ theory.Meth Mol Biol. 1997; 78: 169-186PubMed Google Scholar) in 0.5% agarose and 0.75% tryptone brought to ebullition and cooled to 43°C, and then mixed with 100 μl of bacterial suspension and poured into a 10 cm Petri dish. A series of small wells (diameter, 3 mm) were punched in the plate after the agarose solidified. Two microliters of test samples were applied in each well. Plates were incubated at 37°C overnight to allow visible growth of bacteria. Antibacterial activity was indicated by the clear zone (no bacterial growth) around the well. The colony-forming assay (CFU) was performed as described previously (Valore et al., 1996Valore E.V. Martin E. Harwig S.S. Ganz T. Intramolecular inhibition of human defensin HNP-1 by its propiece.J Clin Invest. 1996; 97: 1624-1629Crossref PubMed Scopus (119) Google Scholar). Briefly, bacterial cultures (E. coli or MRSA) were collected at the logarithmic phase of growth in TSB, washed twice with phosphate-buffered saline, pH 7.4, and diluted to 104 CFU per ml in 10 mM phosphate buffer, pH 7.4, Na2HPO4/NaH2PO4–1% TSB (g per l). Forty-five microliters of bacterial suspension were mixed with 5 μl of H2O (control) or with 5 μl of different concentrations of cathelin-like proteins, and the mixture was incubated at 37°C. Every 30 min, a 10 μl aliquot of the reaction mixture was plated directly onto a TSB agar plate and then incubated at 37°C overnight for enumeration of CFU. Data are reported as growth index=final CFU/CFU in initial inoculum. Full-length hCAP18/LL-37 recombinant protein (10 ng) was incubated with 10 mU of human neutrophil elastase (Calbiochem) at 37°C for 30 min. The sample was subsequently boiled in Laemmli sample buffer and run by SDS-PAGE followed by immmunoblot analysis with anticathelin-like and anti-LL37 antibodies. In this study, cathelin-like protein and full-length cathelicidin hCAP18/LL-37 proteins were required for functional analysis. In order to achieve efficient expression of these proteins in E. coli, cDNA encoding full-length hCAP18/LL-37 or cathelin-like domain alone were cloned into the pET 28a(+) expression vector system (Figure 1a). This system generates fusion proteins with an N-terminal peptide of 5 kDa containing a His(6) purification tag. Through primer design, a sequence corresponding to an enterokinase cleavage site, pentapeptide (Asp)4-Lys, was inserted after the fusion domain and before the cDNA of interest. Cultures of BL21 (DE3) bacteria transformed with either pET-hCAP18 or pET-Cath were then used for expression following IPTG induction. Over 70% of recombinant cathelin-like proteins were found in the soluble fraction. Only about 40% of full-length cathelicidin proteins were soluble, however. The remaining protein was found in the insoluble fraction. The expression of cathelicidin proteins with His-tag sequence at their N-terminus allowed for convenient purification from other soluble bacterial proteins using immobilized metal affinity chromatography. Proteins were further purified to homogeneity using size exclusion chromatography. The purity of recombinant cathelin-like protein after elution was first checked by SDS-PAGE (Figure 1b). A band of approximately 16 kDa, which corresponds to the cathelin-like region and the fusion sequence (5 kDa), was detected by Coomassie blue staining (Figure 1b). The identity of the bands of expected size was confirmed by Western blot using antibodies against the cathelin-like domain (Figure 1c). A single band of approximately 32 kDa was also detected that probably corresponds to a homodimer of cathelin-like protein. These results demonstrated the effectiveness of the expression system used and recovery after purification (10–15 mg per l). Full-length hCAP18/LL-37 was identically purified and confirmed by identical techniques (data not shown). Cathelicidin proteins were removed from the N-terminal tag by cleavage with enterokinase. Optimization studies found that enterokinase treatment yielded complete cleavage when digestion was carried out at 37°C overnight. No nonspecific cleavage or degradation was observed during this period as confirmed by Coomassie blue staining of the gel (Figure 1c, lane 2). Following enterokinase cleavage, cathelicidin proteins were further purified by size exclusion chromatography, fractions were analyzed by SDS-PAGE, and then identity and purity were confirmed by mass spectrometry (Figure 1d). Approximately 50% of the preparations of recombinant cathelin-like protein displayed the expected peak at 11167.5 mass units by matrix-assisted laser desorption/ionization in agreement with theoretical mass and full disulfide bond formation of this protein. To further confirm the identity of hCAP18/LL-37, the recombinant protein was treated with elastase to observe if this protein was processed similarly to the native cathelicidin. Elastase treatment generated a band migrating at the same size as synthetic LL-37, and a band at 14 kDa as seen by Western blot using anti-LL-37 antibodies (data not shown). This profile is similar to that observed with the previously reported experiments on native human cathelicidins isolated from neut
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