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Kallikrein-Mediated Proteolysis Regulates the Antimicrobial Effects of Cathelicidins in Skin

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Kallikrein-Mediated Proteolysis Regulates the Antimicrobial Effects of Cathelicidins in Skin The FASEB Journal • Research Communication Kallikrein-mediated proteolysis regulates the antimicrobial effects of cathelicidins in skin Kenshi Yamasaki,* Ju¨rgen Schauber,* Alvin Coda,* Henry Lin,* Robert A. Dorschner,* Norman M. Schechter,† Chrystelle Bonnart,‡ Pascal Descargues,‡ Alain Hovnanian,‡,§ and Richard L. Gallo*,1 *Division of Dermatology, University of California, San Diego, and VA San Diego Health Care System, San Diego, California, USA; †Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, USA; ‡Inserm, U563, Toulouse, F-31300, France; Universite´ Paul-Sabatier, Toulouse, France, and §CHU Purpan, Department of Genetics, France ABSTRACT The presence of cathelicidin antimicro- the presence of cathelicidin correlates with the ability bial peptides provides an important mechanism for of the host to effectively mount a defense against prevention of infection against a wide variety of micro- infection. Cathelicidin deficiency has also been re- bial pathogens. The activity of cathelicidin is controlled ported in Kostmann syndrome and is associated with by enzymatic processing of the proform (hCAP18 in severe congenial neutropenia and frequent oral infec- humans) to a mature peptide (LL-37 in human neutro- tion (9). Although antimicrobial peptides such as the phils). In this study, elements important to the process- cathelicidins represent an evolutionarily ancient form ing of cathelicidin in the skin were examined. Unique of immune response, current evidence strongly sup- cathelicidin peptides distinct from LL-37 were identi- ports the conclusion that these molecules are an inte- fied in normal skin. Through the use of selective gral element of human immunity (10). Antimicrobial inhibitors, SELDI-TOF-MS, Western blot, and siRNA, peptides such as cathelicidins act both to kill microbes the serine proteases stratum corneum tryptic enzyme and to initiate or modify other cellular immune events. (SCTE, kallikrein 5) and stratum corneum chymotryptic As such, these molecules have been alternatively re- protease (SCCE, kallikrein 7) were shown to control ferred to as “alarmins” (11). Due to this activity on the activation of the human cathelicidin precursor protein host, strict control of cathelicidin expression and func- hCAP18 and also influence further processing to tion is necessary to restrict activity of the peptide to smaller peptides with alternate biological activity. The conditions where maximal defense against microbial importance of this serine protease activity to antimicro- invasion is required. bial activity in vivo was illustrated in SPINK5-deficent The nascent cathelicidin protein is inactive and mice that lack the serine protease inhibitor LEKTI. consists of an N-terminal cathelin domain that is con- Epidermal extracts of these animals show a significant served among mammalian species and a C-terminal increase in antimicrobial activity compared with con- domain encoding the mature cathelicidin peptide. A trols, and immunoabsorption of cathelicidin dimin- comparison of cathelicidin peptides purified from var- ished antimicrobial activity. These observations demon- ious species or predicted by their respective cDNA strate that the balance of proteolytic activity at an sequences has revealed that these potent antimicrobial epithelial interface will control innate immune de- molecules are variable sequences of 20 to 40 amino fense.—Yamasaki, K., Schauber, J., Coda, A., Lin, H., acids in the C-terminal domain. In humans and mice Dorschner, R. A., Schechter, N. M., Bonnart, C., Des- the cathelicidin peptides are alpha-helical, cationic, cargues, P., Hovnanian, A., Gallo, R. L. Kallikrein- and amphipathic. These properties enable association mediated proteolysis regulates the antimicrobial effects and integration with negatively charged cell mem- of cathelicidins in skin. FASEB J. 20, 2068–2080 (2006) branes. Expression of cathelicidins, such as the human LL-37 Key Words: antimicrobial peptide ⅐ stratum corneum tryptic released from neutrophils, is regulated by transcrip- enzyme ⅐ stratum corneum chymotryptic protease ⅐ lympho-epi- tional and post-transcriptional processing. Cathelicidin thelial Kazal-type related inhibitor ⅐ SELDI-TOF-MS transcript and total protein abundance in vivo is in- duced by infection (12), inflammation (13), wounding (14), and differentiation (15, 16). In humans, 1,25- Cathelicidins are an antimicrobial gene family iden- dihydroxyvitamin D is a direct inducer of cathelicidin tified in mammals (1), birds (2), and fish (3). They are 3 expression (17) whereas in mice the cathelicidin best known for their action as innate antimicrobials that protect the host against infection by Gram-positive bacteria (4), Gram-negative bacteria (5, 6), and some 1Correspondence: MC 9111B, 3350 La Jolla Village Dr., San viruses (7). Analysis of several animal models (4), and Diego, CA 92161, USA. E-mail: [email protected] human clinical conditions (8), have demonstrated that doi: 10.1096/fj.06-6075com 2068 0892-6638/06/0020-2068 © FASEB Downloaded from www.fasebj.org by (2600:1700:3900:b30:347e:6b13:5753:a1f7) on February 21, 2020. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 2068-2080. mCRAMP (1) (mouse cathelin-related antimicrobial extracted with 100 ␮lof1ϫ radio-immunoprecipitation assay peptide) expression is dependent on HIF-1a (hypoxia- (RIPA) buffer (50 mM HEPES, 150 mM NaCl, 0.05% SDS, inducible factor 1, alpha) (18). Although these stimuli 0.25% deoxycholate, 0.5% Nonidet P-40, pH 7.4) with pro- teinase inhibitor mixture (complete EDTA-free; Roche, Indi- modify cathelicidin mRNA abundance, factors that anapolis, IN, USA). Samples were kept in –20°C until further regulate the translation and final activation of catheli- analysis. All sample acquisitions, including the skin biopsies, cidin are unclear. Recent observations of cathelicidins were approved by the Committee on Investigations Involving in vivo suggest that the final proteolytic processing of Human Subjects of the University of California, San Diego. cathelicidin dictates antimicrobial or alarmin activity Protein chips (RS100 protein chip array, Ciphergen Bio- ␮ (19). systems, Fremont, CA, USA) were coated with 4 l of rabbit The nascent human cathelicidin gene product is anti-LL-37 antibody (Ab) (0.73 mg/ml) for2hatroom temperature, followed by blocking with 0.5 M etahnolamine designated hCAP18 (human 18-kDa cationic antimicro- in PBS (pH 8.0). After washing three times with PBS/0.5% bial protein) (20). hCAP18 has been shown to be Triton X, protein chips were assembled in the Bioprocessor™ processed to the antimicrobial peptide LL-37 by pro- reservoir, and samples (50 ␮l) were applied and incubated for teinase-3 in neutrophils (21). The prostate-derived pro- 2 h at room temperature. Protein chips were washed three tease gastricsin can also process hCAP18 to ALL-38 times with 1 ϫ RIPA buffer, twice with PBS/0.5% Triton (22). At the skin surface, unidentified proteases present X-100, and three times with PBS, followed by soaking in 10 in human sweat can cleave LL-37 to smaller peptides mM HEPES buffer, then air dried. A half microliter of energy absorbance molecule (50%-saturated alpha-cyano-4-hydroxy such as RK-31, KS-30, and KR-20 (23). The native cinnamic acid in 50% acetonitrile, 0.5% trifluoric acid) was cathelicidin peptides at the skin surface are unknown, applied twice, and all spots were completely dried up. Sam- but these smaller peptides generated in vitro have more ples were analyzed on a SELDI mass analyzer PBS II with a potent antimicrobial activity than LL-37. Analysis of linear time-of-flight mass spectrometer (Ciphergen Biosys- alternatively processed human cathelicidin peptides tems) using time-lag focusing. Specificity and accuracy in this has further shown that processing alters the immuno- system were confirmed by several synthetic cathelicidin pep- tides as standards (19, 23). Synthetic LL-37 and KR-20 pep- stimulatory capacity of cathelicidins, eliminating their tides were used as an internal reference. Skin extracts from ability to stimulate interleukin (IL)-8 release (19). three individuals were analyzed and showed similar patterns These observations demonstrate the importance of of cathelicidin peptides. proteolytic processing to the activity of cathelicidins and, by extension, their importance to immune defense Collection and assay of skin proteases in general. Since the presence of cathelicidin in skin is critical for normal microbial defense and innate immunity, we Protease activity at the skin surface was evaluated in sweat sought in the present study to identify the cathelicidins collected as described for collection of human sweat (24). Activity was monitored by EnzCheck® Protease Assay Kit in human skin and define the proteases responsible for green fluorescence (Molecular Probes, Inc., Eugene, OR, cathelicidin activation. We show that novel cathelicidin USA) according to the manufacturer’s instructions. Briefly, peptide forms are present at the skin surface and that 100 ␮l of the aqueous solution collected from the skin surface kallikreins, a family of serine proteases known for their was mixed with 100 ␮l of BODIPY FL casein substrate and influence on the development of the epidermis, are incubated at 37°C for designated periods. Protease activity responsible for their generation. Moreover, we show was monitored as increased fluorescence with SpectraMax GEMINI EM (Molecular Devices Corp., Sunnyvale, CA, USA). that cathelicidin processing is altered
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