Inhibition of Serine Proteinases Plasmin, Trypsin, Subtilisin A, Cathepsin G, and Elastase by LEKTI: A Kinetic Analysis † Kenji Mitsudo, ‡ Arumugam Jayakumar, ‡ Ying Henderson, ‡ Mitchell J. Frederick, ‡ Ya’an Kang, ‡ Mary Wang, ‡ Adel K. El-Naggar, § and Gary L. Clayman* ,‡,| Departments of Head and Neck Surgery, Pathology, and Cancer Biology, The UniVersity of Texas M. D. Anderson Cancer Center, 1515 Holcombe BouleVard, Houston, Texas 77030-4095 ReceiVed October 18, 2002; ReVised Manuscript ReceiVed January 27, 2003 ABSTRACT: The human LEKTI gene encodes a putative 15-domain serine proteinase inhibitor and has been linked to the inherited disorder known as Netherton syndrome. In this study, human recombinant LEKTI (rLEKTI) was purified using a baculovirus/insect cell expression system, and the inhibitory profile of the full-length rLEKTI protein was examined. Expression of LEKTI in Sf9 cells showed the presence of disulfide bonds, suggesting the maintenance of the tertiary protein structure. rLEKTI inhibited the serine proteinases plasmin, subtilisin A, cathepsin G, human neutrophil elastase, and trypsin, but not chymotrypsin. Moreover, rLEKTI did not inhibit the cysteine proteinase papain or cathepsin K, L, or S. Further, rLEKTI inhibitory activity was inactivated by treatment with 20 mM DTT, suggesting that disulfide bonds are important to LEKTI function. The inhibition of plasmin, subtilisin A, cathepsin G, elastase, and trypsin by rLEKTI occurred through a noncompetitive-type mechanism, with inhibitory constants (K i ) of 27 ( 5, 49 ( 3, 67 ( 6, 317 (36, and 849 ( 55 nM, respectively. Thus, LEKTI is likely to be a major physiological inhibitor of multiple serine proteinases. We have been employing oligonucleotide microarray analysis to identify differences in gene expression between primary head and neck squamous cell carcinomas and matched nonmalignant biopsy specimens. One of the genes identified as being downregulated in the tumors is LEKTI 1 (SPINK5), which encodes a putative 15-domain serine proteinase inhibitor (1, 2) that has been linked to the autosomal recessive genetic disorder known as Netherton syndrome (2-8). LEKTI was initially cloned following sequence identifica- tion of two polypeptides, HF6478 and HF7556, which were isolated from human blood filtrates (1). The polypeptides turned out to be fragments of a much larger precursor protein, LEKTI, which contains two Kazal-type domains containing six conserved cysteine residues and 13 other domains containing only four of the six cysteines. One of the isolated polypeptides, HF7556, which corresponded to domain 6, inhibited trypsin but not chymotrypsin, elastase, or plasmin (1, 9). No inhibitory function could be assigned to the other polypeptide, HF6478, which corresponded to domain 1 (10). Because of the presence of Kazal-type domains in the translated protein and its pattern of expression in different organs, the gene encoding these polypeptides was named lympho-epithelial Kazal-type-related inhibitor (LEKTI) (1, 9). More recently, a 30 kDa protein of unknown function was isolated from human epidermal keratinocytes and shown to have an N-terminal sequence corresponding to LEKTI domain 8 (11). LEKTI (SPINK5) was also independently cloned by Chavanas et al. (2) as the genetic locus responsible for Netherton syndrome. Mutations in the LEKTI gene cause Netherton syndrome (2-8), which is characterized by congenital ichthyosis, hair shaft abnormalities, immune deficiency, elevated IgE levels, and failure to thrive. Currently, the actual biologic function(s) of LEKTI protein is a subject of speculation. LEKTI mRNA localizes to the upper epidermis and pilosebaceous units of skin (5). Patients with Netherton syndrome have elevated levels of trypsin- like hydrolytic activity in their stratum corneum associated with overdesquamation of corneocytes (5). These findings have led to the hypothesis that LEKTI plays an important † This work was supported in part by National Institutes of Health Independent Award R01 DE-13954 (G.L.C.), National Institutes of Health Specialized Program of Research Excellence (SPORE) grant in Head and Neck Cancer (1P50-CA-97007), M. D. Anderson Cancer Center Support Grant 5P30 CA 16672, the Michael A. O’Bannon Endowment for Cancer Research, the Betty Berry Cancer Research Fund, and funds from the M. D. Anderson Cancer Center’s Tobacco Research Program of the Texas Legislative Tobacco Settlement. The Nitto Foundation of Nagoya, Japan, supports K.M. * To whom correspondence and reprint requests should be ad- dressed: Department of Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center, Box 441, 1515 Holcombe Blvd., Houston, TX 77030-4095. Telephone: (713) 792-6920. Fax: (713) 794- 4662. E-mail: gclayman@mdanderson.org. ‡ Department of Head and Neck Surgery. § Department of Pathology. | Department of Cancer Biology. 1 Abbreviations: LEKTI, ; SPINK5, ; IgE, immunoglobulin E; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide electrophoresis; BSA, bovine serum albumin; DTT, dithiothreitol; RT-PCR, reverse tran- scriptase polymerase chain reaction; PFU, plaque-forming units; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethyl- ammonio]-1-propanesulfonate; EDTA, ethylenediaminotetraacetic acid; TBS, Tris-buffered saline; mAb, monoclonal antibody; IgG, immuno- globulin G; MALDI-MS, matrix-assisted laser desorption ionization mass spectroscopy; IC 50, median inhibitory concentration; SEM, standard error of the mean. 3874 Biochemistry 2003, 42, 3874-3881 10.1021/bi027029v CCC: $25.00 © 2003 American Chemical Society Published on Web 03/14/2003