LEKTI is a 15-domain serine proteinase inhibitor whose defective expression underlies the severe autosomal recessive ichthyosiform skin disease, Netherton syndrome. Here, we show that LEKTI is produced as a precursor rapidly cleaved by furin, generating a variety of single or multidomain LEKTI fragments secreted in cultured keratinocytes and in the epidermis. The identity of these biological fragments (D1, D5, D6, D8 -D11, and D9 -D15) was inferred from biochemical analysis, using a panel of LEKTI antibodies. The functional inhibitory capacity of each fragment was tested on a panel of serine proteases. All LEKTI fragments, except D1, showed specific and differential inhibition of human kallikreins 5, 7, and 14. The strongest inhibition was observed with D8 -D11, toward KLK5. Kinetics analysis revealed that this interaction is rapid and irreversible, reflecting an extremely tight binding complex. We demonstrated that pH variations govern this interaction, leading to the release of active KLK5 from the complex at acidic pH. These results identify KLK5, a key actor of the desquamation process, as the major target of LEKTI. They disclose a new mechanism of skin homeostasis by which the epidermal pH gradient allows precisely regulated KLK5 activity and corneodesmosomal cleavage in the most superficial layers of the stratum corneum.

Desquamation of the stratum corneum is a serine proteasedependent process. Two members of the human tissue kallikrein (KLK) family of (chymo)tryptic-like serine proteases, KLK5 and KLK7, are implicated in desquamation by digestion of (corneo)desmosomes and inhibition by desquamation-related serine protease inhibitors (SPIs). However, the epidermal localization and specificity of additional KLKs also supports a role for these enzymes in desquamation. This study aims to delineate the probable contribution of KLK1, KLK5, KLK6, KLK13, and KLK14 to desquamation by examining their interactions, in vitro, with: 1) colocalized SPI, lympho-epithelial Kazal-type-related inhibitor (LEKTI, four recombinant fragments containing inhibitory domains 1-6 (rLEKTI(1-6)), domains 6 -8 and partial domain 9 (rLEKTI(6 -9)), domains 9 -12 (rLEKTI(9 -12)), and domains 12-15 (rLEKTI(12-15)), secretory leukocyte protease inhibitor, and elafin and 2) their ability to digest the (corneo)desmosomal cadherin, desmoglein 1. KLK1 was not inhibited by any SPI tested. KLK5, KLK6, KLK13, and KLK14 were potently inhibited by rLEKTI(1-6), rLEKTI(6 -9), and rLEKTI(9 -12) with K i values in the range of 2.3-28.4 nM, 6.1-221 nM, and 2.7-416 nM for each respective fragment. Only KLK5 was inhibited by rLEKTI(12-15) (K i ‫؍‬ 21.8 nM). No KLK was inhibited by secretory leukocyte protease inhibitor or elafin. Apart from KLK13, all KLKs digested the ectodomain of desmoglein 1 within cadherin repeats, Ca 2؉ binding sites, or in the juxtamembrane region. Our study indicates that multiple KLKs may participate in desquamation through cleavage of desmoglein 1 and regulation by LEKTI. These findings may have clinical implications for the treatment of skin disorders in which KLK activity is elevated.As the outermost layer of the skin, the stratum corneum functions as the body's main protective barrier against physical and chemical damage, dehydration, and microbial pathogens. Inter-corneocyte cohesion within the stratum corneum depends primarily on corneodesmosomes, structurally modified desmosomes (1-3). Akin to classical desmosomes, corneodesmosomes maintain tissue integrity and mediate cell adhesion via calcium-dependent interactions between two families of desmosomal cadherins, the desmogleins (DSG1-4) 2 and desmocollins 1-3 (4, 5). The most abundant isoforms in the stratum corneum include DSG1, DSG4, and desmocollin-1 (6, 7). As specialized desmosomes, corneodesmosomes also contain a unique glycoprotein constituent, corneodesmosin (3).During normal stratum corneum desquamation, the most superficial corneocytes are shed from the skin surface. This process requires proteolysis of the corneodesmosomal adhesion molecules DSG1 (8, 9), desmocollin-1 (10), and corneodesmosin (11) likely mediated by both trypsin-like and chymotrypsin-like serine proteases (9, 12). To date, serine protease activity in the stratum corneum has been attributed to human tissue kallikreins (KLK; encoded by KLK genes (EC 3.4.21)), a subgroup of 15 secreted serine proteases with (chymo)trypsi...

BRAK/CXCL14 is a CXC chemokine constitutively expressed at the mRNA level in certain normal tissues but absent from many established tumor cell lines and human cancers. Although multiple investigators cloned BRAK, little is known regarding the physiologic function of BRAK or the reason for decreased expression in cancer. To understand the possible significance associated with loss of BRAK mRNA in tumors, we examined the pattern of BRAK protein expression in normal and tumor specimens from patients with squamous cell carcinoma (SCC) of the tongue and used recombinant BRAK (rBRAK) to investigate potential biological functions. Using a peptide-specific antiserum, abundant expression of BRAK protein was found in suprabasal layers of normal tongue mucosa but consistently was absent in tongue SCC. Consistent with previous in situ mRNA studies, BRAK protein also was expressed strongly by stromal cells adjacent to tumors. In the rat corneal micropocket assay, BRAK was a potent inhibitor of in vivo angiogenesis stimulated by multiple angiogenic factors, including interleukin 8, basic fibroblast growth factor, and vascular endothelial growth factor. In vitro, rBRAK blocked endothelial cell chemotaxis at concentrations as low as 1 nmol/L, suggesting this was a major mechanism for angiogenesis inhibition. Although only low affinity receptors for BRAK could be found on endothelial cells, human immature monocyte-derived dendritic cells (iDCs) bound rBRAK with high affinity (i.e., K d , ϳ2 nmol/L). Furthermore, rBRAK was chemotactic for iDCs at concentrations ranging from 1 to 10 nmol/L. Our findings support a hypothesis that loss of BRAK expression from tumors may facilitate neovascularization and possibly contributes to immunologic escape.

We have cloned and sequenced the cDNA coding for human HepG2 acetyl-CoA carboxylase (ACC; EC 6.4

Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the 13-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The synthesis of long-chain fatty acids from acetyl-CoA, malonyl-CoA, and NADPH is a complex process catalyzed by the fatty acid synthase (FAS). In animal tissues, the active synthase is a homodimer of a multifunctional protein that is organized in a head-to-tail fashion, generating two active catalytic centers (1). The seven partial activities and the site for the prosthetic group, 4'-phosphopantetheine (acyl carrier protein), are arranged on the multifunctional protein subunit from the amino to carboxyl termini in the following order: f3-ketoacyl synthase, acetyl-CoA and malonyl-CoA transacylases, dehydratase, enoyl reductase, ketoacyl reductase, acyl carrier protein, and thioesterase (1). Most information about the synthase is derived from nonhuman animal studies, so little is known about the human synthase. Investigators who isolated the enzyme from human biopsy tissues (2-5) or cell lines (6) have shown that, except for the enzyme from the SKBR3 cell line (6), all the human FAS preparations had lower activity than the FASs of other animals. This lower activity of the human FAS is comparable to the activities of related human enzymes that are involved in lipogenesis (e.g., pyruvate dehydrogenase, citrate lyase, and glucose-6-phosphate dehydrogenase), which are lower (by a factor of 4-7) in human tissues than in other animal tissues, implying that lipogenesis in humans is highly repressed (7-9).Here we describe the purification and catalytic properties of Assays of FAS and Its Partial Activities. The synthase activity was assayed by measuring the rate of oxidation of NADPH or the incorporation of radiolabeled acetyl-CoA or malonyl-CoA into palmitate (10). The partial activities of FAS were assayed as described earlier: the acetyl/malonyl transacylases (11), dehydratase (12), ,B-ketoacyl synthase (12), f3-ketoacyl reductase (13), ,B-hydroxyacyl enoyl reductase (12), and thioesterase (14). The f3-ketoacyl synthase activity was also determined by measuring the increase in absorbance at 280 nm due to formation of triacetic acid lactone (15).4'-Phosphopantetheine Content. To determine the 4'-phosphopantetheine content of FAS, we developed a simple and sensitive method based on spectrophotometric measurement of the phenylthiocarbamoyl (PTC) derivative of taurine released after performic acid oxidation and hydrolysis of the protein. PTC-taurine can be separated from other PTC-amino acids by using reverse-phase high-performance liquid chromatography (HPLC) and following the absorbance at 254 nm. In this system,...