A potential role for tissue kallikrein-related peptidases in human cervico-vaginal physiology

Human tissue kallikrein-related peptidases (KLK) are a family of 15 genes located on chromosome 19q13.4 that encode secreted serine proteases with trypsin- and/or chymotrypsin-like activity. Relatively large levels of many KLKs are present in human cervico-vaginal fluid (CVF) and in the supernatant of cultured human vaginal epithelial cells. Many KLKs are also hormonally regulated in vaginal epithelial cells, particularly by glucocorticoids and estrogens. The physiological role of KLK in the vagina is currently unknown; however, analysis of the CVF proteome has revealed clues for potential KLK functions in this environment. Here, we detail potential roles for KLKs in cervico-vaginal physiology. First, we suggest that KLKs play a role in the vagina similar to their role in skin physiology: (1) in the desquamation of vaginal epithelial cells, similar to their activity in the desquamation of skin corneocytes; and (2) in their ability to activate antimicrobial proteins in CVF as they do in sweat. Consequently, we hypothesize that dysregulated KLK expression in the vagina could lead to the development of pathological conditions such as desquamative inflammatory vaginitis. Second, we propose that KLKs may play a role in premature rupture of membranes and pre-term birth through their cleavage of fetal membrane extracellular matrix proteins.     

Human kallikrein-related peptidase 14 (KLK14) is a new activator component of the KLK proteolytic cascade. Possible function in seminal plasma and skin

Human kallikrein-related peptidases (KLKs) are a family of 15 serine proteases mainly known for their biomarker utility in various neoplastic and non-neoplastic diseases. Despite significant progress in understanding their clinical application, little is known about the activation mechanism(s) of this important family of enzymes. Emerging evidence indicates that KLKs are activated in a stepwise manner, which is a characteristic of proteolytic cascades. Thus far, KLK cascades have been implicated in semen liquefaction and skin desquamation. Many members of the KLK family have been reported to be active in seminal plasma and/or skin, suggesting their involvement in common proteolytic cascades. KLK14, in particular, is highly active and has recently been proposed as one of the key trypsin-like proteases involved in skin desquamation. This study aims to elucidate a probable cascade-mediated role of KLK14 by 1) examining KLK14-mediated cleavage of a heptapeptide library encompassing activation sites of the 15 KLKs and 2) verifying activation of certain candidate downstream targets of KLK14 (i.e. pro-KLK1, -KLK3, and -KLK11). Heptapeptides encompassing activation motifs of KLK2, -3, -5, and -11 were cleaved with a high (> or =85%) cleavage efficiency. Activation of these candidates was confirmed using full-length recombinant proteins. Pro-KLK11, -KLK3, and -KLK1 were rapidly activated in a concentration-dependent manner. Pro-KLK3 regulation was bidirectional because activation was followed by inactivation via internal cleavage of active KLK3. We are proposing a putative cascade model, operating through multiple KLKs. Identification of novel members of such proteolytic cascades will aid in further defining mechanisms involved in seminal/skin homeostasis.     

Proteomic analysis of human cervico-vaginal fluid

Human cervico-vaginal fluid (CVF) is a mixture of fluids originating from the vagina, cervix, endometrium, and oviduct. CVF has been shown to play an important role in protecting the vagina from infection. We used "bottom-up" proteomic approaches to characterize the protein repertoire of human CVF. We applied two different sample prefractionation methods, one-dimensional-SDS-PAGE (1D-SDS-PAGE) and strong cation-exchange chromatography, followed by LC-MS/MS and bioinformatic analysis. We identified a total of 685 proteins. Strong cation-exchange chromatography prefractionation resulted in a larger number of proteins identified when compared with 1D-SDS-PAGE. Extracellular or membrane proteins made up 30% of the proteins identified, according to Genome Ontology (GO) classifications. We confirmed the presence of defense-related proteins, such as haptoglobin, defensins, and lactoferrin; and identified new ones such as azurocidin and dermcidin. We also identified many serine and cysteine proteases, including 6 members of the kallikrein family (KLKs 6, 7, 10, 11, 12, and 13). The same KLKs were also confirmed quantitatively by ELISA assays. Knowledge of the CVF proteome will aid in the discovery of potential biomarkers for gynecological malignancies and infections and provide additional clues for its physiological functions.     

A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation

Desquamation of the stratum corneum is a serine protease-dependent 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 Ki 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) (Ki = 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, Ca2+ 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.     

Tissue kallikrein proteolytic cascade pathways in normal physiology and cancer

Human tissue kallikreins (KLKs or kallikrein-related peptidases) are a subgroup of extracellular serine proteases that act on a wide variety of physiological substrates, while they display aberrant expression patterns in certain types of cancer. Differential expression patterns lead to the exploitation of these proteins as new cancer biomarkers for hormone-dependent malignancies, in particular. The prostate-specific antigen or kallikrein-related peptidase 3 (PSA/KLK3) is an established tumor marker for the diagnosis and monitoring of prostate cancer. It is well documented that specific KLK genes are co-expressed in tissues and in various pathologies suggesting their participation in complex proteolytic cascades. Here, we review the currently established knowledge on the involvement of KLK proteolytic cascades in the regulation of physiological and pathological processes in prostate tissue and in skin. It is well established that the activity of KLKs is often regulated by auto-activation and subsequent autolytic internal cleavage leading to enzymatic inactivation, as well as by inhibitory serpins or by allosteric inhibition by zinc ions. Redistribution of zinc ions and alterations in their concentration due to physiological or pathological reasons activates specific KLKs initiating the kallikrein cascade(s). Recent studies on kallikrein substrate specificity allowed for the construction of a kallikrein interaction network involved in semen liquefaction and prostate cancer, as well as in skin pathologies, such as skin desquamation, psoriasis and cancer. Furthermore, we discuss the crosstalks between known proteolytic pathways and the kallikrein cascades, with emphasis on the activation of plasmin and its implications in prostate cancer. These findings may have clinical implications for the underlying molecular mechanism and management of cancer and other disorders in which KLK activity is elevated.     

Kallikrein-5 promotes cleavage of desmoglein-1 and loss of cell-cell cohesion in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) ranks among the top 8 causes of cancer death worldwide, with only a 60% 5-year survival rate, highlighting the need for discovery of novel biomarkers and therapeutic targets. We have previously reported that expression of a panel of serine proteinase kallikreins (KLK 5, 7, 8, and 10) is correlated with formation of more aggressive OSCC tumors in a murine orthotopic OSCC model and is elevated in human OSCC. Current studies focus on understanding the potential role of KLK5 in OSCC progression. In initial studies, KLK levels in malignant OSCC cells (SCC25) were compared with cells from normal oral mucosa (OKF/6) and pre-malignant oral keratinocytes (pp126) using qPCR. A marked elevation of all KLKs was observed in aggressive SCC25 cells relative to OKF/6 cells. In normal skin, KLKs are involved in desquamation during epidermal differentiation via proteolytic cleavage of the desmosomal cadherin component desmoglein 1 (Dsg1). As loss of cell-cell cohesion is prevalent in tumor metastasis, Dsg1 integrity was evaluated. Results show that SCC25 cells exhibit cleavage of Dsg1, which is blocked by proteinase inhibitor treatment as well as by siRNA silencing of KLK5 expression. Furthermore, cell-cell aggregation assays demonstrate that silencing of KLK5 enforces cell-cell adhesion; conversely, overexpression of KLK5 in normal oral mucosal cells (OKF/6) enhances cell dispersal. These data suggest that KLK5 may promote metastatic dissemination of OSCC by promoting loss of junctional integrity through cleavage of desmoglein 1.     

Identification of Lympho-Epithelial Kazal-Type Inhibitor 2 in Human Skin as a Kallikrein-Related Peptidase 5-Specific Protease Inhibitor

Kallikreins-related peptidases (KLKs) are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI). Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink)5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5.     

Isolation of SPINK6 in Human Skin: SELECTIVE INHIBITOR OF KALLIKREIN-RELATED PEPTIDASES*

Kallikrein-related peptidases (KLKs) play a central role in skin desquamation. They are tightly controlled by specific inhibitors, including the lymphoepithelial Kazal-type inhibitor (LEKTI) encoded by SPINK5 and LEKTI-2 encoded by SPINK9. Herein, we identify SPINK6 as a selective inhibitor of KLKs in the skin. Unlike LEKTI but similar to LEKTI-2, SPINK6 possesses only one typical Kazal domain. Its mRNA was detected to be expressed at low levels in several tissues and was induced during keratinocyte differentiation. Natural SPINK6 was purified from human plantar stratum corneum extracts. Immunohistochemical analyses revealed SPINK6 expression in the stratum granulosum of human skin at various anatomical localizations and in the skin appendages, including sebaceous glands and sweat glands. SPINK6 expression was decreased in lesions of atopic dermatitis. Using KLK5, KLK7, KLK8, KLK14, thrombin, trypsin, plasmin, matriptase, prostasin, mast cell chymase, cathepsin G, neutrophil elastase, and chymotrypsin, inhibition with recombinant SPINK6 was detected only for KLK5, KLK7, and KLK14, with apparent K_i values of 1.33, 1070, and 0.5 nm, respectively. SPINK6 inhibited desquamation of human plantar callus in an ex vivo model. Our findings suggest that SPINK6 plays a role in modulating the activity of KLKs in human skin. A selective inhibition of KLKs by SPINK6 might have therapeutic potential when KLK activity is elevated.     

Human tissue kallikreins as promiscuous modulators of homeostatic skin barrier functions

Human tissue kallikreins (KLKs) are the largest family of secreted serine protease endopeptidases encoded by 15 genes clustered on chromosome 19q13.4. Multiple KLK enzymes are co-localized in the upper stratum granulosum and stratum corneum of human epidermis, and in associated appendages such as hair follicle epithelia and sweat glands. Until recently, kallikrein proteolytic activity in the skin was exclusively attributed to KLK5 and KLK7. However, wider cutaneous roles of kallikreins became evident in recent years as the proposal of KLK proteolytic activation cascades emerged. We postulate that these proteolytic enzymes may serve as promiscuous mediators of different skin barrier functions, since they are capable of proteolysing different substrates that govern skin desquamation, antimicrobial defense, and lipid permeability. Growing evidence now attests to potential kallikrein involvement in skin inflammation, pigmentation, and tumor suppression via their ability to target proteinase-activated receptor signaling pathways. Current knowledge on kallikrein roles in skin physiology and pathobiology is described in this review.     

Activation profiles of human kallikrein-related peptidases by proteases of the thrombostasis axis

The human kallikrein-related peptidases (KLKs) comprise 15 members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their N-terminal pro-peptide. This processing is a key step in the regulation of KLK function. Much recent work has been devoted to elucidating the potential for activation cascades between members of the KLK family, with physiologically relevant KLK regulatory cascades now described in skin desquamation and semen liquefaction. Despite this expanding knowledge of KLK regulation, details regarding the potential for functional intersection of KLKs with other regulatory proteases are essentially unknown. To elucidate such interaction potential, we have characterized the ability of proteases associated with thrombostasis to hydrolyze the pro-peptide sequences of the KLK family using a previously described pro-KLK fusion protein system. A subset of positive hydrolysis results were subsequently quantified with proteolytic assays using intact recombinant pro-KLK proteins. Pro-KLK6 and 14 can be activated by both plasmin and uPA, with plasmin being the best activator of pro-KLK6 identified to date. Pro-KLK11 and 12 can be activated by a broad-spectrum of thrombostasis proteases, with thrombin exhibiting a high degree of selectivity for pro-KLK12. The results show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, demonstrating the potential for important regulatory interactions between these two major protease families.     

Evolutionary history of tissue kallikreins

The gene family of human kallikrein-related peptidases (KLKs) encodes proteins with diverse and pleiotropic functions in normal physiology as well as in disease states. Currently, the most widely known KLK is KLK3 or prostate-specific antigen (PSA) that has applications in clinical diagnosis and monitoring of prostate cancer. The KLK gene family encompasses the largest contiguous cluster of serine proteases in humans which is not interrupted by non-KLK genes. This exceptional and unique characteristic of KLKs makes them ideal for evolutionary studies aiming to infer the direction and timing of gene duplication events. Previous studies on the evolution of KLKs were restricted to mammals and the emergence of KLKs was suggested about 150 million years ago (mya). In order to elucidate the evolutionary history of KLKs, we performed comprehensive phylogenetic analyses of KLK homologous proteins in multiple genomes including those that have been completed recently. Interestingly, we were able to identify novel reptilian, avian and amphibian KLK members which allowed us to trace the emergence of KLKs 330 mya. We suggest that a series of duplication and mutation events gave rise to the KLK gene family. The prominent feature of the KLK family is that it consists of tandemly and uninterruptedly arrayed genes in all species under investigation. The chromosomal co-localization in a single cluster distinguishes KLKs from trypsin and other trypsin-like proteases which are spread in different genetic loci. All the defining features of the KLKs were further found to be conserved in the novel KLK protein sequences. The study of this unique family will further assist in selecting new model organisms for functional studies of proteolytic pathways involving KLKs.     

Activation of hepatocyte growth factor activator zymogen (pro-HGFA) by human kallikrein 1-related peptidases

Hepatocyte growth factor activator (HGFA) is a serine protease and a potent activator of prohepatocyte growth factor/scatter factor (pro-HGF/SF), a multifunctional growth factor that is critically involved in tissue morphogenesis, regeneration, and tumor progression. HGFA circulates as a zymogen (pro-HGFA) and is activated in response to tissue injury. Although thrombin is considered to be an activator of pro-HGFA, alternative pro-HGFA activation pathways in tumor microenvironments remain to be identified. In this study, we examined the effects of kallikrein 1-related peptidases (KLKs), a family of extracellular serine proteases, on the activation of pro-HGFA. Among the KLKs examined (KLK2, KLK3, KLK4 and KLK5), we identified KLK4 and KLK5 as novel activators of pro-HGFA. Using N-terminal sequencing, the cleavage site was identified as the normal processing site, Arg407-Ile408. The activation of pro-HGFA by KLK5 required a negatively charged substance such as dextran sulfate, whereas KLK4 could process pro-HGFA without dextran sulfate. KLK5 showed more efficient pro-HGFA processing than KLK4, and was expressed in 50% (13/25) of the tumor cell lines examined. HGFA processed by these KLKs efficiently activated pro-HGF/SF, and led to cellular scattering and invasion in vitro. The activities of both KLK4 and KLK5 were strongly inhibited by HGFA inhibitor type 1, an integral membrane Kunitz-type serine protease inhibitor that inhibits HGFA and other pro-HGF/SF-activating proteases. These data suggest that KLK4 and KLK5 mediate HGFA-induced activation of pro-HGF/SF within tumor tissue, which may thereafter trigger a series of events leading to tumor progression via the MET receptor.     

Non-combinatorial library screening reveals subsite cooperativity and identifies new high-efficiency substrates for kallikrein-related peptidase 14

An array of substrates link the tryptic serine protease, kallikrein-related peptidase 14 (KLK14), to physiological functions including desquamation and activation of signaling molecules associated with inflammation and cancer. Recognition of protease cleavage sequences is driven by complementarity between exposed substrate motifs and the physicochemical signature of an enzyme's active site cleft. However, conventional substrate screening methods have generated conflicting subsite profiles for KLK14. This study utilizes a recently developed screening technique, the sparse matrix library, to identify five novel high-efficiency sequences for KLK14. The optimal sequence, YASR, was cleaved with higher efficiency (k(cat)/K(m)=3.81 ± 0.4 × 10(6) M(-1) s(-1)) than favored substrates from positional scanning and phage display by 2- and 10-fold, respectively. Binding site cooperativity was prominent among preferred sequences, which enabled optimal interaction at all subsites as indicated by predictive modeling of KLK14/substrate complexes. These simulations constitute the first molecular dynamics analysis of KLK14 and offer a structural rationale for the divergent subsite preferences evident between KLK14 and closely related KLKs, KLK4 and KLK5. Collectively, these findings highlight the importance of binding site cooperativity in protease substrate recognition, which has implications for discovery of optimal substrates and engineering highly effective protease inhibitors.     

Major role of human KLK14 in seminal clot liquefaction

Liquefaction of human semen involves proteolytic degradation of the seminal coagulum and release of motile spermatozoa. Several members of human kallikrein-related peptidases (KLKs) have been implicated in semen liquefaction, functioning through highly regulated proteolytic cascades. Among these, KLK3 (also known as prostate-specific antigen) is the main executor enzyme responsible for processing of the primary components of semen coagulum, semenogelins I and II. We have recently identified KLK14 as a potential activator of KLK3 and other KLKs. This study aims to elucidate the cascade-mediated role of KLK14 ex vivo. KLK14 expression was significantly lower (p = 0.0252) in individuals with clinically delayed liquefaction. Concordantly, KLK14 expression was significantly (p = 0.0478) lower in asthenospermic cases. Specific inhibition of KLK14 activity by the synthetic inhibitor ACT(G9) resulted in a significant delay in semen liquefaction, a drop in the "early" (30 min postejaculation) "chymotrypsin-like" and KLK1 activity, and an increase in the "late" (90 min postejaculation) chymotrypsin-like activity. Conversely, the addition of recombinant active KLK14 facilitated the liquefaction process, augmented the early chymotrypsin-like activity, and lowered late chymotrypsin-like activity. Given that the observed chymotrypsin-like activity was almost completely attributed to KLK3 activity, KLK3 seems to be regulated bidirectionally. Accordingly, a higher level of KLK3 fragmentation was observed in KLK14-induced coagula, suggesting an inactivation mechanism via internal cleavage. Finally, semenogelins I and II were directly cleaved by KLK14. Semenogelins were also able to reverse KLK14 inhibition by Zn2+, providing a novel regulatory mechanism for KLK14 activity. Our results show that KLK14 exerts a significant and dose-dependent effect in the process of semen liquefaction.     

Discovery of Antimicrobial Peptides in Cervical‐Vaginal Fluid from Healthy Nonpregnant Women via an Integrated Proteome and Peptidome Analysis

Cervical‐vaginal fluid (CVF) covers the lower part of the female reproductive system and functions in the homeostasis and immunity of the surrounding tissues. In contrast to the CVF proteome of both nonpregnant and pregnant women, the CVF peptidome has not been reported to date. In the current study, we identified 1087 proteins in CVF, of which 801 proteins were not previously identified in CVF proteomes. The presence of the tissue‐specific proteins oviductal glycoprotein 1 and tubulin polymerization–promoting protein family member 3 strongly suggests that the tissues of the upper female reproductive tract contribute to the protein composition of CVF. The tremendous catalytic potential of CVF was highlighted by the identification of 85 proteases and the detection of pH‐dependent trypsin‐like proteolytic activity. Over 1000 endogenous peptides were detected in the CVF peptidome, and 39 peptides are predicted to have antimicrobial activity. The detailed proteomic and peptidomic analysis of CVF will further aid in the delineation of pathways related to reproduction, immunity and host defense, and assist in developing new biomarkers for malignant and other diseases of the female reproductive tract. Data are available via ProteomeXchange with identifiers PXD004450 (CVF peptidome) and PDX004363 (CVF proteome).     

Novel Biological Substrates of Human Kallikrein 7 Identified through Degradomics

Kallikrein-related peptidases (KLKs) are a group of serine proteases widely expressed in various tissues and involved in a wide range of physiological and pathological processes. Although our understanding of the pathophysiological roles of most KLKs has blossomed in recent years, identification of the direct endogenous substrates of human KLKs remains an unmet objective. In this study we employed a degradomics approach to systemically investigate the endogenous substrates of KLK7 in an effort to understand the molecular pathways underlying KLK7 action in skin. We identified several previously known as well as novel protein substrates. Our most promising candidates were further validated with the use of targeted quantitative proteomics (selected reaction monitoring methods) and in vitro recombinant protein digestion assays. Our study revealed midkine, CYR61, and tenascin-C as endogenous substrates for KLK7. Interestingly, some of these substrates (e.g. midkine) were prone to proteolytic cleavage only by KLK7 (and not by other skin-associated KLKs), whereas others (e.g. CYR61 and tenascin-C) could be digested by several KLKs. Furthermore, using melanoma cell line, we show that KLK7-mediated cleavage of midkine results in an overall reduction in the pro-proliferative and pro-migratory effect of midkine. An inverse relation between KLK7 and midkine is also observed in human melanoma tissues. In summary, our degradomics approach revealed three novel endogenous substrates for KLK7, which may shed more light on the pathobiological roles of KLK7 in human skin. Similar substrate screening approaches could be applied for the discovery of biological substrates of other protease.     

Structural characteristics of the ceramides of neutral glycosphingolipids in the human female genital tract--their menstrual cycle-associated change in the cervical epithelium and uterine endometrium, and their dissociation in the mucosa of the fallopian tube with the menstrual cycle.

In human cervical epithelium, uterine endometrium, and mucosa of the fallopian tubes, neutral glycosphingolipids were exclusively represented by the globo-series glycosphingolipids, such as CMH, LacCer, Gb3Cer and Gb4Cer, but the molecular species of their ceramide moieties were characteristically altered in the cervical epithelium and uterine endometrium during the menstrual cycle. Individual neutral glycosphingolipids in the cervical epithelium and the uterine endometrium at the follicular phase gave two bands on TLC, whereas those at the luteal phase displayed three bands, the third being the lower migrating one. Neutral glycosphingolipids migrating to the same positions as these lower-migrating bands were constantly detected in the mucosa of the fallopian tubes, independent of the menstrual cycle. The lower-migrating bands for the cervical epithelium and the uterine endometrium at the luteal phase were due to molecules mainly constructed of phytosphingosine with alpha-hydroxy fatty acids having chain lengths of 18-24 and 4-sphingenine with alpha-hydroxy fatty acids having chain lengths of 16-22, whereas those in the mucosa of the fallopian tubes were exclusively N-alpha-hydroxypalmitoyl 4-sphingenine.     

DNA content and the endometrial cellular mitotic activity in the phase of menstrual bleeding

Endometrium obtained during menses from 46 healthy women in reproductive age was investigated morphologically and cytospectrophotometrically in order to solve the problem on the source of the cells reepithelizing the uterine mucous membrane after desquamation. It was stated that desquamation takes place not in the whole functional layer of endometrium, some mucous fragments, covered with persisting luminal epithelium, are always preserved. During endometrial regeneration the cells of the luminal and glandular epithelia and those of endometrial stroma are predominantly diploid. The amount of premitotic cells in population is so small that they cannot secure any intensive cellular proliferation. Mitogenesis in endometrium is stimulated only after a complete restoration of the epithelial layer. It is suggested that persisting luminal epithelium is the source of cells for reepithelization; they migrate towards endometrial "wounds" and repair defects in the uterine mucosa during the regeneration phase.     

Defining the extended substrate specificity of kallikrein 1-related peptidases

Human kallikrein 1-related peptidases (KLKs) form a subfamily of 15 extracellular (chymo)tryptic-like serine proteases. KLKs 4, 5, 13 and 14 display altered expression/activity in diverse pathological conditions, including cancer. However, their distinct (patho)physiological roles remain largely uncharacterized. As a step toward distinguishing their proteolytic functions, we attempt to define their primary and extended substrate specificities and identify candidate biological targets. Heterologously expressed KLKs 4, 5, 13 and 14 were screened against fluorogenic 7-amino-4-carbamoylmethylcoumarin positional scanning-synthetic combinatorial libraries with amino acid diversity at the P1-P4 positions. Our results indicate that these KLKs share a P1 preference for Arg. However, each KLK exhibited distinct P2-P4 specificities, attributable to structural variations in their surface loops. The preferred P4-P1 substrate recognition motifs based on optimal subsite occupancy were as follows: VI-QSAV-QL-R for KLK4; YFWGPV-RK-NSFAM-R for KLK5; VY-R-LFM-R for KLK13; and YW-KRSAM-HNSPA-R for KLK14. Protein database queries using these motifs yielded many extracellular targets, some of which represent plausible KLK substrates. For instance, cathelicidin, urokinase-type plasminogen activator, laminin and transmembrane protease serine 3 were retrieved as novel putative substrates for KLK4, 5, 13 and 14, respectively. Our findings may facilitate studies on the role of KLKs in (patho)physiology and can be used in the development of selective KLK inhibitors.     

Kallikrein-related peptidase-8 (KLK8) is an active serine protease in human epidermis and sweat and is involved in a skin barrier proteolytic cascade

Kallikrein-related peptidase-8 (KLK8) is a relatively uncharacterized epidermal protease. Although proposed to regulate skin-barrier desquamation and recovery, the catalytic activity of KLK8 was never demonstrated in human epidermis, and its regulators and targets remain unknown. Herein, we elucidated for the first time KLK8 activity in human non-palmoplantar stratum corneum and sweat ex vivo. The majority of stratum corneum and sweat KLK8 was catalytically active, displaying optimal activity at pH 8.5 and considerable activity at pH 5. We also showed that KLK8 is a keratinocyte-specific protease, not secreted by human melanocytes or dermal fibroblasts. KLK8 secretion increased significantly upon calcium induction of terminal keratinocyte differentiation, suggesting an active role for this protease in upper epidermis. Potential activators, regulators, and targets of KLK8 activity were identified by in vitro kinetic assays using pro-KLK8 and mature KLK8 recombinant proteins produced in Pichia pastoris. Mature KLK8 activity was enhanced by calcium and magnesium ions and attenuated by zinc ions and by autocleavage after Arg(164). Upon screening KLK8 cleavage of a library of FRET-quenched peptides, trypsin-like specificity was observed with the highest preference for (R/K)(S/T)(A/V) at P1-P1'-P2'. We also demonstrated that KLK5 and lysyl endopeptidase activate latent pro-KLK8, whereas active KLK8 targets pro-KLK11, pro-KLK1, and LL-37 antimicrobial peptide activation in vitro. Together, our data identify KLK8 as a new active serine protease in human stratum corneum and sweat, and we propose regulators and targets that augment its involvement in a skin barrier proteolytic cascade. The implications of KLK8 elevation and hyperactivity in desquamatory and inflammatory skin disease conditions remain to be studied.