Kallikrein-related peptidases.

Kallikrein 1 (KLK1), a key component of the kallikrein-kinin system, originates from a locus on the long arm of chromosome 19 that contains several related serine endopeptidases. The biological role of these kallikrein-related peptidases is not clear, but emerging evidence suggests that they might be important in several physiological systems, e.g., in male reproduction, skin homeostasis, tooth enamel formation and neural development and plasticity. The kallikrein locus has undergone some major evolutionary events. Most spectacular are relatively recent duplications of KLK1 that have created 13 and 9 functional genes that are unique to the mouse and the rat, respectively. Human paralogs are KLK2 and KLK3: the latter encoding the cancer biomarker prostate-specific antigen. In this review on kallikrein-related peptidases, the focus is on their evolution, their role in skin homeostasis and semen liquefaction, and their utility as cancer biomarkers.

hK5 and hK7, two serine proteinases abundant in human skin, are inhibited by LEKTI domain 6.

BACKGROUND: Several skin diseases and atopic disorders including Netherton syndrome and atopic dermatitis have been associated with mutations and deviations of expression of SPINK5, the gene encoding the human 15-domain serine proteinase inhibitor LEKTI. The biochemical mechanisms underlying this phenomenon have not yet been fully clarified. OBJECTIVES: To identify target proteinases of LEKTI important for processes of desquamation and inflammation of the skin which will enable the development of specific drugs. METHODS: The inhibitory activities of LEKTI domains 6 and 15 were tested on a number of commercially available serine proteinases and also on the purified kallikreins hK5 and hK7. In addition, recombinant hK5 was used. RESULTS: LEKTI domain 6 is a potent inhibitor of hK5 and hK7, whereas LEKTI domain 15 exhibits inhibitory activity on plasmin. hK5 and hK7 in particular are relevant to skin disorders. CONCLUSIONS: The inhibition of hK5 and hK7 by LEKTI domain 6 indicates an important regulatory role of LEKTI in processes of skin desquamation and inflammation, which may explain the severe pathological symptoms associated with abnormalities of SPINK5 and/or its expression. Thus, LEKTI represents a potential drug for the treatment of these disorders.

Stratum corneum tryptic enzyme in normal epidermis: a missing link in the desquamation process?

Stratum corneum chymotryptic enzyme may be important in desquamation. It has also been suggested that other proteases, especially stratum corneum tryptic enzyme, may be involved. Stratum corneum tryptic enzyme has been purified and its cDNA has been cloned. Results from expression analyses indicate that stratum corneum tryptic enzyme is as skin specific as stratum corneum chymotryptic enzyme. In this work we have produced and characterized antibodies specific for stratum corneum tryptic enzyme. We have also by means of biochemical, immunochemical, and immunohistochemical methods performed studies on stratum corneum tryptic enzyme in normal human epidermis. Antibodies against bacterial recombinant stratum corneum tryptic enzyme were produced and purified by affinity chromatography. Two types of antibodies were obtained: one reacting only with pro-stratum corneum tryptic enzyme and one specific for the catalytically active part of stratum corneum tryptic enzyme. Immunohistochemistry with the antibodies reacting with pro-stratum corneum tryptic enzyme showed a staining pattern similar to stratum corneum chymotryptic enzyme-specific antibodies, i.e., the expression was confined to cornifying epithelia with a need of desquamation-like processes. Extracts of tape strips with superficial human stratum corneum were found to contain precursors as well as active forms of stratum corneum tryptic enzyme and stratum corneum chymotryptic enzyme. The enzymes had maximal activity at pH 8, but both had considerable activity also at pH 5.5. The results were compatible for a role of stratum corneum tryptic enzyme in desquamation. Stratum corneum tryptic enzyme may act in concert with stratum corneum chymotryptic enzyme and/or function as a stratum corneum chymotryptic enzyme-activating enzyme. The presence in normal superficial stratum corneum of precursors as well as of active forms of stratum corneum chymotryptic enzyme and stratum corneum tryptic enzyme, and the activity of both enzymes over a broad range of pH-values, suggest some possible ways by which the desquamation may be regulated.

Purification, molecular cloning, and expression of a human stratum corneum trypsin-like serine protease with possible function in desquamation.

A new human 33-kDa serine protease was purified from human epidermis, and its cDNA was cloned from a keratinocyte library, from mRNA from a human keratinocyte line (HaCat) and from mRNA from human skin. Polyclonal antibodies specific for the new protein detected three groups of proteins in partially purified extracts of cornified eptihelium of human plantar skin. The three components are proposed to correspond to proenzyme, active enzyme, and proteolytically modified active enzyme. After N-deglycosylation, there was a decrease in apparent molecular mass of all detected components. Expression of the cloned cDNA in a eukaryotic virus-derived system yielded a recombinant protein that could be converted to an active protease by treatment with trypsin. Polymerase chain reaction analyses of cDNA from a number of human tissues showed high expression of the new enzyme in the skin and low expression in brain, placenta, and kidney. Homology searches yielded the highest score for porcine enamel matrix protease (55% amino acid sequence homology). High scores were also obtained for human and mouse neuropsin and for human stratum corneum chymotryptic enzyme. The function of this new protease, tentatively named stratum corneum tryptic enzyme, may be related to stratum corneum turnover and desquamation in the epidermis.

Molecular cloning and tissue expression of the murine analog to human stratum corneum chymotryptic enzyme.

Human stratum corneum chymotryptic enzyme (SCCE) may play a central part in epidermal homeostasis. Its proposed function is to catalyze the degradation of intercellular structures, including desmosomes, in the stratum corneum as part of the desquamation process. In order to facilitate physiologic and pathophysiologic studies on SCCE we have looked for the corresponding murine enzyme. A cDNA obtained by reverse transcription-polymerase chain reaction with total RNA prepared from mouse tails as starting material was cloned, and the expression of the corresponding mRNA studied. The murine cDNA showed 77% homology to human SCCE cDNA. It had an open-reading frame encoding a protein comprising 249 amino acids with 82% amino acid sequence homology to human SCCE including the conserved sequences of the catalytic traid of mammalian serine proteases. The murine protein was deduced to have a 21 amino acid signal peptide and a four amino acid propeptide ending with a tryptic cleavage site, followed by a sequence motif identical to the N-terminal amino acid sequence of native active human SCCE. As in human SCCE the P2 position of the propeptide was occupied by an acidic amino acid residue, and the position corresponding to the suggested bottom of the primary substrate specificity pouch occupied by an asparagine residue. Analyses of mouse tissues by reverse transcriptase-polymerase chain reaction showed high expression in the skin, low expression in lung, kidney, brain, heart, and spleen, and no expression in liver or skeletal muscle. In situ hybridization of mouse skin showed expression in high suprabasal keratinocytes and in the luminal parts of hair follicles. Our results strongly suggest that we have cloned the murine analog of human SCCE cDNA.

Expression of stratum corneum chymotryptic enzyme in human sebaceous follicles.

Stratum corneum chymotryptic enzyme (SCCE) may be involved in desquamation, a process necessary for maintaining a normal anatomy at all sites where there is continuous turnover of cornified epithelia. Using immunohistochemistry and in situ hybridization, we have, in this work, analysed SCCE expression in the sebaceous follicle. We found expression of SCCE in luminal parts of the pilary canal, common sebaceous ducts and proximal sebaceous ducts. In addition, SCCE was seen in cells apparently situated within the distal parts of the glandular lobules. Co-expression of SCCE and keratin 10 was seen only in the pilary canal and the common sebaceous ducts. The results give further support for SCCE being involved in desquamation-like processes. The association with cornification seems to be more general for SCCE than for keratin 10. The possible role of SCCE in diseases involving disturbances in the turnover of cornified cells in the sebaceous follicle, such as acne vulgaris, is a question for future studies.

Purification and characterization of interleukin 1 beta from human plantar stratum corneum. Evidence of interleukin 1 beta processing in vivo not involving interleukin 1 beta convertase.

The major interleukin 1 beta (IL-1 beta) species from human plantar stratum corneum was purified and found to have an N-terminal amino acid sequence homologous to a stretch of the human IL-1 beta precursor, starting with His115. Whereas SDS-polyacrylamide gel electrophoresis followed by immunoblotting revealed only one component in plantar stratum corneum with IL-1 beta-like immunoreactivity, and with an apparent molecular mass around 18 kDa, isoelectric focusing under non-denaturing conditions showed one major component with isoelectric point around 6.1 and two minor components isoelectric at pH 6.3 and 6.9, respectively. Digestion of recombinant human IL-1 beta precursor with chymotrypsin, producing a C-terminal fragment with N-terminal Yal114, yielded a component with IL-1 beta-like immunoreactivity isoelectric at pH 6.3. Recombinant bacterial variants of human IL-1 beta with N-terminal amino acids corresponding to Val114, His115 and Ala117 were isoelectric at pH 6.3, 6.1 and 6.9, respectively. Cloning and subsequent nucleotide sequencing of IL-1 beta precursor cDNA from a human keratinocyte line showed total identify with the sequence previously published for the human monocyte IL-1 beta precursor. The authors conclude that the IL-1 beta species present in plantar stratum corneum have isoelectric points determined by their respective amino acid sequences, and that there is a mechanism for IL-1 beta activation in human epidermis not involving interleukin 1 beta convertase.