Characterization of human gamma-tryptases, novel members of the chromosome 16p mast cell tryptase and prostasin gene families

Previously, this laboratory identified clusters of alpha-, beta-, and mast cell protease-7-like tryptase genes on human chromosome 16p13.3. The present work characterizes adjacent genes encoding novel serine proteases, termed gamma-tryptases, and generates a refined map of the multitryptase locus. Each gamma gene lies between an alpha1H Ca2+ channel gene (CACNA1H) and a betaII- or betaIII-tryptase gene and is approximately 30 kb from polymorphic minisatellite MS205. The tryptase locus also contains at least four tryptase-like pseudogenes, including mastin, a gene expressed in dogs but not in humans. Genomic DNA blotting results suggest that gammaI- and gammaII-tryptases are alleles at the same site. betaII- and betaIII-tryptases appear to be alleles at a neighboring site, and alphaII- and betaI-tryptases appear to be alleles at a third site. gamma-Tryptases are transcribed in lung, intestine, and in several other tissues and in a mast cell line (HMC-1) that also expresses gamma-tryptase protein. Immunohistochemical analysis suggests that gamma-tryptase is expressed by airway mast cells. gamma-Tryptase catalytic domains are approximately 48% identical with those of known mast cell tryptases and possess mouse homologues. We predict that gamma-tryptases are glycosylated oligomers with tryptic substrate specificity and a distinct mode of activation. A feature not found in described tryptases is a C-terminal hydrophobic domain, which may be a membrane anchor. Although the catalytic domains contain tryptase-like features, the hydrophobic segment and intron-exon organization are more closely related to another recently described protease, prostasin. In summary, this work describes gamma-tryptases, which are novel members of chromosome 16p tryptase/prostasin gene families. Their unique features suggest possibly novel functions.     

Identification of a subgroup of glycosylphosphatidylinositol-anchored tryptases

The tryptase locus on mouse chromosome 17A3.3 contains 13 genes that encode enzymatically active serine proteases with different tissue expression profiles and substrate specificities. Mouse mast cell protease (mMCP) 6, mMCP-7, mMCP-11/protease serine member S (Prss) 34, tryptase 6/Prss33, tryptase ε/Prss22, implantation serine protease (Isp) 1/Prss28, and Isp-2 are constitutively exocytosed enzymes. We now demonstrate that tryptase 5/Prss32, pancreasin/Prss27, and testis serine protease-1 are inserted into plasma membranes via glycosylphosphatidylinositol (GPI) anchors analogous to Prss21, and that these serine proteases can be released from the cell’s surface by a phosphatidylinositol-specific phospholipase C. These data suggest that the C-terminal residues play key roles in determining where tryptases compartmentalize in cells. GPI-anchored proteins are targeted to lipid rafts. Thus, our identification of a number of GPI-anchored tryptases whose genes reside at mouse chromosome 17A3.3 also implicates important biological functions for this new family of serine proteases on the surfaces of cells.     

Mouse DESC1 is located within a cluster of seven DESC1-like genes and encodes a type II transmembrane serine protease that forms serpin inhibitory complexes

We report the identification and functional analysis of a type II transmembrane serine protease encoded by the mouse differentially expressed in squamous cell carcinoma (DESC) 1 gene, and the definition of a cluster of seven homologous DESC1-like genes within a 0.5-Mb region of mouse chromosome 5E1. This locus is syntenic to a region of human chromosome 4q13.3 containing the human orthologues of four of the mouse DESC1-like genes. Bioinformatic analysis indicated that all seven DESC1-like genes encode functional proteases. Direct cDNA cloning showed that mouse DESC1 encodes a multidomain serine protease with an N-terminal signal anchor, a SEA (sea urchin sperm protein, enterokinase, and agrin) domain, and a C-terminal serine protease domain. The mouse DESC1 mRNA was present in epidermal, oral, and male reproductive tissues and directed the translation of a membrane-associated 60-kDa N-glycosylated protein with type II topology. Mouse DESC1 was synthesized in insect cells as a zymogen that could be activated by exposure to trypsin. The purified activated DESC1 hydrolyzed synthetic peptide substrates, showing a preference for Arg in the P1 position. DESC1 proteolytic activity was abolished by generic inhibitors of serine proteases but not by other classes of protease inhibitors. Most interestingly, DESC1 formed stable inhibitory complexes with both plasminogen activator inhibitor-1 and protein C inhibitor that are expressed in the same tissues with DESC1, suggesting that type II transmembrane serine proteases may be novel targets for serpin inhibition. Together, these data show that mouse DESC1 encodes a functional cell surface serine protease that may have important functions in the epidermis, oral, and reproductive epithelium.     

The serine protease marapsin is expressed in stratified squamous epithelia and is up-regulated in the hyperproliferative epidermis of psoriasis and regenerating wounds

The trypsin-like serine protease marapsin is a member of the large protease gene cluster at human chromosome 16p13.3, which also contains the structurally related proteases testisin, tryptase epsilon, tryptase gamma, and EOS. To gain insight into the biological functions of marapsin, we undertook a detailed gene expression analysis. It showed that marapsin expression was restricted to tissues containing stratified squamous epithelia and was absent or only weakly expressed in all other tissues, including the pancreas. Marapsin was constitutively expressed in nonkeratinizing stratified squamous epithelia of human esophagus, tonsil, cervix, larynx, and cornea. In the keratinizing stratified squamous epidermis of skin, however, its expression was induced only during epidermal hyperproliferation, such as in psoriasis and in murine wound healing. In fact, marapsin was the second most strongly up-regulated protease in psoriatic lesions, where expression was localized to the upper region of the hyperplastic epidermis. Similarly, in the hyperproliferative epithelium of regenerating murine skin wounds, marapsin localized to the suprabasal layers, where keratinocytes undergo squamous differentiation. The transient up-regulation of marapsin, which closely correlated with re-epithelialization, was virtually absent in a genetic mouse model of delayed wound closure. These results suggested a function during the process of re-epithelialization. Furthermore, in reconstituted human epidermis, a model system of epidermal differentiation, members of the IL-20 subfamily of cytokines, such as IL-22, induced marapsin expression. Consistent with a physiologic role in marapsin regulation, IL-22 was also strongly expressed in re-epithelializing skin wounds. Marapsin's restricted expression, localization, and cytokine-inducible expression suggest a role in the terminal differentiation of keratinocytes in hyperproliferating squamous epithelia.     

Sequencing and expression analysis of the serine protease gene cluster located in chromosome 19q13 region

The human kallikrein gene cluster, located in the chromosome band 19q13, contains several tissue-specific serine protease genes including the prostate-specific KLK2, KLK3 and prostase genes. To further characterize the gene cluster, we have mapped, sequenced, and analyzed the genomic sequence from the region. The results of EST database searches and GENSCAN gene prediction analysis reveal 13 serine protease genes and several pseudogenes in the region. Expression analysis by RT-PCR indicates that most of these protease genes are expressed only in a subset of the 35 different normal tissues that have been examined. Several protease genes expressed in skin show higher expression levels in psoriatic lesion samples than in non-lesional skin samples from the same patient. This suggests that the imbalance of a complex protease cascade in skin may contribute to the pathology of disease. The proteases, excluding the kallikrein genes, share approximately 40% of their sequences suggesting that the serine protease gene cluster on chromosome 19q13 arose from ancient gene duplications.     

Genomic organization, mapping, tissue expression, and hormonal regulation of trypsin-like serine protease (TLSP PRSS20), a new member of the human kallikrein gene family

The cDNA for the trypsin-like serine protease gene (TLSP, HGMW-approved symbol PRSS20) has been recently identified. TLSP is expressed in brain and skin tissues but little else is known about this new serine protease gene. In this paper, we describe the complete genomic organization and precise mapping of the TLSP gene. This gene spans 5.3 kb of genomic sequence on chromosome 19q13.3-q13. 4. The gene consists of six exons, the first of which is untranslated. All splice junctions follow the GT/AG rule, and the intron phases are identical to those of other kallikrein-like genes, including zyme (PRSS9), NES1 (PRSSL1), and neuropsin (PRSS19). Fine-mapping of the area indicates that TLSP lies downstream from the PSA, zyme, neuropsin, and NES1 genes. Significant sequence homologies were found between TLSP and other human kallikreins. Furthermore, there is conservation of the catalytic triad (histidine, aspartic acid, serine) and of the number of coding exons (five; the same in all members of the kallikrein gene family). We thus suggest that TLSP is a new member of the human kallikrein gene family. TLSP is expressed in many tissues including cerebellum, prostate, salivary glands, stomach, lung, thymus, small intestine, spleen, liver, and uterus. TLSP expression appears to be regulated by steroid hormones in the breast carcinoma cell line BT-474.     

Chromosome localization of two human serine protease genes to region 14q11.2----q12 by in situ hybridization

Two human serine protease genes have been cloned. One corresponds to CTLA1, the human equivalent of the mouse cytotoxic cell protease gene Ctla-1, and the other is novel. Both genes were localized to 14q11.2----q12 by in situ hybridization. This result confirms the assignment of human CTLA1 to 14q11.2----q12 and provides new mapping data for another human serine protease gene located in the same chromosome region.     

Molecular cloning and characterization of the human beta-like globin gene cluster

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.     

Structure based design of 4-(3-aminomethylphenyl)piperidinyl-1-amides: novel, potent, selective, and orally bioavailable inhibitors of betaII tryptase

Tryptase is a serine protease found almost exclusively in mast cells. It has trypsin-like specificity, favoring cleavage of substrates with an arginine (or lysine) at the P1 position, and has optimal catalytic activity at neutral pH. Current evidence suggests tryptase beta is the most important form released during mast cell activation in allergic diseases. It is shown to have numerous pro-inflammatory cellular activities in vitro, and in animal models tryptase provokes broncho-constriction and induces a cellular inflammatory infiltrate characteristic of human asthma. Screening of in-house inhibitors of factor Xa (a closely related serine protease) identified beta-amidoester benzamidines as potent inhibitors of recombinant human betaII tryptase. X-ray structure driven template modification and exchange of the benzamidine to optimize potency and pharmacokinetic properties gave selective, potent and orally bioavailable 4-(3-aminomethyl phenyl)piperidinyl-1-amides.