Cathepsin P, a novel protease in mouse placenta (original) (raw)
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FEBS Letters, 1999
A novel mouse cysteine protease of the papain family was identified by searching the dbEST database. A 1.28 kb fulllength cDNA was obtained which contains an open reading frame of 999 nucleotides and encodes a predicted polypeptide of 333 amino acids. The deduced polypeptide exhibits features characteristic of cysteine proteases of the papain type including the highly conserved residues of the catalytic triad, and was hence named cathepsin J. Cathepsin J represents the murine homologue of a previously described rat cathepsin L-related protein. Mature cathepsin J shows 59.3% identity to mouse cathepsin L and contains the characteristic ER(F/W)NIN motif within the propeptide indicating that this protease belongs to the subgroup of cathepsin L-like cysteine proteases. Northern blot analysis of various tissues revealed a placenta-restricted expression. This expression pattern may suggest a role of cathepsin J in embryo implantation and/or placental function. Ctsj was mapped to mouse chromosome 13 in the vicinity of cathepsin L suggesting that cathepsin J may have arisen by gene duplication from cathepsin L or a common ancestral gene.
Cathepsin Q, a Novel Lysosomal Cysteine Protease Highly Expressed in Placenta
Biochemical and Biophysical Research Communications, 2000
The complete nucleotide sequence of a novel cathepsin cDNA derived from rat placenta was determined and is termed cathepsin Q. The predicted protein of 343 amino acid is a member of the family C1A protease related to cathepsin L. Rat cathepsin Q and its mouse counterpart were found highly expressed in placenta, whereas no detectable levels were found in lung, spleen, heart, brain, kidney, thymus, testicle, liver, or embryonic tissues. It is predicted that cathepsin Q will differ in catalytic specificity to another placentalspecific protease, cathepsin P, indicating that these enzymes will have unique proteolytic functions in extra-embryonic tissues.
The importance of cysteine cathepsin proteases for placental development
Journal of Molecular Medicine, 2006
The typically lysosomal family of cysteine cathepsin proteases has been implicated in the development of the placenta, in particular from studies in the mouse. Here, we analysed overall expression, regulation and presence of transcript isoforms of cysteine cathepsins during human extra-embryonic development. All 11 family members are expressed in human placental tissues, and many are differentially regulated during gestation. Several cysteine cathepsins exhibit deregulated expression levels in placentas from pregnancies complicated by pre-eclampsia. The localization of cathepsin B predominantly in placental and decidual macrophages suggests a role in the physiological functions of these cells in mediating villous angiogenesis and decidual apoptosis. Cathepsin L levels are highest in a subpopulation of invasive cytotrophoblasts. Reflecting the expression pattern of two murine cathepsins, these data give insights into the evolutionary conservation of cathepsin function that is not necessarily exhibited by gene pairs defined by highest sequence similarity. Furthermore, cathepsin L protein localization in uterine epithelial cells demonstrates the in vivo occurrence of intranuclear cathepsin L isoforms. The zonally restricted expression of cathepsins in the syncytiotrophoblast may be important for the metabolic breakdown of maternal nutrients. Overall, the distribution and abnormal expression levels in pre-eclamptic placentas indicate that cysteine cathepsins may play important roles during normal placentation and in the etiology of pre-eclampsia.
Evolution of placentally expressed cathepsins
Biochemical and Biophysical Research Communications, 2002
Species and strain variants of a family of placentally expressed cathepsins (PECs) were cloned and sequenced in order to identify evolutionary conserved structural characteristics of this large family of cysteine proteases. Cathepsins M, P, Q, and R, are conserved in mice and rats but homologs of these genes are not found in human or rabbit placenta, showing that this family of proteases are probably restricted to rodents. Species-specific gene duplications have given rise to variants of cathepsin M in mice, and cathepsin Q in rats. Although the PECs have diverged at a greater rate than the other lysosomal cathepsins, residues around the specificity sub-sites of the individual enzymes are conserved. Strain-specific polymorphisms show that the evolutionary rate of divergence of cathepsins M and 3, the most recently duplicated pair of mouse genes, is even higher than the other PECs. In human placenta, critical functions of the PECs are probably performed by broader specificity proteases such as cathepsins B and L.
Expression and characterization of cathepsin P
Biochemical Journal, 2004
The mouse genome contains a family of clan C1A proteases that appear to be restricted to rodents within Eutherian (placental) mammals. mRNA analysis has shown that these genes are expressed exclusively in placenta. Sequence analysis predicts that the expressed proteins will be functional and consequently it was proposed that this family of proteases may have evolved to perform subspecialized functions of the closely related protease, cathepsin L, that is expressed in placental tissues of all mammalian species. In the present study, it was shown that cathepsin P can be expressed in Pichia pastoris as an inactive zymogen that can be activated with proteinase K, chymotrypsin or pancreatic elastase at neutral pH. Unlike other mammalian cathepsins, cathepsin P could also be autoactivated at neutral pH, but not at acidic pH. The activated enzyme was capable of hydrolysing peptidyl substrates and the protein substrates azocasein and transferrin, with optimal activity at pH 6.5-7.5. Little activity could be detected at pH 5.0 and below. Salts such as Na 2 SO 4 and hyaluronate stimulated the activity of the protease against peptidyl substrates. The properties of cathepsin P appear to be quite distinct from those of cathepsin L, indicating that the duplication that gave rise to cathepsin P has probably not yielded an enzyme that provides a subfunction of cathepsin L in rodents. It seems probable that cathepsin P has evolved to perform a function that is performed by an evolutionarily unrelated protease in other mammalian species.
Development of a specific inhibitor for the placental protease, cathepsin P
Archives of Biochemistry and Biophysics, 2007
Gene duplications in rodents have given rise to a family of proteases that are expressed exclusively in placenta. To define the biological role of these enzymes specific inhibitors are needed to differentiate their activities from other more ubiquitously expressed proteases, such as cathepsins B and L. Libraries of peptidyl inhibitors based upon a 4-cyclohexanone pharmacophore were screened for inhibition of cathepsins P, L, and B. The tightest binding dipeptidyl inhibitor for cathepsin P contained Tyr in P 2 and Trp in P 0 2 , consistent with the specificity of this enzyme for hydrophobic amino acids at these sites in synthetic substrates. An inhibitor containing Trp in both P 2 and P 0 2 provided better discrimination between cathepsin P and cathepsins B and L. Extension of the inhibitors to include P 3 , and P 0 3 amino acids identified an inhibitor with Trp in P 2 , P 0 2 , and P 3 , and Phe in P 0 3 that bound to cathepsin P with a K i of 32 nM. This specificity for inhibitors with hydrophobic aromatic amino acids in these four positions is unique among the lysosomal cysteine proteases. This inhibitor bound to cathepsin P an order of magnitude tighter than to mouse and human cathepsin L and two orders of magnitude tighter than to human cathepsin B. Cbz-Trp-Trp-4-cyclohexanone-Trp-Phe-OMe can discriminate cathepsin P from cathepsins B and L and consequently can be used to specifically inhibit and identify cathepsin P in cellular systems.
Mouse cathepsin F: cDNA cloning, genomic organization and chromosomal assignment of the gene
Gene, 2000
A murine cysteine protease of the papain family was identified by dbEST-database search. A 1.87 kb full-length cDNA encoding a predicted polypeptide of 462 amino acids was sequenced. Since the encoded polypeptide shows more than 80% sequence identity with human cathepsin F, it is most likely that this cDNA represents the murine homologue of cathepsin F, and it was therefore named accordingly. Murine cathepsin F exhibits a domain structure typical for papain-like cysteine proteases, a 20 amino acid N-terminal hydrophobic signal sequence followed by an extraordinarily long propeptide of 228 amino acids and the domain of the mature protease comprising 214 amino acids. The mature region contains all features characteristic of a papainlike cysteine protease, including the highly conserved cysteine, histidine and asparagine residues of the 'catalytic triad'. Genomic clones covering the murine cathepsin F gene were isolated. The mouse cathepsin F gene consists of 14 exons and 13 introns and spans 5.8 kb. Murine cathepsin F was mapped to chromosome 19, a region with synteny homology to a region of human chromosome 11 to which human cathepsin F has been mapped previously. Northern blot analysis of RNA from multiple tissues revealed a ubiquitous expression of cathepsin F in mouse and man.
Expression of Cysteine Proteases in Extraembryonic Tissues during Mouse Embryogenesis
Archives of Biochemistry and Biophysics, 1999
The expression of cathepsin B-and L-specific mRNAs as well as active forms of the enzymes was determined in mouse placenta and visceral yolk sac from 7.5 through 17.5 days postconception, a period marked by major anatomic transitions in the mouse conceptus. The level of specific mRNA was determined relative to the 28S ribosomal RNA in a series of multiprobe ribonuclease protection assays using high-specific-activity antisense cathepsin B and L riboprobes. The molecular forms of active cysteine proteases present in the tissues at the time of extraction were detected using a membrane-permeant radiolabeled active site-specific inhibitor, Fmoc-[ 125 I 2 ]Tyr-Ala-CHN 2 . The results of this study show that the expression of active cathepsin L relative to active cathepsin B is significantly higher in visceral yolk sac than in placenta, consistent with a higher proteolytic requirement for the former tissue. Active cathepsin L was highest at Day 9.5 in visceral yolk sac, a stage at which it has been shown that proteolysis in this organ is required for production of amino acids for embryonic protein synthesis. Cathepsin L mRNA was also elevated in the Day 9.5 placenta, but paradoxically this did not result in an increase in cellular active enzyme. An unknown protein, termed p14, highly expressed in placenta, also reacted with the inhibitor. Expression of this protein was highest early during gestation in the ectoplacental cone, suggesting that p14 may be important in the implantation process.
Zoological Science, 1998
The levels of cathepsins D and E in various rat tissues during development were determined with the sensitive assay method we have developed. The level of cathepsin D increased gradually in each tissue during fetal development suggesting the gradual maturation of the lysosomal system in a cell. The level of cathepsin E differed significantly between tissues at various developmental stages. The level in liver increased rapidly from 13-day-gestation fetal stage and decreased gradually at later fetal stages. The level in other tissues such as stomach and spleen began to increase at later fetal stages or the infant stage. Cathepsin E was found in fetal hepatocytes and its gene was hypomethylated when the expression of the gene was elevated. The enzyme was found to be present mainly as a proform suggesting that, after working, an active form is rapidly inactivated.
Molecular Human Reproduction, 1999
The aim of this study was to identify genes involved in human placentation. To do this, differential gene expression was assessed in the decidua (placental bed) from pre-eclamptic and normotensive pregnancies using the polymerase chain reaction (PCR)-based subtractive technique of representational difference analysis. A novel aspartyl protease (cathepsin D-like) cDNA sequence was isolated by virtue of its over-expression in the pre-eclamptic decidual sample tested. It was designated DAP-1 (for Decidual Aspartyl Protease 1). Using DAP-1 primer sequences a second cDNA (DAP-2) was subsequently isolated from decidual RNA by reverse transcription (RT)-PCR and found to be identical to DAP-1 apart from 80 additional and consecutive base pairs in the N-terminal coding region. In DAP-2, a stop codon within the unique 80 bp sequence was predicted to terminate translation immediately before the consensus active site residues. While Southern blotting was used to show that there are two loci with homology to DAP-1 in the human genome, it is postulated that alternative pre-mRNA splicing of the 80 bp exon is involved in the regulated expression of active (DAP-1) and inactive (DAP-2) forms of this novel protease; a mechanism similar to that involved in the regulated expression of Caspase-2, a protease involved in apoptosis. In other systems the regulation of alternative splicing is indicated by tissue specificity and developmental stage specificity of the various spliced products. In this context it was demonstrated that whereas DAP-1 was the major transcript expressed in decidua, the pattern was reversed in the adjacent placental tissue. It is proposed that tissue and developmental stage-specific expression of the DAP protease are important for the normal development and function of the uteroplacental tissues and that dysregulation of the control of DAP gene splicing may play a role in abnormal placentation, like that seen in pre-eclampsia.