Involvement of EDTP, an egg-derived tyrosine phosphatase, in the early development of Drosophila melanogaster

Previously, we purified a novel protein tyrosine phosphatase from eggs of the flesh fly, Sarcophaga peregrina. This protein tyrosine phosphatase, named egg-derived tyrosine phosphatase (EDTP), is expressed during oogenesis and early embryogenesis but is rapidly degraded in middle embryogenesis by lysosomal cathepsin L. Here, we demonstrate the requirement of EDTP in the development of the fruit fly, Drosophila melanogaster. Deletion of the Drosophila EDTP gene using transposase-catalyzed imprecise excision resulted in homozygous lethals during embryogenesis. Additionally, germline clones generated using the FLP-FRT-ovo(D) system showed severe defects in ovarian development during oogenesis. These results indicate that the Drosophila EDTP gene is crucial in oogenesis and embryogenesis.     

Cloning of cDNA for cathepsin B mRNA 3'-untranslated-region-binding protein (CBBP), and characterization of recombinant CBBP

Previously, we purified the cathepsin B mRNA 3'-untranslated-region-binding protein (CBBP) from Sarcophaga and suggested its participation in the translational regulation of cathepsin B mRNA in this insect. In this study, we isolated a full length cDNA for CBBP. CBBP was an RNA-binding protein that contained four RGG boxes and four zinc finger motifs required for RNA binding. CBBP was shown to be localized in both the nuclei and cytoplasm of Sarcophaga hemocytes. Recombinant CBBP bound to the entire region of cathepsin B mRNA and repressed its translation in vitro.     

Two subunits of the insect 26/29-kDa proteinase are probably derived from a common precursor protein

We previously identified the 26/29-kDa proteinase in the hemocytes of Sarcophaga peregrina (flesh fly) that appears to participate in elimination of foreign proteins in this insect [Eur. J. Biochem. 209, 939-944 (1992)]. Here, we report the cDNA cloning of this proteinase. The cDNA encodes a protein which includes both the 26- and 29-kDa subunit, strongly suggesting that the both subunits are derived from a single precursor protein. The 26- and 29-kDa subunit located at the amino-terminal and carboxyl-terminal of the precursor protein. The 29-kDa subunit itself appeared to be a proteinase, for this subunit had 52% sequence identity with Sarcophaga cathepsin L, while 26-kDa subunit had no significant similarity. We also showed that 26/29-kDa proteinase was insensitive to specific inhibitors of cathepsin L. These results indicate that this proteinase is a novel member of the papain family. We isolated similar cDNAs from Drosophila melanogaster and Periplaneta americana (cockroach), suggesting that this proteinase is conserved in a wide variety of insects and participates in their defense mechanisms.     

Molecular cloning and characterization of a protein tyrosine phosphatase enriched in testis, a putative murine homologue of human PTPMEG

Protein tyrosine phosphorylation is regulated by protein tyrosine kinase and protein tyrosine phosphatase activities. These two counteracting proteins are implicated in cell growth and transformation. Using polymerase chain reaction with degenerate primers, we have identified a novel mouse protein tyrosine phosphatase (PTP). This cDNA contains a single open reading frame of the predicted 926 amino acids. Those predicted amino acids showed significant identity with human megakaryocyte protein-tyrosine phosphatase by 91% in nucleotide sequences and 94% in amino acid sequences. We have identified that expression of this PTP is highly enriched in the testis in mouse and human and has been termed here as a 'testis-enriched phosphatase' (TEP). Northern analysis detected two mRNA species of 3.7 and 3.2kb for this PTP in mouse testis and the expression of TEP is regulated during development. The recombinant phosphatase domain possesses protein tyrosine phosphatase activity when expressed in Escherichia coli. Immunohistochemical analysis of the cellular localization of TEP on mouse testis sections showed that this PTP is specifically expressed in spermatocytes and spermatids within seminiferous tubules, suggesting an important role in spermatogenesis.     

Inorganic polyphosphate inhibits an aspartic protease‐like activity in the eggs of Rhodnius prolixus (Stahl) and impairs yolk mobilization in vitro

Inorganic polyphosphate (poly P) is a polymer of phosphate residues that has been shown to act as modulator of some vertebrate cathepsins. In the egg yolk granules of Rhodnius prolixus, a cathepsin D is the main protease involved in yolk mobilization and is dependent on an activation by acid phosphatases. In this study, we showed a possible role of poly P stored inside yolk granules on the inhibition of cathepsin D and arrest of yolk mobilization during early embryogenesis of these insects. Enzymatic assays detected poly P stores inside the eggs of R. prolixus. We observed that micromolar poly P concentrations inhibited cathepsin D proteolytic activity using both synthetic peptides and homogenates of egg yolk as substrates. Poly P was a substrate for Rhodnius acid phosphatase and also a strong competitive inhibitor of a pNPPase activity. Fusion events have been suggested as important steps towards acid phosphatase transport to yolk granules. We observed that poly P levels in those compartments were reduced after in vitro fusion assays and that the remaining poly P did not have the same cathepsin D inhibition activity after fusion. Our results are consistent with the hypothesis that poly P is a cathepsin D inhibitor and a substrate for acid phosphatase inside yolk granules. It is possible that, once activated, acid phosphatase might degrade poly P, allowing cathepsin D to initiate yolk proteolysis. We, therefore, suggest that degradation of poly P might represent a new step toward yolk mobilization during embryogenesis of R. prolixus. J. Cell. Physiol. 222: 606–611, 2010. © 2009 Wiley‐Liss, Inc.     

Sequence analysis, tissue distribution, and expression of rat cathepsin S.

Cysteine proteases are involved in many diverse cellular processes ranging from processing of precursor proteins to intracellular degradation. In an effort to identify novel cysteine proteases, we used the polymerase chain reaction and primers directed against the catalytic sites of previously cloned cysteine proteases. From rat brain mRNA, a 600-base pair band was amplified; cloning and partial sequence analysis of this band resulted in the identification of cathepsins B and L and five novel sequences. The novel cDNAs contained a number of residues conserved in lysosomal cysteine proteases, including the active site residue His159 (papain numbering). In addition, the amino acid homology between the novel sequences and either cathepsins B, L, or H, ranged from 63 to 32%. The insert with highest homology was used to screen a rat brain cDNA library; a 1334-base pair cDNA was isolated and the nucleotide sequence determined. This sequence encodes an open reading frame of 330 amino acids which is 82% homologous to human cathepsin S, suggesting that this sequence represents rat cathepsin S. Northern blot analysis for rat cathepsin S revealed tissue-specific expression distinct from the distribution of cathepsin B and L. The regulation of expression of rat cathepsin S mRNA in response to thyroid-stimulating hormone was studied in a rat thyroid cell line FRTL-5. The level of cathepsin S mRNA was substantially increased in response to thyroid-stimulating hormone, whereas cathepsin B and cathepsin L mRNA levels were not altered by this treatment. A portion of cDNA encoding the predicted mature protein of rat cathepsin S was expressed as a glutathione S-transferase-fusion protein. The affinity-purified protein exhibited proteolytic activity with properties similar to bovine cathepsin S. Taken together, these results imply highly specific functions for cathepsin S.     

Contribution of cathepsin L to secretome composition and cleavage pattern of mouse embryonic fibroblasts

The endolysosomal cysteine endoprotease cathepsin L is secreted from cells in a variety of pathological conditions such as cancer and arthritis. We compared the secretome composition and extracellular proteolytic cleavage events in cell supernatants of cathepsin L-deficient and wild-type mouse embryonic fibroblasts (MEFs). Quantitative proteomic comparison of cell conditioned media indicated that cathepsin L deficiency affects, albeit in a limited manner, the abundances of extracellular matrix (ECM) components, signaling proteins, and further proteases as well as endogenous protease inhibitors. Immunodetection corroborated that cathepsin L deficiency results in decreased abundance of the ECM protein periostin and elevated abundance of matrix metalloprotease (MMP)-2. While mRNA levels of MMP-2 were not affected by cathepsin L ablation, periostin mRNA levels were reduced, potentially indicating a downstream effect. To characterize cathepsin L contribution to extracellular proteolysis, we performed terminal amine isotopic labeling of substrates (TAILS), an N-terminomic technique for the identification and quantification of native and proteolytically generated protein N-termini. TAILS identified >1500 protein N-termini. Cathepsin L deficiency predominantly reduced the magnitude of collagenous cleavage sites C-terminal to a proline residue. This contradicts cathepsin L active site specificity and indicates altered activity of further proteases as a result of cathepsin L ablation.     

Cathepsin L is essential for embryogenesis and development of Caenorhabditis elegans.

Cysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterized Ce-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1 activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential role for cpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP) was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/degradation of cuticular or other proteins. Similar localization of a related cathepsin L in the filarial nematode Onchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes.     

An Active 32-kDa Cathepsin L Is Secreted Directly from HT 1080 Fibrosarcoma Cells and Not via Lysosomal Exocytosis

Cathepsin L [EC 3.4.22.15] is secreted via lysosomal exocytosis by several types of cancer cells, including prostate and breast cancer cells. We previously reported that human cultured fibrosarcoma (HT 1080) cells secrete cathepsin L into the medium; this secreted cathepsin is 10-times more active than intracellular cathepsin. This increased activity was attributed to the presence of a 32-kDa cathepsin L in the medium. The aim of this study was to examine how this active 32-kDa cathepsin L is secreted into the medium. To this end, we compared the secreted active 32-kDa cathepsin L with lysosomal cathepsin L by using a novel gelatin zymography technique that employs leupeptin. We also examined the glycosylation and phosphorylation status of the proteins by using the enzymes endoglycosidase H [EC 3.2.1.96] and alkaline phosphatase [EC 3.1.3.1]. Strong active bands corresponding to the 32-kDa and 34-kDa cathepsin L forms were detected in the medium and lysosomes, respectively. The cell extract exhibited strong active bands for both forms. Moreover, both forms were adsorbed onto a concanavalin A-agarose column. The core protein domain of both forms had the same molecular mass of 30 kDa. The 32-kDa cathepsin L was phosphorylated, while the 34-kDa lysosomal form was dephosphorylated, perhaps because of the lysosomal marker enzyme, acid phosphatase. These results suggest that the active 32-kDa form does not enter the lysosomes. In conclusion, our results indicate that the active 32-kDa cathepsin L is secreted directly from the HT 1080 cells and not via lysosomal exocytosis.     

RPTPmu and protein tyrosine phosphorylation regulate K(+) channel mRNA expression in adult cardiac myocytes

Previously, we reported that cell-cell contact regulates K(+) channel mRNA expression in cultured adult rat cardiac myocytes. Here we show that exposing cardiac myocytes to tyrosine kinase inhibitors (genistein, tyrphostin A25), but not inactive analogs, prevents downregulation of Kv1.5 mRNA and upregulation of Kv4.2 mRNA normally observed when they are cultured under low-density conditions. Furthermore, cardiac myocytes cocultured with cells that endogenously (Mv 1 Lu) or heterologously (Chinese hamster ovary cells) express the receptor-type protein tyrosine phosphatase mu (RPTPmu) display Kv1.5 mRNA levels paralleling that which was observed in myocytes cultured under high-density conditions and in intact tissue. In contrast, myocytes cocultured with control cells failed to produce this response. Finally, it is shown that Kv4.2 mRNA expression is unaffected by RPTPmu. These findings reveal that multiple tyrosine phosphorylation-dependent mechanisms control cardiac myocyte K(+) channel genes. Furthermore, we conclude that RPTPmu specifically regulates cardiac myocyte Kv1.5 mRNA expression. Thus this receptor protein tyrosine phosphatase may be important in responses to pathological conditions associated with the loss of cell-cell interactions in the heart.     

Structure of Xenopus laevis ribosomal protein L32 and its expression during development.

cDNA clones for Xenopus laevis ribosomal protein L32 have been isolated and sequenced. The deduced amino acid sequence indicates that L32 is a basic protein of 110 amino acids, has a molecular weight of 12,603 and is homologous to the rat ribosomal protein L35. Using the cDNA clone as a probe to follow the expression of this gene during Xenopus development, it has been shown that the pattern of accumulation of this mRNA follows the one previously described for other ribosomal protein mRNAs during oogenesis and embryogenesis. The analysis of the utilization of L32 mRNA during embryogenesis shows that this is controlled by the translational regulation typical of other ribosomal protein mRNAs.     

Ursolic acid and its derivative inhibit protein tyrosine phosphatase 1B, enhancing insulin receptor phosphorylation and stimulating glucose uptake

Protein tyrosine phosphatase 1B (PTP1B) is a key element in the negative regulation of the insulin signaling pathway and may play an important role in diabetes and obesity. We identified ursolic acid, a natural pentacyclic triterpenoid that occurs widely in traditional Chinese medicinal herbs, as an inhibitor of PTP1B by screening an extract library of the traditional Chinese medicinal herbs used a diabetes clinic. By modifying urosolic acid, we designed and synthesized a derivative with a K(i) of 283 nM. As competitive inhibitors of PTP1B, ursolic acid and its derivative also inhibit T-cell protein tyrosine phosphatase and src homology phosphatase-2 but not leucocyte antigen-related phosphatase or protein tyrosine phosphatase alpha and epsilon, which are all possibly involved in the insulin pathway. The ursolic acid derivative enhanced insulin receptor phosphorylation in CHO/hIR cells and stimulate glucose uptake in L6 myotubes.     

Purification and characterization of a hemocyte proteinase of Sarcophaga, possibly participating in elimination of foreign substances.

We have reported that foreign protein injected into the abdominal cavity of Sarcophaga peregrina (flesh fly) larvae is degraded in the hemolymph by a proteinase secreted by hemocytes [Suzuki, T. and Natori, S. (1985) Comp. Biochem. Physiol. 81A, 191-193]. Here we report the purification and characterization of a proteinase from larval hemocytes. This enzyme is a cysteine proteinase consisting of 26-kDa and 29-kDa subunits with similar substrate specificity to mammalian cathepsin B. This enzyme was shown to be released from hemocytes into the hemolymph of larvae following injection of sheep red blood cells into the larvae, suggesting that it participates, at least in part, in elimination of foreign substances introduced into the body cavity.     

Purification of a 29-kDa hemocyte proteinase of Sarcophaga peregrina.

Previously, we suggested the participation of a hemocyte proteinase in the dissociation of fat body of Sarcophaga peregrina (flesh fly) at metamorphosis. We have now purified this proteinase to near homogeneity from pupal hemocytes. It is a cysteine proteinase with a molecular mass of 29 kDa and has a unique substrate specificity hydrolyzing both Suc-Leu-Leu-Val-Tyr-MCA and Z-Phe-Arg-MCA (Suc, succinyl; MCA, methylcoumaryl-7-amide; Z, carbobenzoxy), which are substrates for chymotrypsin and cathepsin B, respectively. Partial similarity was found between the amino-terminal sequence of this proteinase and that of cathepsin B, including Pro, Glu and Arg residues conserved in the papain superfamily of enzymes.     

Comparison of cathepsin L synthesized by normal and transformed cells at the gene, message, protein, and oligosaccharide levels

The major excreted protein of transformed mouse fibroblasts (MEP) has recently been identified as the lysosomal cysteine protease, cathepsin L. The synthesis and intracellular trafficking of this protein in mouse fibroblasts are regulated by growth factors and malignant transformation. To further define the basis for this regulation, a cDNA encoding MEP/cathepsin L was isolated from a mouse liver cDNA library and used to compare cathepsin L of normal and Kirsten sarcoma virus-transformed NIH 3T3 fibroblasts. Although cathepsin L message levels were elevated 20-fold in the transformed fibroblasts, normal and transformed cells displayed similar cathepsin L genomic DNA digest patterns and gene copy numbers, and cathepsin L mRNA sequences appeared identical by RNase protection analysis. These findings indicate that (i) cathepsin L is synthesized from the same gene in normal and transformed cells and (ii) cathepsin L polypeptides made by these cells are translated with the same primary sequence. Cathepsin L polypeptides synthesized by quiescent, growing, and transformed cells displayed similar isoelectric focusing patterns, suggesting similar post-translational modification. Site-directed mutagenesis of the mouse liver cDNA and expression in COS monkey cells was used to examine the glycosylation of mouse cathepsin L. The results indicated that only one of the two potential N-linked glycosylation sites (the one at Asn221) is glycosylated. Analysis by ion exchange chromatography on QAE-Sephadex, and affinity chromatography on mannose 6-phosphate receptor-Affi-Gel 10, indicated that the cathepsin L oligosaccharide was phosphorylated similarly in normal and transformed cells. Although several phosphorylated oligosaccharide species were observed, the major species contained two phosphomonoester moieties and bound efficiently to the receptor. These findings suggest that cathepsin L made by normal and transformed mouse fibroblasts are identical and substantiate the hypothesis that trafficking of cathepsin L in these cells is regulated by growth-induced changes in the lysosomal protein transport system.