Frédéric Esnard

Rat Seminal Vesicle FAD-dependent Sulfhydryl Oxidase BIOCHEMICAL CHARACTERIZATION AND MOLECULAR CLONING OF A MEMBER OF THE NEW SULFHYDRYL OXIDASE/QUIESCIN Q6 GENE FAMILY*

Rat FAD-dependent sulfhydryl oxidase was purified; partial sequencing indicated that it was homologous to human quiescin Q6. A cDNA (GenBank™ accession no. AF285078) was cloned from rat seminal vesicles, and active recombinant sulfhydryl oxidase was expressed in Chinese hamster ovary epithelial cells. This 2472-nucleotide cDNA has an open reading frame of 1710 base pairs, encoding a protein of 570 amino acids including a 32-amino acid leader sequence and two potential sites forN-glycosylation. One of them is used and the 64,000M r purified protein was transformed to 61,000 by the action of endoglycosidase F. Northern blotting and reverse transcription-polymerase chain reaction analyses showed that there were small amounts of sulfhydryl oxidase in the rat testis, prostate, lung, heart, kidney, spleen, and liver, and that the gene was highly expressed in seminal vesicles and epididymis. Rat sulfhydryl oxidase cDNA corresponds to the human cell growth inhibiting factor cDNA, which could be a differently spliced form of quiescin Q6. Comparing sulfhydryl oxidase sequences with those of human quiescin Q6 and mammalian and Caenorhabditis elegans quiescin Q6-related genes established the existence of a new family of FAD-dependent sulfhydryl oxidase/quiescin Q6-related genes containing protein-disulfide isomerase-type thioredoxin and yeast ERV1 domains.

Two rat homologues of human cystatin C

Two immunochemically related forms of cystatin C‐like inhibitors which differ in their M r app and isoelectric point have been found both in urine and seminal vesicles of rats. Amino‐terminal sequences of these two cystatins are identical within the same fluid and exhibit a high degree of homology with that of human cystatin C. However, cystatins C purified from urine lack eight residues at their amino‐terminal end when compared to those of seminal vesicles. The occurrence of two cystatin C‐like components in rat fluids has been found to be due to the presence of a glycosylated form reported here as cystatin Cg which specifically binds concanavalin A and is susceptible to endo‐β‐N‐acetylglucosaminidase treatment.

A simple method for calibrating collagenase/pronase E ratio to optimize heart cell isolation

Summary— A mixture of crude collagenase and non‐specific proteases has been used to isolate guinea pig ventricular heart cells. Measurements of collagenase activity with Wünschs substrate and protein content with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) suggest that collagenase enzymes do not play a major role in heart cell isolation. On the other hand, an important factor in heart digestion seems to consist of some fractions of the proteases present in crude collagenase. It is also noted that crude collagenases do not present any sensitivity to added calcium but because this ion is important to obtain isolated cells its role is discussed. According to our results, the SDS‐PAGE method can be used to determine the appropriate enzyme concentrations to obtain calcium‐tolerant myocytes. These myocytes have electrophysiological properties as reported in the literature.

A heat stable low molecular weight inhibitor of lysosomal cysteine proteinases in human serum

Abstract A new proteinase inhibitor of lysosomal cysteine proteinases has been isolated from human serum. In spite of its low concentration, kinetics studies indicate that it may be of physiological relevance given the low Ki values found for its interaction with human cathepsin B and cathepsin H.

Rat plasma α1‐inhibitor3: a member of the α‐macroglobulin family

α1 Inhibitor3 α.‐Macroglobulin Rat plasma Thiol ester Heat fragmentation

Purification and physicochemical characterization of a new rat plasma proteinase inhibitor, α1-inhibitor III

A three-stage procedure was used to isolate an additional proteinase inhibitor in rat plasma tentatively called alpha 1-inhibitor III. A 20% yield was obtained after two successive gel filtrations on Ultrogel Ac-A. 33-4 followed by an ion-exchange chromatography on DEAE-cellulose. This method was chosen since it permits further study of the enzyme binding properties of the isolated molecule. The purified material was first controlled to retain an inhibiting capacity towards serine proteinases using bovine chymotrypsin. The isolated molecule has an apparent molecular weight of 215 000, a pI of 4.65, an E1%1cm, 280 nm of 7.50 and a sedimentation coefficient of 8.6 S. It contains approx. 15% carbohydrates and is made up of a single peptidic chain. Study of the periodic structure by circular dichroism has demonstrated a low alpha-helix content (4--5%) whereas the beta-sheet conformation accounts for approx. 30% of the peptidic moiety. Tryptophan residues have been shown to be mainly responsible for the molecular fluorescence most of them being non-accessible to the solvent since only 25% of the tryptophanyl fluorescence was quenched in presence of I.

Production of the cysteine proteinase inhibitor cystatin C by rat Sertoli cells

The Sertoli cells of the rat testis produce cystatin C, a cysteine proteinase inhibitor. Primary culture of Sertoli cells secreted both unglycosylated and glycosylated forms of rat cystatin C. Despite the low concentration of cystatin C in rete testis fluid, equilibrium dissociation constants (K 1) for the interaction between cystatin C and lysosomal cathepsins indicate that this molecule could be involved in the local regulation of testicular cysteine proteinase activity which may be necessary for spermatogenesis and spermiogenesis.

High expression of QSOX1 reduces tumorogenesis, and is associated with a better outcome for breast cancer patients

IntroductionThe gene quiescin/sulfhydryl oxidase 1, QSOX1, encodes an enzyme directed to the secretory pathway and excreted into the extracellular space. QSOX1 participates in the folding and stability of proteins and thus could regulate the biological activity of its substrates in the secretory pathway and/or outside the cell. The involvement of QSOX1 in oncogenesis has been studied primarily in terms of its differential expression in systemic studies. QSOX1 is overexpressed in prostate cancers and in pancreatic adenocarcinoma. In contrast, QSOX1 gene expression is repressed in endothelial tumors. In the present study, we investigated the role of QSOX1 in breast cancer.MethodsWe analyzed QSOX1 mRNA expression in a cohort of 217 invasive ductal carcinomas of the breast. Moreover, we investigated QSOX1s potential role in regulating tumor growth and metastasis using cellular models in which we overexpressed or extinguished QSOX1 and xenograft experiments.ResultsWe showed that the QSOX1 expression level is inversely correlated to the aggressiveness of breast tumors. Our results show that QSOX1 leads to a decrease in cell proliferation, clonogenic capacities and promotes adhesion to the extracellular matrix. QSOX1 also reduces the invasive potential of cells by reducing cell migration and decreases the activity of the matrix metalloproteinase, MMP-2, involved in these mechanisms. Moreover, in vivo experiments show that QSOX1 drastically reduces the tumor development.ConclusionsTogether, these results suggest that QSOX1 could be posited as a new biomarker of good prognosis in breast cancer and demonstrate that QSOX1 inhibits human breast cancer tumorogenesis.

Posttranslational folding of alpha 1-inhibitor 3. Evidence for a compaction process.

α1-Inhibitor 3 (α1I3) is a rodent-specific proteinase inhibitor of about 190 kDa belonging to the α2-macroglobulin family. It consists of five globular domains, three of which are connected by disulfide bridges, and contains an intramolecular thiol ester which can react with attacking proteinases. To explore the folding of newly synthesized α1I3, we have used rat hepatocytes and pulse-chase experiments. In one of the analyses, the radiolabeled protein was isolated from cell lysates by immunoprecipitation and its Asp-Pro bonds cleaved by treatment with formic acid. The size of the major fragment, as assessed by electrophoresis under nonreducing conditions, was found to increase from 100 to 150 kDa upon the chasing. This result, together with knowledge of the positions of the cleavage sites and the disulfide arrangement, indicates that one of the interdomain disulfide bonds is formed after the synthesis of the polypeptide. Analysis of the same material by limited proteolysis and by velocity centrifugation showed that the folded regions became larger and that the protein became more compact; the thiol ester was found to be formed after these conformational changes. These results suggest that the domains of α1I3 are only partially developed directly after the synthesis of the polypeptide and that they acquire their final structure as the protein condenses and the domains interact with one another.

The flavo-oxidase QSOX1 supports vascular smooth muscle cell migration and proliferation: Evidence for a role in neointima growth.

Quiescin sulfhydryl oxidase 1 (QSOX1) is a flavoenzyme largely present in the extracellular milieu whose physiological functions and substrates are not known. QSOX1 has been implicated in the regulation of tumor cell survival, proliferation and migration, in addition to extracellular matrix (ECM) remodeling. However, data regarding other pathophysiological conditions are still lacking. Arterial injury by balloon catheter is an established model of post-angioplasty restenosis. This technique induces neointima formation due to migration and proliferation of vascular smooth muscle cells (VSMC), followed by ECM synthesis and remodeling. Here, we show that QSOX1 knockdown inhibited VSMC migration and proliferation in vitro. In contrast, QSOX1 overexpression stimulated these processes. While migration could be induced by the incubation of cells with the active recombinant QSOX1, proliferation was induced by addition of the active and also of an inactive mutant QSOX1 protein. The proliferation induced by both recombinants was independent of intracellular hydrogen peroxide and dependent of the MEK/ERK pathway. To recapitulate in vivo VSMC pathophysiology, balloon-induced arterial injury was performed. The expression of QSOX1 in the neointimal layer of balloon-injured rat carotids was high and peaked at 14 days post-injury. In vivo QSOX1 knockdown led to a significant decrease in PCNA expression at day 14 post-injury and a decreased intima/media area ratio at day 21 post-injury, compared with scrambled siRNA transfection. In summary, our findings demonstrate that QSOX1 induces VSMC migration and proliferation in vitro and contributes to neointima thickening in balloon-injured rat carotids.