11 results on '"Sánchez LM"'
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2. Withdrawal: Matriptase-2, a membrane-bound mosaic serine proteinase predominantly expressed in human liver and showing degrading activity against extracellular matrix proteins.
- Author
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Velasco G, Cal S, Quesada V, Sánchez LM, and López-Otín C
- Published
- 2019
- Full Text
- View/download PDF
3. Withdrawal: Identification and characterization of human and mouse ovastacin: A novel metalloproteinase similar to hatching enzymes from arthropods, birds, amphibians, and fish.
- Author
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Quesada V, Sánchez LM, Álvarez J, and López-Otín C
- Published
- 2019
- Full Text
- View/download PDF
4. Identification of human aminopeptidase O, a novel metalloprotease with structural similarity to aminopeptidase B and leukotriene A4 hydrolase.
- Author
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Díaz-Perales A, Quesada V, Sánchez LM, Ugalde AP, Suárez MF, Fueyo A, and López-Otín C
- Subjects
- Amino Acid Sequence, Aminopeptidases classification, Aminopeptidases genetics, Angiotensin III metabolism, Animals, Armadillo Domain Proteins, Base Sequence, Brain enzymology, Epoxide Hydrolases genetics, Epoxide Hydrolases metabolism, Humans, Molecular Sequence Data, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Sequence Alignment, Tissue Distribution, Trans-Activators chemistry, Trans-Activators genetics, Aminopeptidases chemistry, Aminopeptidases metabolism, Epoxide Hydrolases chemistry, Protein Conformation
- Abstract
We have cloned and characterized a human brain cDNA encoding a new metalloprotease that has been called aminopeptidase O (AP-O). AP-O exhibits a series of structural features characteristic of aminopeptidases, including a conserved catalytic domain with a zinc-binding site (HEXXHX18E) that allows its classification in the M1 family of metallopeptidases or gluzincins. The structural complexity of AP-O is further increased by the presence of an additional C-terminal domain 170 residues long, which is predicted to have an ARM repeat fold originally identified in the Drosophila segment polarity gene product Armadillo. This ARM repeat domain is also present in aminopeptidase B, aminopeptidase B-like, and leukotriene A4 hydrolase and defines a novel subfamily of aminopeptidases that we have called ARM aminopeptidases. Northern blot analysis revealed that AP-O is mainly expressed in the pancreas, placenta, liver, testis, and heart. Human AP-O was produced in Escherichia coli, and the purified recombinant protein hydrolyzed synthetic substrates used for assaying aminopeptidase activity. This activity was abolished by general inhibitors of metalloproteases and specific inhibitors of aminopeptidases. Recombinant AP-O also cleaved angiotensin III to generate angiotensin IV, a bioactive peptide of the renin-angiotensin pathway with multiple actions on diverse tissues, including brain, testis, and heart. On the basis of these results we suggest that AP-O could play a role in the proteolytic processing of bioactive peptides in those tissues where it is expressed.
- Published
- 2005
- Full Text
- View/download PDF
5. Identification and characterization of human and mouse ovastacin: a novel metalloproteinase similar to hatching enzymes from arthropods, birds, amphibians, and fish.
- Author
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Quesada V, Sánchez LM, Alvarez J, and López-Otín C
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Blastocyst metabolism, Blotting, Northern, Blotting, Southern, Catalytic Domain, Cell Line, Tumor, Cloning, Molecular, Conserved Sequence, DNA, Complementary metabolism, Databases as Topic, Escherichia coli metabolism, Female, Humans, In Situ Hybridization, Mice, Molecular Sequence Data, Ovarian Neoplasms metabolism, Phylogeny, Protein Structure, Tertiary, Recombinant Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Software, Time Factors, Metalloproteases biosynthesis, Metalloproteases chemistry, Ovary metabolism
- Abstract
We have cloned and characterized human and mouse ovary cDNAs encoding a new protein of the astacin family of metalloproteinases, called ovastacin because of its predominant expression in ovarian tissues. Human and mouse ovastacins exhibit the same domain organization as other astacins, including signal sequence, propeptide, and metalloproteinase domain. However, ovastacins show an additional C-terminal domain of about 150 amino acids with no similarity to other ancillary domains present in the equivalent region of most astacins. Northern blot analysis of human tissues and cell lines revealed that ovastacin is only detected at significant levels in leukemia and lymphoma cells of different origin. In addition, RT-PCR analysis demonstrated that ovastacin is expressed in human and mouse ovary, in unfertilized mouse oocytes, and in 1.5-day-postcoitum preimplantation embryos. Further studies showed that superovulation caused a dramatic increase in the expression of mouse ovastacin, indicating that the production of this enzyme is under hormonal regulation. Human ovastacin was expressed in Escherichia coli and the purified recombinant protein hydrolyzed synthetic substrates used for assaying metalloproteinases. These activities were abolished by inhibitors of metalloproteinases, but not by inhibitors of other classes of proteases. On the basis of these results, we suggest that ovastacin could play in mammals a physiological function similar to that performed by hatching proteases in evolutionary distant species from arthropods to fish.
- Published
- 2004
- Full Text
- View/download PDF
6. Matriptase-2, a membrane-bound mosaic serine proteinase predominantly expressed in human liver and showing degrading activity against extracellular matrix proteins.
- Author
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Velasco G, Cal S, Quesada V, Sánchez LM, and López-Otín C
- Subjects
- Amino Acid Sequence, Base Sequence, Cell Membrane metabolism, DNA Primers, Fetus, Gene Library, Humans, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes metabolism, Liver embryology, Membrane Proteins chemistry, Molecular Sequence Data, Polymerase Chain Reaction, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sequence Alignment, Sequence Homology, Amino Acid, Serine Endopeptidases chemistry, Substrate Specificity, Trypsin chemistry, Extracellular Matrix Proteins metabolism, Liver enzymology, Membrane Proteins genetics, Membrane Proteins metabolism, Serine Endopeptidases genetics, Serine Endopeptidases metabolism, Trypsin genetics, Trypsin metabolism
- Abstract
We have identified and cloned a fetal liver cDNA encoding a new serine proteinase that has been called matriptase-2. This protein exhibits a domain organization similar to other members of an emerging family of membrane-bound serine proteinases known as type II transmembrane serine proteinases. Matriptase-2 contains a short cytoplasmic domain, a type II transmembrane sequence, a central region with several modular structural domains including two CUB (complement factor C1s/C1r, urchin embryonic growth factor, bone morphogenetic protein) domains and three low density lipoprotein receptor tandem repeats, and finally, a C-terminal catalytic domain with all typical features of serine proteinases. The human matriptase-2 gene maps to 22q12-q13, a location that differs from all type II transmembrane serine proteinase genes mapped to date. Immunofluorescence and Western blot analysis of COS-7 cells transfected with the isolated cDNA confirmed that matriptase-2 is anchored to the cell surface. Matriptase-2 was expressed in Escherichia coli, and the purified recombinant protein hydrolyzed synthetic substrates used for assaying serine proteinases and endogenous proteins such as type I collagen, fibronectin, and fibrinogen. Matriptase-2 could also activate single-chain urokinase plasminogen activator, albeit with low efficiency. These activities were abolished by inhibitors of serine proteinases but not by inhibitors of other classes of proteolytic enzymes. Northern blot analysis demonstrated that matriptase-2 transcripts are only detected at significant levels in both fetal and adult liver, suggesting that this novel serine proteinase may play a specialized role in matrix remodeling processes taking place in this tissue during development or in adult tissues.
- Published
- 2002
- Full Text
- View/download PDF
7. Structural and enzymatic characterization of Drosophila Dm2-MMP, a membrane-bound matrix metalloproteinase with tissue-specific expression.
- Author
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Llano E, Adam G, Pendás AM, Quesada V, Sánchez LM, Santamariá I, Noselli S, and López-Otín C
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Catalytic Domain, Cloning, Molecular, DNA, Complementary chemistry, Matrix Metalloproteinases metabolism, Molecular Sequence Data, Organ Specificity, Cell Membrane enzymology, Drosophila enzymology, Matrix Metalloproteinases chemistry
- Abstract
We report the isolation and characterization of a cDNA encoding Dm2-MMP, the second matrix metalloproteinase (MMP) identified in the Drosophila melanogaster genome. The cloned cDNA codes for a polypeptide of 758 residues that displays a domain organization similar to that of other MMPs, including signal peptide, propeptide, catalytic, and hemopexin domains. However, the structure of Dm2-MMP is unique because of the presence of an insertion of 214 amino acids between the catalytic and hemopexin domains that is not present in any of the previously described MMPs. Dm2-MMP also contains a C-terminal extension predicted to form a cleavable glycosylphosphatidylinositol anchor site. Western blot and immunofluorescence analysis of S2 cells transfected with the isolated cDNA confirmed that Dm2-MMP is localized at the cell surface. Production of the catalytic domain of Dm2-MMP in Escherichia coli and analysis of its enzymatic activity revealed that this proteinase cleaves several synthetic peptides used for analysis of vertebrate MMPs. This proteolytic activity was abolished by MMP inhibitors such as BB-94, confirming that the isolated cDNA codes for an enzymatically active metalloproteinase. Reverse transcription-PCR analysis showed that Dm2-MMP is expressed at low levels in all of the developmental stages of Drosophila as well as in adult flies. However, detailed in situ hybridization at the larval stage revealed a strong tissue-specific expression in discrete regions of the brain and eye imaginal discs. According to these results, we propose that Dm2-MMP plays both general proteolytic functions during Drosophila development and in adult tissues and specific roles in eye development and neural tissues through the degradation and remodeling of the extracellular matrix.
- Published
- 2002
- Full Text
- View/download PDF
8. Identification and enzymatic characterization of two diverging murine counterparts of human interstitial collagenase (MMP-1) expressed at sites of embryo implantation.
- Author
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Balbín M, Fueyo A, Knäuper V, López JM, Alvarez J, Sánchez LM, Quesada V, Bordallo J, Murphy G, and López-Otín C
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Blotting, Northern, Chromosome Mapping, Cloning, Molecular, Collagen metabolism, Collagenases genetics, DNA, Complementary metabolism, Female, Gene Expression Regulation, Developmental, Gene Library, Genetic Vectors, Humans, In Situ Hybridization, Fluorescence, Matrix Metalloproteinase 9 chemistry, Matrix Metalloproteinase 9 metabolism, Matrix Metalloproteinases genetics, Mice, Models, Molecular, Molecular Sequence Data, Multigene Family, Open Reading Frames, Phylogeny, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Uterus enzymology, Uterus metabolism, Collagenases chemistry, Embryo Implantation, Embryo, Mammalian enzymology, Matrix Metalloproteinase 1 chemistry, Matrix Metalloproteinases chemistry
- Abstract
Remodeling of fibrillar collagen in mouse tissues has been widely attributed to the activity of collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the main collagenase identified in this species. This proposal has been largely based on the repeatedly unproductive attempts to detect the presence in murine tissues of interstitial collagenase (MMP-1), a major collagenase in many species, including humans. In this work, we have performed an extensive screening of murine genomic and cDNA libraries using as probe the full-length cDNA for human MMP-1. We report the identification of two novel members of the MMP gene family which are contained within the cluster of MMP genes located at murine chromosome 9. The isolated cDNAs contain open reading frames of 464 and 463 amino acids and are 82% identical, displaying all structural features characteristic of archetypal MMPs. Comparison for sequence similarities revealed that the highest percentage of identities was found with human interstitial collagenase (MMP-1). The new proteins were tentatively called Mcol-A and Mcol-B (Murine collagenase-like A and B). Analysis of the enzymatic activity of the recombinant proteins revealed that both are catalytically autoactivable but only Mcol-A is able to degrade synthetic peptides and type I and II fibrillar collagen. Both Mcol-A and Mcol-B genes are located in the A1-A2 region of mouse chromosome 9, Mcol-A occupying a position syntenic to the human MMP-1 locus at 11q22. Analysis of the expression of these novel MMPs in murine tissues revealed their predominant presence during mouse embryogenesis, particularly in mouse trophoblast giant cells. According to their structural and functional characteristics, we propose that at least one of these novel members of the MMP family, Mcol-A, may play roles as interstitial collagenase in murine tissues and could represent a true orthologue of human MMP-1.
- Published
- 2001
- Full Text
- View/download PDF
9. Human cathepsin O. Molecular cloning from a breast carcinoma, production of the active enzyme in Escherichia coli, and expression analysis in human tissues.
- Author
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Velasco G, Ferrando AA, Puente XS, Sánchez LM, and López-Otín C
- Subjects
- Amino Acid Sequence, Base Sequence, Cathepsin K, Cathepsins biosynthesis, Cathepsins metabolism, Cloning, Molecular, Cysteine Endopeptidases biosynthesis, Cysteine Endopeptidases metabolism, DNA, Complementary genetics, Escherichia coli genetics, Female, Humans, Molecular Sequence Data, Polymerase Chain Reaction, Recombinant Proteins biosynthesis, Recombinant Proteins metabolism, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Tissue Distribution, Breast Neoplasms enzymology, Carcinoma enzymology, Cathepsins genetics, Cysteine Endopeptidases genetics
- Abstract
A cDNA encoding a novel member of the cysteine proteinase family of proteins has been cloned from a human breast carcinoma cDNA library, by using a polymerase chain reaction-based cloning strategy. The isolated cDNA contains an open reading frame coding for a polypeptide of 321 amino acids that has been tentatively called cathepsin O. This protein presents all the structural features characteristic of the different cysteine proteinases identified to date, including the active site cysteine residue that is involved in covalent intermediate formation during peptide hydrolysis. The cathepsin O cDNA was expressed in Escherichia coli, and after purification and refolding, the recombinant protein was able to degrade the synthetic peptides benzyloxycarbonyl-Phe-Arg-7-amido-4- methylcoumarin and benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin widely used as substrates for cysteine proteinases. Cathepsin O proteolytic activity was abolished by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), an inhibitor of this subclass of proteolytic enzymes, thus providing additional evidence that the isolated cDNA codes for an authentic cysteine proteinase. Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues demonstrated that cathepsin O is expressed in all examined tissues, which is consistent with a putative role of this protein as a proteolytic enzyme involved in normal cellular protein degradation and turnover.
- Published
- 1994
10. Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas.
- Author
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Freije JM, Díez-Itza I, Balbín M, Sánchez LM, Blasco R, Tolivia J, and López-Otín C
- Subjects
- Amino Acid Sequence, Base Sequence, Breast Neoplasms pathology, Cloning, Molecular, Collagenases chemistry, Collagenases genetics, Conserved Sequence, Escherichia coli, Female, Gene Expression, Gene Library, Humans, Immunohistochemistry, Matrix Metalloproteinase 13, Matrix Metalloproteinase 3, Metalloendopeptidases chemistry, Metalloendopeptidases genetics, Molecular Sequence Data, Oligodeoxyribonucleotides, Polymerase Chain Reaction methods, RNA, Neoplasm isolation & purification, RNA, Neoplasm metabolism, Sequence Homology, Amino Acid, Breast Neoplasms enzymology, Collagenases biosynthesis
- Abstract
A cDNA coding for a new human matrix metalloproteinase (MMP) has been cloned from a cDNA library derived from a breast tumor. The isolated cDNA contains an open reading frame coding for a polypeptide of 471 amino acids. The predicted protein sequence displays extensive similarity to the previously known MMPs and presents all the structural features characteristic of the members of this protein family, including the well conserved PRCGXPD motif, involved in the latency of the enzyme and the zinc-binding domain (HEXGHXXXXXHS). In addition, this novel human MMP contains in its amino acid sequence several residues specific to the collagenase subfamily (Tyr-214, Asp-235, and Gly-237) and lacks the 9-residue insertion present in the stromelysins. According to these structural characteristics, the MMP described herein has been tentatively called collagenase-3, since it represents the third member of this subfamily, composed at present of fibroblast and neutrophil collagenases. The collagenase-3 cDNA was expressed in a vaccinia virus system, and the recombinant protein was able to degrade fibrillar collagens, providing support to the hypothesis that the isolated cDNA codes for an authentic collagenase. Northern blot analysis of RNA from normal and pathological tissues demonstrated the existence in breast tumors of three different mRNA species, which seem to be the result of the utilization of different polyadenylation sites present in the 3'-noncoding region of the gene. By contrast, no collagenase-3 mRNA was detected either by Northern blot or RNA polymerase chain reaction analysis with RNA from other human tissues, including normal breast, mammary fibroadenomas, liver, placenta, ovary, uterus, prostate, and parotid gland. On the basis of the increased expression of collagenase-3 in breast carcinomas and the absence of detectable expression in normal tissues, a possible role for this metalloproteinase in the tumoral process is proposed.
- Published
- 1994
11. Isolation and characterization of a pepsin C zymogen produced by human breast tissues.
- Author
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Sánchez LM, Freije JP, Merino AM, Vizoso F, Foltmann B, and López-Otín C
- Subjects
- Amino Acid Sequence, Breast cytology, Breast pathology, Breast Neoplasms enzymology, Breast Neoplasms pathology, Cathepsin D genetics, DNA genetics, DNA isolation & purification, Enzyme Precursors genetics, Enzyme Precursors isolation & purification, Female, Fibrocystic Breast Disease pathology, Gastric Mucosa cytology, Gastric Mucosa enzymology, Humans, Molecular Sequence Data, Pepsin A genetics, Pepsinogens genetics, Pepsinogens isolation & purification, Polymerase Chain Reaction, RNA analysis, RNA genetics, Sequence Homology, Amino Acid, Breast enzymology, Enzyme Precursors analysis, Fibrocystic Breast Disease enzymology, Pepsin A analysis, Pepsinogens analysis
- Abstract
An aspartic proteinase present in cyst fluid from women with gross cystic breast disease was purified by a procedure involving affinity chromatography on pepstatin-agarose and size-exclusion high performance liquid chromatography. The amino-terminal sequence of the purified breast proteinase was identical to that corresponding to gastric pepsinogen C. Additional data on cleavage specificity, pH optimum, and immunological properties supported the close relationship between both molecules. Northern blot analysis and polymerase chain reaction amplification studies performed on RNAs obtained from normal and pathological breast tissues demonstrated that the protein is produced by mammary carcinomas and cysts, but not by the normal resting mammary gland. Immunohistochemical staining of paraffin-embedded tissue sections confirmed the existence of a subset of tumors that have the ability to synthesize and secrete this pepsin zymogen. On the basis of these results, we suggest that pepsinogen C expression by human mammary epithelium may be involved in the development of breast diseases, being also of potential interest as a biochemical marker of the hormonal imbalance underlying these pathologies.
- Published
- 1992
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