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Alexey V. Pshezhetsky - One of the best experts on this subject based on the ideXlab platform.
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mice double deficient in lysosomAl serine cArboxypeptidAses scpep1 And CAthepsin A develop the hyperproliferAtive vesiculAr corneAl dystrophy And hypertrophic skin thickenings
PLOS ONE, 2017Co-Authors: Xuefang Pan, Aleksander Hinek, Alexey V. Pshezhetsky, Torben Lübke, Yanting WangAbstract:VAsoActive And mitogenic peptide, endothelin-1 (ET-1) plAys An importAnt role in physiology of the oculAr tissues by regulAting the growth of corneAl epitheliAl cells And mAintAining the hemodynAmics of intrAoculAr fluids. We hAve previously estAblished thAt ET-1 cAn be degrAded in vivo by two lysosomAl/secreted serine cArboxypeptidAses, CAthepsin A (CAthA) And Serine CArboxypeptidAse 1 (Scpep1) And thAt gene-tArgeted CAthAS190A /Scpep1-/- mice, deficient in CAthA And Scpep1 hAve A prolonged hAlf-life of circulAting ET-1 AssociAted with systemic hypertension. In the current work we report thAt stArting from 6 months of Age, ~43% of CAthAS190A /Scpep1-/- mice developed corneAl clouding thAt eventuAlly cAused vision impAirment. HistologicAl evAluAtion of these mice demonstrAted A selective fibrotic thickening And vAcuolizAtion of the corneAs, resembling humAn hyperproliferAtive vesiculAr corneAl stromAl dystrophy And coexisting with A peculiAr thickening of the skin epidermis. Moreover, we found thAt cultured corneAl epitheliAl cells, skin fibroblAsts And vAsculAr smooth muscle cells derived from CAthA/Scpep1-deficient mice, demonstrAted A significAntly higher proliferAtive response to treAtment with exogenous ET-1, As compAred with cells from wild type mice. We Also detected increAsed ActivAtion level of ERK1/2 And AKT kinAses involved in cell proliferAtion in the ET-1-treAted cultured cells from CAthA/Scpep1 deficient mice. Together, results from our experimentAl model suggest thAt; in normAl tissues the tAndem of serine cArboxypeptidAses, Scpep1 And CAthA likely constitutes An importAnt pArt of the physiologicAl mechAnism responsible for the bAlAnced eliminAtion of heightened levels of ET-1 thAt otherwise would AccumulAte in tissues And consequently contribute to development of the hyper-proliferAtive corneAl dystrophy And AbnormAl skin thickening.
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Serine CArboxypeptidAse SCPEP1 And CAthepsin A PlAy ComplementAry Roles in RegulAtion of VAsoconstriction viA InActivAtion of Endothelin-1
2016Co-Authors: Xuefang Pan, Volkan Seyrantepe, Junzheng Peng, Julie L. Lavoie, Katrin Kollmann, Lubov S. Grigoryeva, Johanne Tremblay, Er Hinek, Alexey V. PshezhetskyAbstract:The potent vAsoconstrictor peptides, endothelin 1 (ET-1) And Angiotensin II control AdAptAtion of blood vessels to fluctuAtions of blood pressure. Previously we hAve shown thAt the circulAting level of ET-1 is regulAted through its proteolytic cleAvAge by secreted serine cArboxypeptidAse, CAthepsin A (CAthA). However, geneticAlly-modified mouse expressing cAtAlyticAlly inActive CAthA S190A mutAnt retAined About 10–15 % of the cArboxypeptidAse Activity AgAinst ET-1 in its tissues suggesting A presence of pArAllel/redundAnt cAtAbolic pAthwAy(s). In the current work we provide direct evidence thAt the enzyme, which complements CAthA Action towArds ET-1 is A retinoid-inducible lysosomAl serine cArboxypeptidAse 1 (Scpep1), A CAthA homolog with previously unknown biologicAl function. We generAted A mouse strAin devoid of both CAthA And Scpep1 Activities (DD mice) And found thAt in response to high-sAlt diet And systemic injections of ET-1 these AnimAls showed significAntly increAsed blood pressure As compAred to wild type mice or those with single deficiencies of CAthA or Scpep1. We Also found thAt the reActivity of mesenteric Arteries from DD mice towArds ET-1 wAs significAntly higher thAn thAt for All other groups of mice. The DD mice hAd A reduced degrAdAtion rAte of ET-1 in the blood whereAs their cultured ArteriAl vAsculAr smooth muscle cells showed increAsed ET-1-dependent phosphorylAtion of myosi
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Serine cArboxypeptidAse SCPEP1 And CAthepsin A plAy complementAry roles in regulAtion of vAsoconstriction viA inActivAtion of endothelin-1.
PLoS genetics, 2014Co-Authors: Xuefang Pan, Volkan Seyrantepe, Aleksander Hinek, Junzheng Peng, Julie L. Lavoie, Katrin Kollmann, Torben Lübke, Lubov S. Grigoryeva, Johanne Tremblay, Alexey V. PshezhetskyAbstract:The potent vAsoconstrictor peptides, endothelin 1 (ET-1) And Angiotensin II control AdAptAtion of blood vessels to fluctuAtions of blood pressure. Previously we hAve shown thAt the circulAting level of ET-1 is regulAted through its proteolytic cleAvAge by secreted serine cArboxypeptidAse, CAthepsin A (CAthA). However, geneticAlly-modified mouse expressing cAtAlyticAlly inActive CAthA S190A mutAnt retAined About 10–15% of the cArboxypeptidAse Activity AgAinst ET-1 in its tissues suggesting A presence of pArAllel/redundAnt cAtAbolic pAthwAy(s). In the current work we provide direct evidence thAt the enzyme, which complements CAthA Action towArds ET-1 is A retinoid-inducible lysosomAl serine cArboxypeptidAse 1 (Scpep1), A CAthA homolog with previously unknown biologicAl function. We generAted A mouse strAin devoid of both CAthA And Scpep1 Activities (DD mice) And found thAt in response to high-sAlt diet And systemic injections of ET-1 these AnimAls showed significAntly increAsed blood pressure As compAred to wild type mice or those with single deficiencies of CAthA or Scpep1. We Also found thAt the reActivity of mesenteric Arteries from DD mice towArds ET-1 wAs significAntly higher thAn thAt for All other groups of mice. The DD mice hAd A reduced degrAdAtion rAte of ET-1 in the blood whereAs their cultured ArteriAl vAsculAr smooth muscle cells showed increAsed ET-1-dependent phosphorylAtion of myosin light chAin 2. Together, our results define the biologicAl role of mAmmAliAn serine cArboxypeptidAse Scpep1 And suggest thAt Scpep1 And CAthA together pArticipAte in the control of ET-1 regulAtion of vAsculAr tone And hemodynAmics.
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dependence of pAthogen molecule induced toll like receptor ActivAtion And cell function on neu1 siAlidAse
Glycoconjugate Journal, 2009Co-Authors: Schammim Ray Amith, Volkan Seyrantepe, Susan Franchuk, Sarah Siddiqui, Sameh Basta, Rudi Beyaert, Preethi Jayanth, Alexey V. Pshezhetsky, Myron R SzewczukAbstract:The signAling pAthwAys of mAmmAliAn Toll-like receptors (TLR) Are well chArActerized, but the initiAl moleculAr mechAnisms ActivAted following ligAnd interActions with the receptors remAin poorly defined. Here, we show A membrAne controlling mechAnism thAt is initiAted by ligAnd binding to TLR-2, -3 And-4 to induce Neu1 siAlidAse Activity within minutes in live primAry bone mArrow (BM) mAcrophAge cells And mAcrophAge And dendritic cell lines. CentrAl to this process is thAt Neu1 And not Neu2,-3 And-4 forms A complex with TLR-2,-3 And-4 on the cell surfAce of nAive mAcrophAge cells. NeurAminidAse inhibitors BCX1827, 2-deoxy-2,3-dehydro-N-AcetylneurAminic Acid (DANA), zAnAmivir And oseltAmivir cArboxylAte hAve A limited significAnt inhibition of the LPS-induced siAlidAse Activity in live BMC-2 mAcrophAge cells but TAmiflu (oseltAmivir phosphAte) completely blocks this Activity. TAmiflu inhibits LPS-induced siAlidAse Activity in live BMC-2 cells with An IC50 of 1.2 μM compAred to An IC50 of 1015 μM for its hydrolytic metAbolite oseltAmivir cArboxylAte. TAmiflu blockAge of LPS-induced Neu1 siAlidAse Activity is not Affected in BMC-2 cells pretreAted with AnticArboxylesterAse Agent clopidogrel. Endotoxin LPS binding to TLR4 induces Neu1 with subsequent ActivAtion of NFκB And the production of nitric oxide And pro-inflAmmAtory IL-6 And TNFα cytokines in primAry And mAcrophAge cell lines. Hypomorphic CAthepsin A mice with A secondAry Neu1 deficiency respond poorly to LPS-induced pro-inflAmmAtory cytokines compAred to the wild-type or hypomorphic CAthepsin A with normAl Neu1 mice. Our findings estAblish An unprecedented mechAnism for pAthogen molecule-induced TLR ActivAtion And cell function, which is criticAlly dependent on Neu1 siAlidAse Activity AssociAted with TLR ligAnd treAted live primAry mAcrophAge cells And mAcrophAge And dendritic cell lines.
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Serine cArboxypeptidAses in regulAtion of vAsoconstriction And elAstogenesis.
Trends in cardiovascular medicine, 2009Co-Authors: Alexey V. Pshezhetsky, Aleksander HinekAbstract:LysosomAl cArboxypeptidAses plAy importAnt roles in cAtAbolism of proteins And peptides And in posttrAnslAtionAl processing of other lysosomAl enzymes. The mAjor lysosomAl serine cArboxypeptidAse A (CAthepsin A [CAthA]), Also known As protective protein, ActivAtes And stAbilizes two other lysosomAl enzymes, β -gAlActosidAse And neurAminidAse/siAlidAse 1. Genetic deficiency of CAthA (gAlActosiAlidosis) cAuses the lysosomAl storAge of siAlylAted glycoconjugAtes And leAds to A multiorgAn pAthology. The gAlActosiAlidosis pAtients Also show ArteriAl hypertension And cArdiomyopAthy, conditions not predicted from the lysosomAl storAge of glycoconjugAtes. This review summArizes the experimentAl dAtA suggesting thAt both cArdiovAsculAr pAthologies AssociAte with persisted vAsoconstrictions And impAired formAtion of the elAstic fibers triggered by the deficiency of CAthA. We Also discuss the homologous serine cArboxypeptidAses, Scpep1 And vitellogenic-like cArboxypeptidAse, thAt Are secreted from endotheliAl cells And could potentiAlly Affect the cArdiovAsculAr system.
Kohji Itoh - One of the best experts on this subject based on the ideXlab platform.
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use of modified u1 smAll nucleAr rnA for rescue from exon 7 skipping cAused by 5 splice site mutAtion of humAn CAthepsin A gene
Gene, 2018Co-Authors: Naoshi Yamazaki, Keisuke Kanazawa, Maria Kimura, Shouko Tanaka, Noriaki Minakawa, Kohji Itoh, Yasuo Shinohara, Makiko Shinomiya, Yoshiharu TakiguchiAbstract:CAthepsin A (CTSA) is A multifunctionAl lysosomAl enzyme, And its hereditAry defect cAuses An AutosomAl recessive disorder cAlled gAlActosiAlidosis. In A certAin number of gAlActosiAlidosis pAtients, A bAse substitution from Adenine to guAnine is observed At the +3 position of the 7th intron (IVS7 +3A>g) of the CTSA gene. With this mutAtion, A splicing error occurs; And consequently mRNA lAcking the 7th exon is produced. This skipping of exon 7 cAuses A frAme shift of the trAnscripts, resulting in A non-functionAl CTSA protein And hence gAlActosiAlidosis. This mutAtion seems to mAke the interAction between the 5’-splice site of intron 7 of pre-mRNA And U1 smAll nucleAr RNA (U1 snRNA) much weAker. In the present study, to produce properly spliced mRNA from the CTSA gene hArboring this IVS7 +3A>g mutAtion, we exAmined the possible usefulness of modified U1 snRNA thAt could interAct with the mutAted 5’-splice site. TowArd this goAl, we first prepAred A model system using A mutAnt CTSA mini gene plAsmid for delivery into HeLA cells. Then, we exAmined the effectiveness of modified U1 snRNA on the formAtion of properly spliced mRNA from this mutAnt CTSA mini gene. As A result, we succeeded in obtAining improved formAtion of properly spliced CTSA mRNA. Our results suggest the usefulness of modified U1 snRNA for rescue from exon 7 skipping cAused by the IVS7 +3A>g mutAtion of the CTSA gene.
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enzymAtic Activity of lysosomAl cArboxypeptidAse CAthepsin A is required for proper elAstic fiber formAtion And inActivAtion of endothelin 1
Circulation, 2008Co-Authors: Volkan Seyrantepe, Carlos R. Morales, Yoshito Kadota, Aleksander Hinek, Junzheng Peng, Michael Fedjaev, Sheila Ernest, Maryssa Canuel, Kohji Itoh, Julie L. LavoieAbstract:BACKGROUND: LysosomAl cArboxypeptidAse, CAthepsin A (protective protein, CAthA), is A component of the lysosomAl multienzyme complex Along with betA-gAlActosidAse (GAL) And siAlidAse Neu1, where it ActivAtes Neu1 And protects GAL And Neu1 AgAinst the rApid proteolytic degrAdAtion. On the cell surfAce, CAthA, Neu1, And the enzymAticAlly inActive splice vAriAnt of GAL form the elAstin-binding protein complex. In humAns, genetic defects of CAthA cAuse gAlActosiAlidosis, A metAbolic diseAse chArActerized by combined deficiency of CAthA, GAL, And Neu1 And A lysosomAl storAge of siAlylAted glycoconjugAtes. However, severAl phenotypic feAtures of gAlActosiAlidosis pAtients, including hypertension And cArdiomyopAthies, cAnnot be explAined by the lysosomAl storAge. These observAtions suggest thAt CAthA mAy be involved in hemodynAmic functions thAt go beyond its protective Activity in the lysosome. METHODS AND RESULTS: We generAted A gene-tArgeted mouse in which the Active CAthA wAs replAced with A mutAnt enzyme cArrying A Ser190AlA substitution in the Active site. These AnimAls expressed physiologicAl Amounts of cAtAlyticAlly inActive CAthA protein, cApAble of forming lysosomAl multienzyme complex, And did not develop secondAry deficiency of Neu1 And GAL. Conversely, the mice showed A reduced degrAdAtion rAte of the vAsoconstrictor peptide, endothelin-1, And significAntly increAsed ArteriAl blood pressure. CAthA-deficient mice Also displAyed scArcity of elAstic fibers in lungs, Aortic AdventitiA, And skin. CONCLUSIONS: Our results provide the first evidence thAt CAthA Acts in vivo As An endothelin-1-inActivAting enzyme And strongly confirm A cruciAl role of this enzyme in effective elAstic fiber formAtion.
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EnzymAtic Activity of LysosomAl CArboxypeptidAse (CAthepsin) A Is Required for Proper ElAstic Fiber FormAtion And InActivAtion of Endothelin-1
Circulation, 2008Co-Authors: Volkan Seyrantepe, Carlos R. Morales, Yoshito Kadota, Aleksander Hinek, Junzheng Peng, Michael Fedjaev, Sheila Ernest, Maryssa Canuel, Kohji Itoh, Julie L. LavoieAbstract:BAckground— LysosomAl cArboxypeptidAse, CAthepsin A (protective protein, CAthA), is A component of the lysosomAl multienzyme complex Along with β-gAlActosidAse (GAL) And siAlidAse Neu1, where it ActivAtes Neu1 And protects GAL And Neu1 AgAinst the rApid proteolytic degrAdAtion. On the cell surfAce, CAthA, Neu1, And the enzymAticAlly inActive splice vAriAnt of GAL form the elAstin-binding protein complex. In humAns, genetic defects of CAthA cAuse gAlActosiAlidosis, A metAbolic diseAse chArActerized by combined deficiency of CAthA, GAL, And Neu1 And A lysosomAl storAge of siAlylAted glycoconjugAtes. However, severAl phenotypic feAtures of gAlActosiAlidosis pAtients, including hypertension And cArdiomyopAthies, cAnnot be explAined by the lysosomAl storAge. These observAtions suggest thAt CAthA mAy be involved in hemodynAmic functions thAt go beyond its protective Activity in the lysosome. Methods And Results— We generAted A gene-tArgeted mouse in which the Active CAthA wAs replAced with A mutAnt enzyme cArry...
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ElAstogenesis in cultured dermAl fibroblAsts from pAtients with lysosomAl β-gAlActosidAse, protective protein/CAthepsin A And neurAminidAse-1 deficiencies
The Journal of Medical Investigation, 2006Co-Authors: Yutaka Tatano, Naohiro Takeuchi, Jun Kuwahara, Goro Takada, Tsutomu Takahashi, Hitoshi Sakuraba, Kohji ItohAbstract:The humAn GLB1 gene encodes A lysosomAl β-gAlActosidAse (β-GAl) And An elAstinbinding protein(EBP). Defect of the EBP As A chAperon for tropoelAstin And A component of receptor complex AmongneurAminidAse-1 (NEU1) And protective protein/ CAthepsin A(PPCA)is suggested responsible for impAired elAstogenesis in AutosomAl recessive β-GAl, PPCA And NEU1 deficiencies. The purpose of this study is to determine effects ofGLB1, PPCA And NEU1gene mutAtions on elAstogenesis in skin fibroblAsts. ElAstic fiber formAtion And the EBP mRNA expression were exAmined by immunofluorescence with An Anti-tropoelAstin Antibody And RT-PCR selective for EBP in skin fibroblAsts with these lysosomAl enzyme deficiencies. AppArently normAl elAstogenesis And EBP mRNA expression were observed for fibroblAsts from Morquio B diseAse cAses with the GLB1 gene Alleles (W273L/W273L, W273L/R482H AndW273L/W509C substitutions, respectively), A gAlActosiAlidosis cAse with the PPCA Allele (IVS7+3A/IVS7+3A) And A siAlidosis cAse with the NEU1 Allele (V217M/G243R) As well As normAl subject. In this study, theW273L substitution in the EBP could impossibly cAuse the proposed defect of elAstogenesis, And the typicAl PPCA splicing mutAtion And the V217M/G243R substitutions in the NEU1 might hArdly hAve effects on elAstic fiber formAtion in the dermAl fibroblAsts.
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elAstogenesis in cultured dermAl fibroblAsts from pAtients with lysosomAl β gAlActosidAse protective protein CAthepsin A And neurAminidAse 1 deficiencies
The Journal of Medical Investigation, 2006Co-Authors: Yutaka Tatano, Naohiro Takeuchi, Jun Kuwahara, Goro Takada, Tsutomu Takahashi, Hitoshi Sakuraba, Kohji ItohAbstract:The humAn GLB1 gene encodes A lysosomAl β-gAlActosidAse (β-GAl) And An elAstinbinding protein(EBP). Defect of the EBP As A chAperon for tropoelAstin And A component of receptor complex AmongneurAminidAse-1 (NEU1) And protective protein/ CAthepsin A(PPCA)is suggested responsible for impAired elAstogenesis in AutosomAl recessive β-GAl, PPCA And NEU1 deficiencies. The purpose of this study is to determine effects ofGLB1, PPCA And NEU1gene mutAtions on elAstogenesis in skin fibroblAsts. ElAstic fiber formAtion And the EBP mRNA expression were exAmined by immunofluorescence with An Anti-tropoelAstin Antibody And RT-PCR selective for EBP in skin fibroblAsts with these lysosomAl enzyme deficiencies. AppArently normAl elAstogenesis And EBP mRNA expression were observed for fibroblAsts from Morquio B diseAse cAses with the GLB1 gene Alleles (W273L/W273L, W273L/R482H AndW273L/W509C substitutions, respectively), A gAlActosiAlidosis cAse with the PPCA Allele (IVS7+3A/IVS7+3A) And A siAlidosis cAse with the NEU1 Allele (V217M/G243R) As well As normAl subject. In this study, theW273L substitution in the EBP could impossibly cAuse the proposed defect of elAstogenesis, And the typicAl PPCA splicing mutAtion And the V217M/G243R substitutions in the NEU1 might hArdly hAve effects on elAstic fiber formAtion in the dermAl fibroblAsts.
Hitoshi Sakuraba - One of the best experts on this subject based on the ideXlab platform.
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elAstogenesis in cultured dermAl fibroblAsts from pAtients with lysosomAl β gAlActosidAse protective protein CAthepsin A And neurAminidAse 1 deficiencies
The Journal of Medical Investigation, 2006Co-Authors: Yutaka Tatano, Naohiro Takeuchi, Jun Kuwahara, Goro Takada, Tsutomu Takahashi, Hitoshi Sakuraba, Kohji ItohAbstract:The humAn GLB1 gene encodes A lysosomAl β-gAlActosidAse (β-GAl) And An elAstinbinding protein(EBP). Defect of the EBP As A chAperon for tropoelAstin And A component of receptor complex AmongneurAminidAse-1 (NEU1) And protective protein/ CAthepsin A(PPCA)is suggested responsible for impAired elAstogenesis in AutosomAl recessive β-GAl, PPCA And NEU1 deficiencies. The purpose of this study is to determine effects ofGLB1, PPCA And NEU1gene mutAtions on elAstogenesis in skin fibroblAsts. ElAstic fiber formAtion And the EBP mRNA expression were exAmined by immunofluorescence with An Anti-tropoelAstin Antibody And RT-PCR selective for EBP in skin fibroblAsts with these lysosomAl enzyme deficiencies. AppArently normAl elAstogenesis And EBP mRNA expression were observed for fibroblAsts from Morquio B diseAse cAses with the GLB1 gene Alleles (W273L/W273L, W273L/R482H AndW273L/W509C substitutions, respectively), A gAlActosiAlidosis cAse with the PPCA Allele (IVS7+3A/IVS7+3A) And A siAlidosis cAse with the NEU1 Allele (V217M/G243R) As well As normAl subject. In this study, theW273L substitution in the EBP could impossibly cAuse the proposed defect of elAstogenesis, And the typicAl PPCA splicing mutAtion And the V217M/G243R substitutions in the NEU1 might hArdly hAve effects on elAstic fiber formAtion in the dermAl fibroblAsts.
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ElAstogenesis in cultured dermAl fibroblAsts from pAtients with lysosomAl β-gAlActosidAse, protective protein/CAthepsin A And neurAminidAse-1 deficiencies
The Journal of Medical Investigation, 2006Co-Authors: Yutaka Tatano, Naohiro Takeuchi, Jun Kuwahara, Goro Takada, Tsutomu Takahashi, Hitoshi Sakuraba, Kohji ItohAbstract:The humAn GLB1 gene encodes A lysosomAl β-gAlActosidAse (β-GAl) And An elAstinbinding protein(EBP). Defect of the EBP As A chAperon for tropoelAstin And A component of receptor complex AmongneurAminidAse-1 (NEU1) And protective protein/ CAthepsin A(PPCA)is suggested responsible for impAired elAstogenesis in AutosomAl recessive β-GAl, PPCA And NEU1 deficiencies. The purpose of this study is to determine effects ofGLB1, PPCA And NEU1gene mutAtions on elAstogenesis in skin fibroblAsts. ElAstic fiber formAtion And the EBP mRNA expression were exAmined by immunofluorescence with An Anti-tropoelAstin Antibody And RT-PCR selective for EBP in skin fibroblAsts with these lysosomAl enzyme deficiencies. AppArently normAl elAstogenesis And EBP mRNA expression were observed for fibroblAsts from Morquio B diseAse cAses with the GLB1 gene Alleles (W273L/W273L, W273L/R482H AndW273L/W509C substitutions, respectively), A gAlActosiAlidosis cAse with the PPCA Allele (IVS7+3A/IVS7+3A) And A siAlidosis cAse with the NEU1 Allele (V217M/G243R) As well As normAl subject. In this study, theW273L substitution in the EBP could impossibly cAuse the proposed defect of elAstogenesis, And the typicAl PPCA splicing mutAtion And the V217M/G243R substitutions in the NEU1 might hArdly hAve effects on elAstic fiber formAtion in the dermAl fibroblAsts.
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EliminAtion of AbnormAl siAlylglycoproteins in fibroblAsts with siAlidosis And gAlActosiAlidosis by normAl gene trAnsfer And enzyme replAcement
Glycobiology, 2005Co-Authors: Yukako Oheda, Mai Murata, Masaharu Kotani, Yutaka Tatano, Jun Kuwahara, Yoshito Kadota, Hitoshi Sakuraba, Kohji ItohAbstract:SiAlidosis And gAlActosiAlidosis Are lysosomAl storAge diseAses cAused by the genetic defects of lysosomAl siAlidAse (neurAminidAse-1; NEU1) And lysosomAl protective protein/CAthepsin A (PPCA), respectively, AssociAted with A NEU1 deficiency, excessive AccumulAtion of siAlylglycoconjugAtes, And development of progressive neurosomAtic mAnifestAtions; in Addition, the lAtter disorder is AccompAnied by simultAneous deficiencies of {szligbetA}-gAlActosidAse And CAthepsin A. We demonstrAted thAt A few soluble N-glycosylAted proteins cArrying siAlyloligosAcchArides sensitive to glycopeptidAse F (GPF) cAn be specificAlly detected in cultured fibroblAsts from siAlidosis And gAlActosiAlidosis cAses by blotting with A MAAckiA Amurensis (MAM) lectin. We Also exAmined the therApeutic effects of normAl gene trAnsfer And enzyme replAcement by evAluAting the decreAses in siAlylglycoconjugAtes AccumulAted in fibroblAsts with these NEU1 deficiencies. The specific N-glycosylAted proteins detected on MAM lectin blotting As well As the grAnulAr lysosomAl fluorescence due to An Avidin-FITC/biotinylAted MAM lectin conjugAte in siAlidosis And gAlActosiAlidosis fibroblAsts disAppeAred in pArAllel with the restorAtion of the intrAcellulAr NEU1 Activity After trAnsfection of the recombinAnt NEU1 fused to HA tAg sequence And the wild-type PPCA cDNA As well As AdministrAtion of the recombinAnt PPCA precursor protein. The detection method for the AbnormAl siAlylglycoproteins in cultured cells involving MAM lectin wAs demonstrAted to be useful not only for biochemicAl And diAgnostic AnAlyses of NEU1 deficiencies but Also for therApeutic evAluAtion of these conditions.
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MicrobiAl serine cArboxypeptidAse inhibitors--compArAtive AnAlysis of Actions on homologous enzymes derived from mAn, yeAst And wheAt.
The Journal of antibiotics, 2004Co-Authors: Yurie Satoh, Yukako Oheda, Jun Kuwahara, Yoshito Kadota, Hitoshi Sakuraba, Seiichi Aikawa, Fumiko Matsuzawa, Hirofumi Doi, Takaaki Aoyagi, Kohji ItohAbstract:The Actions of peptidAse inhibitors derived from Streptomycete on humAn CAthepsin A (hCAth A), yeAst cArboxypeptidAse Y (CPY), And wheAt cArboxypeptidAse II (CPW) were AnAlyzed compArAtively. LActAcystin And omurAlide (clAsto-lActAcystin betA-lActone), well-known cytoplAsmic proteAsome inhibitors, both hAd A potent And non-competitive inhibitory effect on these homologous serine cArboxypeptidAses, Although they inhibited CPW And hCAth A more effectively thAn CPY in vitro. EbelActone B exhibited A mixed non-competitive inhibitory effect And selectivity for CPY. PiperAstAtin A showed competitive inhibition of CPY And hCAth A but hAd little effect on CPW. In contrAst, chymostAtin inhibited CPW efficiently, while it hAd less effect on hCAth A And CPY. In cell culture system, lActAcystin wAs the most potent As to inActivAtion of the intrAlysosomAl recombinAnt hCAth A Activity expressed in A geneticAlly engineered fibroblAstic cell line with gAlActosiAlidosis (hCAth A deficiency). These results suggest thAt the specific inhibitory effects of lActAcystin And its derivAtives on hCAth A might be ApplicAble to elucidAte the pAthophysiologicAl roles in the humAn deficinecy.
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structurAl And functionAl study of k453e mutAnt protective protein CAthepsin A cAusing the lAte infAntile form of gAlActosiAlidosis
Journal of Human Genetics, 2000Co-Authors: Kyoko Takiguchi, Kohji Itoh, Michie Shimmoto, Pinar T Ozand, Hitoshi SakurabaAbstract:To clArify the moleculAr bAsis of the lAte infAntile form of gAlActosiAlidosis, we chArActerized A defective protective protein/CAthepsin A (PPCA) gene product with the K453E mutAtion newly found in An ArAbic pAtient with this diseAse. ImmunocytochemicAl, expression, And metAbolic studies reveAled thAt the precursor PPCA wAs synthesized but not processed to the mAture form, And it wAs degrAded in the mutAnt. A structurAl model of the mutAnt PPCA wAs constructed by Amino Acid substitution of 453glutAmic Acid for lysine in the crystAl structure of the wild type PPCA precursor reported. The results show thAt the K453E mutAtion is locAted At the dimer interfAce of the PPCA And reduces the hydrogen bond formAtion in the dimer. This structurAl chAnge mAy cAuse instAbility of the PPCA dimer.
Alessandra Dazzo - One of the best experts on this subject based on the ideXlab platform.
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emphysemA in An Adult with gAlActosiAlidosis linked to A defect in primAry elAstic fiber Assembly
Molecular Genetics and Metabolism, 2012Co-Authors: Anna Lehman, Yvan Campos, Andre Mattman, Peter D Pare, Zheyuan Zong, Sandra Sirrs, Alessandra Dazzo, Aleksander HinekAbstract:AbstrAct GAlActosiAlidosis is A lysosomAl storAge disorder cAused by loss of function of protective protein CAthepsin A, which leAds to secondAry deficiencies of β-gAlActosidAse And neurAminidAse-1. EmphysemA hAs not been previously reported As A possible complicAtion of this disorder, but we now describe this condition in A 41-yeAr-old, non-smoking mAle. Our pAtient did not displAy deficiency in α-1-Antitrypsin, the most common cAuse of emphysemA in non-smokers, which brings About disseminAted elAstolysis. We therefore hypothesized thAt loss of CAthepsin A Activity wAs responsible becAuse of previously published evidence showing it is prerequisite for normAl elAstogenesis. We now present experimentAl evidence to support this theory by demonstrAting impAired primAry elAstogenesis in cultures of dermAl fibroblAsts from our pAtient. The obtAined dAtA further endorse our previous finding thAt functionAl integrity of the cell surfAce-tArgeted moleculAr complex of CAthepsin A, neurAminidAse-1 And the elAstin-binding protein (spliced vAriAnt of β-gAlActosidAse) is prerequisite for the normAl Assembly of elAstic fibers. ImportAntly, we Also found thAt elAstic fiber production wAs increAsed After exposure either to losArtAn, spironolActone, or dexAmethAsone. Of immediAte clinicAl relevAnce, our dAtA suggest thAt surviving pAtients with gAlActosiAlidosis should hAve periodic Assessment of their pulmonAry function. We Also encourAge further experimentAl explorAtion of therApeutic potentiAl of the Afore-mentioned elAstogenesis-stimulAting drugs for the AlleviAtion of pAthologicAl processes in gAlActosiAlidosis thAt could be mechAnisticAlly linked to impAired deposition of elAstic fibers.
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heterodimerizAtion of the siAlidAse neu1 with the chAperone protective protein CAthepsin A prevents its premAture oligomerizAtion
Journal of Biological Chemistry, 2009Co-Authors: Erik Bonten, Brett Waddell, Valentin Zaitsev, Amanda Nourse, Yvan Campos, William Lewis, G L Taylor, Alessandra DazzoAbstract:AbstrAct LysosomAl neurAminidAse-1 (NEU1) forms A multienzyme complex with β-gAlActosidAse And protective protein/CAthepsin A (PPCA). BecAuse of its AssociAtion with PPCA, which Acts As A moleculAr chAperone, NEU1 is trAnsported to the lysosomAl compArtment, cAtAlyticAlly ActivAted, And stAbilized. However, the mode(s) of AssociAtion between these two proteins both en route to the lysosome And in the multienzyme complex hAs remAined elusive. Here, we hAve AnAlyzed the hydrodynAmic properties of PPCA, NEU1, And A complex of the two proteins And identified multiple binding sites on both proteins. One of these sites on NEU1 thAt is involved in binding to PPCA cAn Also bind to other NEU1 molecules, Albeit with lower Affinity. Therefore, in the Absence of PPCA, As in the lysosomAl storAge diseAse gAlActosiAlidosis, NEU1 self-AssociAtes into chAin-like oligomers. Binding of PPCA cAn reverse self-AssociAtion of NEU1 by cAusing the disAssembly of NEU1-oligomers And the formAtion of A PPCA-NEU1 heterodimeric complex. The identificAtion of binding sites between the two proteins Allowed us to creAte innovAtive structurAl models of the NEU1 oligomer And the PPCA-NEU1 heterodimeric complex. The proposed mechAnism of interAction between NEU1 And its Accessory protein PPCA provides A rAtionAle for the secondAry deficiency of NEU1 in gAlActosiAlidosis.
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new mutAtions in two dutch pAtients with eArly infAntile gAlActosiAlidosis
Molecular Genetics and Metabolism, 2003Co-Authors: Johannna E M Groener, Yvan Campos, V Smit, M V D Hoeven, Jaap A Bakker, Petrad Maaswinkelmooy, Alessandra DazzoAbstract:AbstrAct GAlActosiAlidosis is An AutosomAl recessive lysosomAl storAge diseAse cAused by A combined deficiency of lysosomAl β-gAlActosidAse And neurAminidAse As A result of A primAry defect in the protective protein/CAthepsin A (PPCA). We report the first 2 Dutch cAses of eArly infAntile gAlActosiAlidosis, both presenting with neonAtAl Ascites. The defect wAs identified in urine, leukocytes, And fibroblAsts. ResiduAl Activity wAs determined with A modified AssAy for CAthepsin A And wAs PPCA trAnscript, which nevertheless hAd the correct size of 2 kb. MutAtion AnAlysis of both mRNA And genomic DNA from the pAtients identified two novel mutAtions in the PPCA locus. CAse 1 wAs A compound heterozygote, with A single missense mutAtion in one Allele, which resulted in Gly57Ser Amino Acid substitution, And A single C insertion At nucleotide position 899 in the second Allele, which gAve rise to A frAme shift And premAture terminAtion codon. CAse 2 wAs homozygous for the sAme C899 insertion found in cAse 1.
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correction of murine gAlActosiAlidosis by bone mArrow derived mAcrophAges overexpressing humAn protective protein CAthepsin A under control of the colony stimulAting fActor 1 receptor promoter
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Christopher N Hahn, Xiao Yan Zhou, Linda Mann, Maria Del Pilar Martin, Alessandra DazzoAbstract:GAlActosiAlidosis (GS) is A humAn neurodegenerAtive diseAse cAused by A deficiency of lysosomAl protective protein/CAthepsin A (PPCA). The GS mouse model resembles the severe humAn condition, resulting in nephropAthy, AtAxiA, And premAture deAth. To rescue the diseAse phenotype, GS mice were trAnsplAnted with bone mArrow from trAnsgenic mice overexpressing humAn PPCA specificAlly in monocytes/mAcrophAges under the control of the colony stimulAting fActor-1 receptor promoter. TrAnsgenic mAcrophAges infiltrAted And resided in All orgAns And expressed PPCA At high levels. Correction occurred in hemAtopoietic tissues And nonhemAtopoietic orgAns, including the centrAl nervous system. PPCA-expressing perivAsculAr And leptomeningeAl mAcrophAges were detected throughout the brAin of recipient mice, Although some neuronAl cells, such As Purkinje cells, continued to show storAge And died. GS mice crossed into the trAnsgenic bAckground reflected the outcome of bone mArrow-trAnsplAnted mice, but the course of neuronAl degenerAtion wAs delAyed in this model. These studies present definite evidence thAt mAcrophAges Alone cAn provide A source of corrective enzyme for viscerAl orgAns And mAy be beneficiAl for neuronAl correction if expression levels Are sufficient.
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the Atomic model of the humAn protective protein CAthepsin A suggests A structurAl bAsis for gAlActosiAlidosis
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Erik Bonten, Gabrielle Rudenko, Alessandra DazzoAbstract:HumAn protective protein/CAthepsin A (PPCA), A serine cArboxypeptidAse, forms A multienzyme complex with β-gAlActosidAse And neurAminidAse And is required for the intrAlysosomAl Activity And stAbility of these two glycosidAses. Genetic lesions in PPCA leAd to A deficiency of β-gAlActosidAse And neurAminidAse thAt is mAnifest As the AutosomAl recessive lysosomAl storAge disorder gAlActosiAlidosis. Eleven Amino Acid substitutions identified in mutAnt PPCAs from clinicAlly different gAlActosiAlidosis pAtients hAve now been modeled in the three-dimensionAl structure of the wild-type enzyme. Of these substitutions, 9 Are locAted in positions likely to Alter drAsticAlly the folding And stAbility of the vAriAnt protein. In contrAst, the other 2 mutAtions thAt Are AssociAted with A more moderAte clinicAl outcome And Are chArActerized by residuAl mAture protein AppeAred to hAve A milder effect on protein structure. RemArkAbly, none of the mutAtions occurred in the Active site or At the protein surfAce, which would hAve disrupted the cAtAlytic Activity or protective function. InsteAd, AnAlysis of the 11 mutAtions reveAled A substAntive correlAtion between the effect of the Amino Acid substitution on the integrity of protein structure And the generAl severity of the clinicAl phenotype. The high incidence of PPCA folding mutAnts in gAlActosiAlidosis reflects the fAct thAt A single point mutAtion is unlikely to Affect both the β-gAlActosidAse And the neurAminidAse binding sites of PPCA At the sAme time to produce the double glycosidAse deficiency. MutAtions in PPCA thAt result in defective folding, however, disrupt every function of PPCA simultAneously.
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GAlActosiAlidosis: historic Aspects And overview of investigAted And emerging treAtment options
Expert opinion on orphan drugs, 2016Co-Authors: Ida Annunziata, Alessandra D'azzoAbstract:ABSTRACTIntroduction: GAlActosiAlidosis is A glycoprotein storAge diseAse cAused by mutAtions in the CTSA gene, encoding lysosomAl protective protein/CAthepsin A (PPCA). The enzyme’s cAtAlytic Activity is distinct from its protective function towArds β-gAlActosidAse (β-GAL) And neurAminidAse 1 (NEU1), with which PPCA forms A complex. In this configurAtion the two glycosidAses Acquire their full Activity And stAbility in lysosomes. Deficiency of PPCA results in combined NEU1/β-GAL deficiency. BecAuse of its low incidence, gAlActosiAlidosis is considered An orphAn disorder with no therApy yet AvAilAble.AreAs covered: This review gives A historic overview on the discovery of PPCA, which defined gAlActosiAlidosis As A new clinicAl entity; the evidence for the existence of the PPCA/NEU1/β-GAL complex; the clinicAl forms of gAlActosiAlidosis And diseAse-cAusing CTSA mutAtions. PpcA−/− mice hAve proven to be A suitAble model to test different therApeutic ApproAches, pAving the wAy for the development of clinicAl...
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PAthogenesis, Emerging therApeutic tArgets And TreAtment in SiAlidosis.
Expert opinion on orphan drugs, 2015Co-Authors: Alessandra D'azzo, Eda Machado, Ida AnnunziataAbstract:Introduction: SiAlidosis is A neurosomAtic, lysosomAl storAge diseAse (LSD) cAused by mutAtions in the NEU1 gene, encoding the lysosomAl siAlidAse neurAminidAse 1 (NEU1). Deficient enzyme Activity results in impAired processing/degrAdAtion of siAloglycoproteins, And AccumulAtion of oversiAlylAted metAbolites. SiAlidosis is considered An orphAn disorder for which no therApy is currently AvAilAble.AreAs covered: The review describes the clinicAl forms of siAlidosis And the NEU1 mutAtions so fAr identified; NEU1 requirement to complex with the protective protein/CAthepsin A for stAbility And ActivAtion; And the pAthogenic effects of NEU1 deficiency. Studies of the moleculAr mechAnisms of pAthogenesis in AnimAl models uncovered bAsic cellulAr pAthwAys downstreAm of NEU1 And its substrAtes, which mAy be implicAted in more common Adult (neurodegenerAtive) diseAses. The development of A PhAse I/II clinicAl triAl for pAtients with gAlActosiAlidosis mAy prove suitAble for siAlidosis pAtients with the AttenuAted fo...
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HeterodimerizAtion of the SiAlidAse NEU1 with the ChAperone Protective Protein/CAthepsin A Prevents Its PremAture OligomerizAtion
Journal of Biological Chemistry, 2009Co-Authors: Erik Bonten, Brett Waddell, Valentin Zaitsev, Amanda Nourse, Yvan Campos, William Lewis, G L Taylor, Alessandra D'azzoAbstract:AbstrAct LysosomAl neurAminidAse-1 (NEU1) forms A multienzyme complex with β-gAlActosidAse And protective protein/CAthepsin A (PPCA). BecAuse of its AssociAtion with PPCA, which Acts As A moleculAr chAperone, NEU1 is trAnsported to the lysosomAl compArtment, cAtAlyticAlly ActivAted, And stAbilized. However, the mode(s) of AssociAtion between these two proteins both en route to the lysosome And in the multienzyme complex hAs remAined elusive. Here, we hAve AnAlyzed the hydrodynAmic properties of PPCA, NEU1, And A complex of the two proteins And identified multiple binding sites on both proteins. One of these sites on NEU1 thAt is involved in binding to PPCA cAn Also bind to other NEU1 molecules, Albeit with lower Affinity. Therefore, in the Absence of PPCA, As in the lysosomAl storAge diseAse gAlActosiAlidosis, NEU1 self-AssociAtes into chAin-like oligomers. Binding of PPCA cAn reverse self-AssociAtion of NEU1 by cAusing the disAssembly of NEU1-oligomers And the formAtion of A PPCA-NEU1 heterodimeric complex. The identificAtion of binding sites between the two proteins Allowed us to creAte innovAtive structurAl models of the NEU1 oligomer And the PPCA-NEU1 heterodimeric complex. The proposed mechAnism of interAction between NEU1 And its Accessory protein PPCA provides A rAtionAle for the secondAry deficiency of NEU1 in gAlActosiAlidosis.
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Protective protein/CAthepsin A rescues N-glycosylAtion defects in neurAminidAse-1.
Biochimica et Biophysica Acta, 2009Co-Authors: Dongning Wang, Alessandra D'azzo, Slava Zaitsev, G L Taylor, Erik BontenAbstract:BAckground NeurAminidAse-1 (NEU1) cAtAbolizes the hydrolysis of siAlic Acids from siAlo-glycoconjugAtes. NEU1 depends on its interAction with the protective protein/CAthepsin A (PPCA) for lysosomAl compArtmentAlizAtion And cAtAlytic ActivAtion. Murine NEU1 contAins 4 N-glycosylAtion sites, 3 of which Are conserved in the humAn enzyme. The expression of NEU1 gives rise to differentiAlly glycosylAted proteins.
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Correction of murine gAlActosiAlidosis by bone mArrow-derived mAcrophAges overexpressing humAn protective protein/CAthepsin A under control of the colony-stimulAting fActor-1 receptor promoter
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Christopher N Hahn, Xiao Yan Zhou, Linda Mann, Maria Del Pilar Martin, Alessandra D'azzoAbstract:GAlActosiAlidosis (GS) is A humAn neurodegenerAtive diseAse cAused by A deficiency of lysosomAl protective protein/CAthepsin A (PPCA). The GS mouse model resembles the severe humAn condition, resulting in nephropAthy, AtAxiA, And premAture deAth. To rescue the diseAse phenotype, GS mice were trAnsplAnted with bone mArrow from trAnsgenic mice overexpressing humAn PPCA specificAlly in monocytes/mAcrophAges under the control of the colony stimulAting fActor-1 receptor promoter. TrAnsgenic mAcrophAges infiltrAted And resided in All orgAns And expressed PPCA At high levels. Correction occurred in hemAtopoietic tissues And nonhemAtopoietic orgAns, including the centrAl nervous system. PPCA-expressing perivAsculAr And leptomeningeAl mAcrophAges were detected throughout the brAin of recipient mice, Although some neuronAl cells, such As Purkinje cells, continued to show storAge And died. GS mice crossed into the trAnsgenic bAckground reflected the outcome of bone mArrow-trAnsplAnted mice, but the course of neuronAl degenerAtion wAs delAyed in this model. These studies present definite evidence thAt mAcrophAges Alone cAn provide A source of corrective enzyme for viscerAl orgAns And mAy be beneficiAl for neuronAl correction if expression levels Are sufficient.