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Judith S Bond - One of the best experts on this subject based on the ideXlab platform.
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Meprin A impAirs epitheliAl bArrier function enhAnces monocyte migrAtion And cleAves the tight junction protein occludin
American Journal of Physiology-renal Physiology, 2013Co-Authors: Jialing Bao, Gail L Matters, Renee Yura, Gaylen S Bradley, Pan Shi, Fang Tian, Judith S BondAbstract:Meprin metAlloproteAses Are highly expressed At the luminAl interfAce of the intestine And kidney And in certAin leukocytes. Meprins cleAve A vAriety of substrAtes in vitro, including extrAcellulAr mAtrix proteins, Adherens junction proteins, And cytokines, And hAve been implicAted in A number of inflAmmAtory diseAses. The linkAge between results in vitro And pAthogenesis, however, hAs not been elucidAted. The present study Aimed to determine whether Meprins Are determinAtive fActors in disrupting the bArrier function of the epithelium. Active Meprin A or Meprin B Applied to MAdin-DArby cAnine kidney (MDCK) cell monolAyers increAsed permeAbility to fluorescein isothiocyAnAte-dextrAn And disrupted immunostAining of the tight junction protein occludin but not clAudin-4. Meprin A, but not Meprin B, cleAved occludin in MDCK monolAyers. Experiments with recombinAnt occludin demonstrAted thAt Meprin A cleAves the protein between Gly100 And Ser101 on the first extrAcellulAr loop. In vivo experiments demonstrAted thAt Meprin A infused into the mouse blAdder increAsed the epithelium permeAbility to sodium fluorescein. Furthermore, monocytes from Meprin knockout mice on A C57BL/6 bAckground were less Able to migrAte through An MDCK monolAyer thAn monocytes from their wild-type counterpArts. These results demonstrAte the cApAbility of Meprin A to disrupt epitheliAl bArriers And implicAte occludin As one of the importAnt tArgets of Meprin A thAt mAy modulAte inflAmmAtion.
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bAlAnce of Meprin A And b in mice Affects the progression of experimentAl inflAmmAtory bowel diseAse
American Journal of Physiology-gastrointestinal and Liver Physiology, 2011Co-Authors: Sanjita Banerjee, Gail L Matters, Gaylen S Bradley, Jacqueline M Crisman, Ge Jin, Ryan D Gailey, Judith S BondAbstract:MEP1A, which encodes the α subunit of Meprin metAlloproteinAses, is A susceptibility gene for inflAmmAtory bowel diseAse (IBD), And decreAsed intestinAl Meprin-α expression is AssociAted with enhAn...
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Meprin A metAlloproteAses enhAnce renAl dAmAge And blAdder inflAmmAtion After lps chAllenge
American Journal of Physiology-renal Physiology, 2009Co-Authors: Renee Yura, Gaylen S Bradley, Ganesan Ramesh, Brian W Reeves, Judith S BondAbstract:Meprin metAlloproteAses, composed of α And/or β subunits, consist of membrAne-bound And secreted forms thAt Are AbundAntly expressed in proximAl tubules of the kidney As well As secreted into the urinAry trAct. Previous studies indicAted thAt Meprin metAlloproteAses plAy A role in pAthologicAl conditions such As ischemic Acute renAl fAilure And urinAry trAct infection. The Aim of this work wAs to exAmine the role of Meprins in endotoxemic Acute renAl fAilure using Meprin α knockout (αKO), Meprin β knockout (βKO), And wild-type (WT) mice. Differences Among the responses of the genotypes were observed As eArly As 1 h After chAllenge with 2.5 mg/kg ip EscherichiA coli LPS, estAblishing roles for Meprins in the endotoxemic response. Meprin αKO mice displAyed lower blood ureA nitrogen levels And decreAsed nitric oxide levels, indicAtive of A decreAsed systemic response to LPS compAred with WT And Meprin βKO mice. Serum cytokine profiles showed lower levels of IL-1β And TNF–α in the Meprin αKO mice within 3 h After LPS chAllenge And confirmed A role for Meprins in the eArly phAses of the host response. Meprin αKO mice were Also hyporesponsive to LPS Administered to the blAdder, exhibiting significAntly less blAdder edemA, leukocyte infiltrAtion, And blAdder permeAbility thAn WT mice. These dAtA indicAte thAt Meprin A contributes to the renAl And urogenitAl pAthogenesis of endotoxicity.
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humAn And mouse homo oligomeric Meprin A metAlloendopeptidAse substrAte And inhibitor specificities
Biological Chemistry, 2007Co-Authors: John E Bylander, Greg P Bertenshaw, Gail L Matters, Simon J Hubbard, Judith S BondAbstract:: Meprin metAlloproteinAses hAve been implicAted in the susceptibility to And progression of diAbetic nephropAthy And inflAmmAtory bowel diseAses. Our studies with experimentAl models of these diseAses in mice Are congruent with the conclusion thAt Meprins modulAte the inflAmmAtory responses And tissue dAmAge. To determine whether the mouse And humAn enzymes differ, recombinAnt forms of Meprin A from the two species were compAred with respect to structure, substrAtes And inhibitors. HumAn homo-oligomeric Meprin A formed oligomers rAnging from 950,000 to 1,500,000 DA vs. 900,000 DA for mouse Meprin A. HumAn And mouse Meprin A exhibited similAr Activity AgAinst AzocAsein, fibronectin, collAgen IV, And peptides such As pArAthyroid hormone, ghrelin, And gAstrin-releAsing peptide. The humAn enzyme hAd lower Activity AgAinst gelAtin, brAdykinin, AlphA-melAnocyte-stimulAting hormone And neurotensin, And higher Activity AgAinst secretin And orcokinin. HumAn Meprin A showed A preference for Acidic residues in the P1' position of the substrAte, unlike mouse Meprin A. SeverAl metAlloproteinAse inhibitors hAd IC(50) vAlues in the nAnomolAr rAnge, but potency rAnged from similAr vAlues to A difference of severAl orders of mAgnitude for Meprins from the two species. This work provides vAluAble dAtA to improve predictAbility for humAn systems bAsed on Meprin functions in mouse models.
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proteAse domAin glycAns Affect oligomerizAtion disulfide bond formAtion And stAbility of the Meprin A metAlloproteAse homo oligomer
Journal of Biological Chemistry, 2006Co-Authors: Susan S Ishmael, Faoud T. Ishmael, Daniel A Jones, Judith S BondAbstract:The Meprin A homo-oligomer is A highly glycosylAted, secreted zinc metAlloproteAse of the AstAcin fAmily And metzincin superfAmily. This isoform of Meprin is composed of disulfide-bonded dimers of α subunits thAt further AssociAte to form lArge, secreted megAdAlton complexes of 10 or more subunits. The Aim of this study wAs to determine the sites of glycAn AttAchment And to Assess their Ability to Affect the formAtion And stAbility of the homo-oligomer. Nine of the ten potentiAl N-linked glycosylAtion sites (Asn-41, Asn-152, Asn-234, Asn-270, Asn-330, Asn-426, Asn-452, Asn-546, And Asn-553) were found to be glycosylAted in recombinAnt mouse Meprin A using chemicAl And enzymAtic deglycosylAtion methods And electrosprAy ionizAtion mAss spectrometry. ChemicAl cross-linking demonstrAted thAt cArbohydrAtes Are At or neAr the noncovAlent subunit interfAce. The removAl of two glycAns in the proteAse domAin At Asn-234 And Asn-270, As well As one in the tumor necrosis fActor receptor-AssociAted fActor domAin At Asn-452, by A deglycosidAse under nondenAturing conditions decreAsed the chemicAl And thermAl stAbility of the homo-oligomer without Affecting quAternAry structure. Site-directed mutAgenesis demonstrAted thAt no single glycAn wAs essentiAl for oligomer formAtion; however, the combined Absence of the glycAns At Asn-152 And Asn-270 in the proteAse domAin hindered intersubunit disulfide bond formAtion, prevented noncovAlent AssociAtions, And Abolished enzymAtic Activity. These studies provide insights into the role of glycAns in the biosynthesis, Activity, And stAbility of this extrAcellulAr proteAse.
Gur P. Kaushal - One of the best experts on this subject based on the ideXlab platform.
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Proteolytic processing And inActivAtion of CCL2/MCP-1 by Meprins.
Biochemistry and biophysics reports, 2016Co-Authors: Christian Herzog, Sudhir V Shah, Randy S Haun, Gur P. KaushalAbstract:Monocyte chemotActic protein 1 (CCL2/MCP-1) is A smAll chemokine involved in the recruitment And trAfficking of mononucleAr immune cells to inflAmmAtion sites. Our studies demonstrAte thAt the metAlloendopeptidAses Meprin A (purified from kidney cortex), recombinAnt Meprin α, And recombinAnt Meprin β cAn All process CCL2/MCP-1. The cleAvAge sites were determined by Amino Acid sequencing And mAss spectrometry AnAlysis of the generAted products, And the biologicAl Activity of the products wAs evAluAted by chemotActic migrAtion AssAy using THP-1 cells. The cleAvAge sites generAted by the Meprin isoforms reveAled thAt Meprin A And Meprin α cleAved the N-terminAl domAin of mouse CCL2/MCP-1 At the Asn6 And AlA7 bond, resulting in significAnt reduction in the chemotActic Activity of the cleAved CCL2/MCP-1. Meprin β wAs unAble to cleAve the N-terminus of mouse CCL2/MCP-1 but cleAved the C-terminAl region between Ser74 And Glu75. HumAn CCL2/MCP-1 thAt lAcks the murine C-terminAl region wAs Also cleAved by Meprin α At the N-terminus resulting in significAnt loss of CCL2/MCP-1 biologicAl Activity, whereAs Meprin β did not Affect the biologicAl Activity. These studies suggest thAt Meprin α And Meprin β mAy plAy importAnt roles in regulAting the CCL2/MCP-1 chemokine Activity during inflAmmAtion.
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AdAm10 is the mAjor sheddAse responsible for the releAse of membrAne AssociAted Meprin A
Journal of Biological Chemistry, 2014Co-Authors: C. Herzog, Andreas Ludwig, Randy S Haun, Sudhir V Shah, Gur P. KaushalAbstract:Meprin A, composed of α And β subunits, is A membrAne-bound metAlloproteinAse in renAl proximAl tubules. Meprin A plAys An importAnt role in tubulAr epitheliAl cell injury during Acute kidney injury (AKI). The present study demonstrAted thAt during ischemiA-reperfusion-induced AKI, Meprin A wAs shed from proximAl tubule membrAnes, As evident from its redistribution towArd the bAsolAterAl side, proteolytic processing in the membrAnes, And excretion in the urine. To identify the proteolytic enzyme responsible for shedding of Meprin A, we generAted stAble HEK cell lines expressing Meprin β Alone And both Meprin α And Meprin β for the expression of Meprin A. Phorbol 12-myristAte 13-AcetAte And ionomycin stimulAted ectodomAin shedding of Meprin β And Meprin A. Among the inhibitors of vArious proteAses, the broAd spectrum inhibitor of the ADAM fAmily of proteAses, tumor necrosis fActor-α proteAse inhibitor (TAPI-1), wAs most effective in preventing constitutive, phorbol 12-myristAte 13-AcetAte-, And ionomycin-stimulAted shedding of Meprin β And Meprin A in the medium of both trAnsfectAnts. The use of differentiAl inhibitors for ADAM10 And ADAM17 indicAted thAt ADAM10 inhibition is sufficient to block shedding. In Agreement with these results, smAll interfering RNA to ADAM10 but not to ADAM9 or ADAM17 inhibited Meprin β And Meprin A shedding. Furthermore, overexpression of ADAM10 resulted in enhAnced shedding of Meprin β from both trAnsfectAnts. Our studies demonstrAte thAt ADAM10 is the mAjor ADAM metAlloproteinAse responsible for the constitutive And stimulAted shedding of Meprin β And Meprin A. These studies further suggest thAt inhibiting ADAM 10 Activity could be of therApeutic benefit in AKI.
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Meprin A metAlloproteinAse And its role in Acute kidney injury
American Journal of Physiology-renal Physiology, 2013Co-Authors: Gur P. Kaushal, Randy S Haun, Christian Herzog, Sudhir V ShahAbstract:Meprin A, composed of α- And β-subunits, is A membrAne-AssociAted neutrAl metAlloendoproteAse thAt belongs to the AstAcin fAmily of zinc endopeptidAses. It wAs first discovered As An AzocAsein And ...
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Actinonin A Meprin A inhibitor protects the renAl microcirculAtion during sepsis
Shock, 2011Co-Authors: Zhen Wang, Neriman Gokden, Gur P. Kaushal, C. Herzog, Philip R MayeuxAbstract:Sepsis-induced Acute renAl injury (AKI) occurs in 20%–50% of septic pAtients And neArly doubles the mortAlity rAte of sepsis. Since treAtment in the septic pAtient is usuAlly only begun After the onset of symptoms, therApy thAt is effective even when delAyed would hAve the greAtest impAct on pAtient survivAl. The metAlloproteinAse Meprin A, An oligomeric complex mAde of α- And β- subunits, is highly expressed At the brush-border membrAnes of the kidney And cApAble of degrAding numerous substrAtes including extrAcellulAr mAtrix proteins And cytokines. The goAl of the present study wAs to compAre the therApeutic potentiAl of Actinonin, An inhibitor of Meprin A when Administered before And After the onset of sepsis. Mice were treAted with Actinonin At 30 min prior to or 7 h post induction of sepsis by cecAl ligAtion And puncture (CLP). IntrAvitAl videomicroscopy wAs utilized to imAge renAl peritubulAr cApillAry perfusion And reActive nitrogen species. Actinonin treAtment 30 min before CLP reduced IL1-β levels And prevented the fAll in renAl cApillAry perfusion At 7 h And 18 h. Actinonin Also prevented the fAll in renAl cApillAry perfusion even when Administered At 7 h post CLP. In Addition, even lAte AdministrAtion of Actinonin preserved renAl morphology And lowered blood ureA nitrogen And serum creAtinine concentrAtions. These dAtA suggest thAt Agents like Actinonin should be evAluAted further As possible therApeutic Agents becAuse tArgeting both the eArly systemic And lAter orgAn-dAmAging effects of sepsis should hAve the highest likelihood of success.
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Actinonin A Meprin A inhibitor protects the renAl microcirculAtion during sepsis
Shock, 2011Co-Authors: Zhen Wang, Neriman Gokden, Gur P. Kaushal, Christian Herzog, Philip R MayeuxAbstract:Sepsis-induced Acute kidney injury occurs in 20% to 50% of septic pAtients And neArly doubles the mortAlity rAte of sepsis. BecAuse treAtment in the septic pAtient is usuAlly begun only After the onset of symptoms, therApy thAt is effective even when delAyed would hAve the greAtest impAct on pAtient survivAl. The metAlloproteinAse Meprin A, An oligomeric complex mAde of α- And β-subunits, is highly expressed At the brush-border membrAnes of the kidney And cApAble of degrAding numerous substrAtes including extrAcellulAr mAtrix proteins And cytokines. The goAl of the present study wAs to compAre the therApeutic potentiAl of Actinonin, An inhibitor of Meprin A, when Administered before And After the onset of sepsis. Mice were treAted with Actinonin At 30 min before or 7 h After induction of sepsis by cecAl ligAtion And puncture (CLP). IntrAvitAl videomicroscopy wAs used to imAge renAl peritubulAr cApillAry perfusion And reActive nitrogen species. Actinonin treAtment 30 min before CLP reduced IL-1β levels And prevented the fAll in renAl cApillAry perfusion At 7 And 18 h. Actinonin Also prevented the fAll in renAl cApillAry perfusion even when Administered At 7 h After CLP. In Addition, even lAte AdministrAtion of Actinonin preserved renAl morphology And lowered blood ureA nitrogen And serum creAtinine concentrAtions. These dAtA suggest thAt Agents such As Actinonin should be evAluAted further As possible therApeutic Agents becAuse tArgeting both the eArly systemic And lAter orgAn-dAmAging effects of sepsis should hAve the highest likelihood of success.
Sudhir V Shah - One of the best experts on this subject based on the ideXlab platform.
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Proteolytic processing And inActivAtion of CCL2/MCP-1 by Meprins.
Biochemistry and biophysics reports, 2016Co-Authors: Christian Herzog, Sudhir V Shah, Randy S Haun, Gur P. KaushalAbstract:Monocyte chemotActic protein 1 (CCL2/MCP-1) is A smAll chemokine involved in the recruitment And trAfficking of mononucleAr immune cells to inflAmmAtion sites. Our studies demonstrAte thAt the metAlloendopeptidAses Meprin A (purified from kidney cortex), recombinAnt Meprin α, And recombinAnt Meprin β cAn All process CCL2/MCP-1. The cleAvAge sites were determined by Amino Acid sequencing And mAss spectrometry AnAlysis of the generAted products, And the biologicAl Activity of the products wAs evAluAted by chemotActic migrAtion AssAy using THP-1 cells. The cleAvAge sites generAted by the Meprin isoforms reveAled thAt Meprin A And Meprin α cleAved the N-terminAl domAin of mouse CCL2/MCP-1 At the Asn6 And AlA7 bond, resulting in significAnt reduction in the chemotActic Activity of the cleAved CCL2/MCP-1. Meprin β wAs unAble to cleAve the N-terminus of mouse CCL2/MCP-1 but cleAved the C-terminAl region between Ser74 And Glu75. HumAn CCL2/MCP-1 thAt lAcks the murine C-terminAl region wAs Also cleAved by Meprin α At the N-terminus resulting in significAnt loss of CCL2/MCP-1 biologicAl Activity, whereAs Meprin β did not Affect the biologicAl Activity. These studies suggest thAt Meprin α And Meprin β mAy plAy importAnt roles in regulAting the CCL2/MCP-1 chemokine Activity during inflAmmAtion.
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AdAm10 is the mAjor sheddAse responsible for the releAse of membrAne AssociAted Meprin A
Journal of Biological Chemistry, 2014Co-Authors: C. Herzog, Andreas Ludwig, Randy S Haun, Sudhir V Shah, Gur P. KaushalAbstract:Meprin A, composed of α And β subunits, is A membrAne-bound metAlloproteinAse in renAl proximAl tubules. Meprin A plAys An importAnt role in tubulAr epitheliAl cell injury during Acute kidney injury (AKI). The present study demonstrAted thAt during ischemiA-reperfusion-induced AKI, Meprin A wAs shed from proximAl tubule membrAnes, As evident from its redistribution towArd the bAsolAterAl side, proteolytic processing in the membrAnes, And excretion in the urine. To identify the proteolytic enzyme responsible for shedding of Meprin A, we generAted stAble HEK cell lines expressing Meprin β Alone And both Meprin α And Meprin β for the expression of Meprin A. Phorbol 12-myristAte 13-AcetAte And ionomycin stimulAted ectodomAin shedding of Meprin β And Meprin A. Among the inhibitors of vArious proteAses, the broAd spectrum inhibitor of the ADAM fAmily of proteAses, tumor necrosis fActor-α proteAse inhibitor (TAPI-1), wAs most effective in preventing constitutive, phorbol 12-myristAte 13-AcetAte-, And ionomycin-stimulAted shedding of Meprin β And Meprin A in the medium of both trAnsfectAnts. The use of differentiAl inhibitors for ADAM10 And ADAM17 indicAted thAt ADAM10 inhibition is sufficient to block shedding. In Agreement with these results, smAll interfering RNA to ADAM10 but not to ADAM9 or ADAM17 inhibited Meprin β And Meprin A shedding. Furthermore, overexpression of ADAM10 resulted in enhAnced shedding of Meprin β from both trAnsfectAnts. Our studies demonstrAte thAt ADAM10 is the mAjor ADAM metAlloproteinAse responsible for the constitutive And stimulAted shedding of Meprin β And Meprin A. These studies further suggest thAt inhibiting ADAM 10 Activity could be of therApeutic benefit in AKI.
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Meprin A metAlloproteinAse And its role in Acute kidney injury
American Journal of Physiology-renal Physiology, 2013Co-Authors: Gur P. Kaushal, Randy S Haun, Christian Herzog, Sudhir V ShahAbstract:Meprin A, composed of α- And β-subunits, is A membrAne-AssociAted neutrAl metAlloendoproteAse thAt belongs to the AstAcin fAmily of zinc endopeptidAses. It wAs first discovered As An AzocAsein And ...
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Meprin A And Meprin α generAte biologicAlly functionAl il 1β from pro il 1β
Biochemical and Biophysical Research Communications, 2009Co-Authors: Christian Herzog, Philip R Mayeux, Randy S Haun, Sudhir V Shah, Varsha Kaushal, Gur P. KaushalAbstract:AbstrAct The present study demonstrAtes thAt both oligomeric metAlloendopeptidAse Meprin A purified from kidney cortex And recombinAnt Meprin α Are cApAble of generAting biologicAlly Active IL-1β from its precursor pro-IL-1β. Amino-Acid sequencing AnAlysis reveAls thAt Meprin A And Meprin α cleAve pro-IL-1β At the His 115 -Asp 116 bond, which is one Amino Acid N-terminAl to the cAspAse-1 cleAvAge site And five Amino Acids C-terminAl to the Meprin β site. The biologicAl Activity of the pro-IL-1β cleAved product produced by Meprin A, determined by proliferAtive response of helper T-cells, wAs 3-fold higher to thAt of the IL-1β product produced by Meprin β or cAspAse-1. In A mouse model of sepsis induced by cecAl ligAtion puncture thAt results in elevAted levels of serum IL-1β, Meprin inhibitor Actinonin significAntly reduces levels of serum IL-1β. Meprin A And Meprin α mAy therefore plAy A criticAl role in the production of Active IL-1β during inflAmmAtion And tissue injury.
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role of Meprin A in renAl tubulAr epitheliAl cell injury
Kidney International, 2007Co-Authors: Christian Herzog, Sudhir V Shah, Rohit Seth, Gur P. KaushalAbstract:Meprins Are zinc-dependent metAlloproteinAses thAt Are highly expressed in the brush-border membrAnes of both the kidney And the intestines. Meprins Are cApAble of proteolyticAlly degrAding extrAcellulAr mAtrix proteins, proteolyticAlly processing bioActive proteins, And plAy A role in inflAmmAtory processes. In this study, the function of Meprin A in the Acute kidney injury (AKI) model of cisplAtin nephrotoxicity wAs exAmined. NormAl lineAr locAlizAtion of Meprin A in the brush border membrAnes of proximAl tubules wAs Altered in AKI. The Meprin A α- subunit wAs detected in the urine of both control And cisplAtin-treAted mice. A cleAved product of the Meprin A β- subunit, undetected in the urine of control mice, wAs found to be significAntly increAsed in the urine during the progression of cisplAtin nephrotoxicity. The excretion of this β -frAgment wAs found to be before the rise in serum creAtinine And blood ureA nitrogen (BUN) suggesting usefulness As A biomArker for AKI. PretreAtment of mice with A Meprin A inhibitor Afforded protection from cisplAtin nephrotoxicity As reflected by significAnt decreAses in serum creAtinine, BUN, And the excretion of kidney injury molecule-1. These decreAses in serum And urine biomArkers were AccompAnied by significAnt decreAses in histologic mArkers such As leukocyte infiltrAtion And Apoptosis. Meprin A AppeArs to be An importAnt therApeutic tArget And urinAry excretion AppeArs to be A potentiAl biomArker of AKI.
Takayuki Tsukuba - One of the best experts on this subject based on the ideXlab platform.
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chAperone interActions of the metAlloproteinAse Meprin A in the secretory or proteAsomAl degrAdAtive pAthwAy
Archives of Biochemistry and Biophysics, 2002Co-Authors: Takayuki Tsukuba, Jeremy A Hengst, Tomoko Kadowaki, Judith S BondAbstract:The secreted form of mouse Meprin A is A homooligomer of Meprin AlphA subunits thAt contAin A prosequence, A cAtAlytic domAin, And three domAins designAted As MAM (Meprin, A5 protein, receptor protein-tyrosine phosphAtAse mu), MATH (Meprin And TRAF homology), And AM (AfterMAth). Previous studies indicAted thAt wild-type mouse Meprin AlphA is predominAntly A secreted protein, while the MAM deletion mutAnt (DeltAMAM) is degrAded intrAcellulArly. The work herein indicAtes thAt the DeltAMAM mutAnt is ubiquitinAted And degrAded viA the proteAsomAl pAthwAy. Both wild-type Meprin AlphA And the DeltAMAM mutAnt interAct with the moleculAr chAperones cAlnexin And cAlreticulin in the endoplAsmic reticulum. The interActions of the chAperones with the DeltAMAM mutAnt were significAntly prolonged in the presence of lActAcystin, A specific inhibitor of the proteAsome, whereAs those with the wild type were not Affected by this inhibitor. Trimming of the Asn-linked core oligosAcchArides of Meprin subunits wAs required for interActions with the chAperones. The dAtA indicAted thAt folding of the wild-type protein wAs AccelerAted by chAperones, whereAs the rAte of dimerizAtion wAs unAffected. Thus, cAlnexin And cAlreticulin Are intimAtely involved in the correct folding And trAnsport of Meprin to the plAsmA membrAne, As well As in retrogrAde trAnsport of the DeltAMAM mutAnt to the ubiquitin-dependent proteAsomAl degrAdAtive pAthwAy in the cytosol.
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n linked oligosAcchArides on the Meprin A metAlloproteAse Are importAnt for secretion And enzymAtic Activity but not for ApicAl tArgeting
Journal of Biological Chemistry, 2000Co-Authors: Tomoko Kadowaki, Greg P Bertenshaw, Takayuki Tsukuba, Judith S BondAbstract:The α And β subunits of Meprins, mAmmAliAn zinc metAlloendopeptidAses, Are extensively glycosylAted; ∼25% of the totAl moleculAr mAss of the subunits is cArbohydrAte. The Aim of this study wAs to investigAte the roles of the N-linked oligosAcchArides on the secreted form of mouse Meprin A. RecombinAnt Meprin α And mutAnts in which one of the 10 potentiAl Asn glycosylAtion sites wAs mutAted to Gln were All secreted And sorted exclusively into the ApicAl medium of polArized MAdin-DArby cAnine kidney cells, indicAting thAt no specific N-linked oligosAcchAride Acts As A determinAnt for ApicAl tArgeting of Meprin α. SeverAl of the mutAnt proteins hAd decreAsed enzymAtic Activity using A brAdykinin AnAlog As substrAte, And deglycosylAtion of the wild-type protein resulted in loss of 75–100% Activity. Some of the mutAnts were Also more sensitive to heAt inActivAtion. In studies with Agents thAt inhibit glycosylAtion processes in vivo, tunicAmycin mArkedly decreAsed secretion of Meprin, whereAs cAstAnospermine And swAinsonine hAd little effect on secretion, sorting, or enzymAtic properties of Meprin. When All the potentiAl glycosylAtion sites on A truncAted form of Meprin α (α-(1–445)) were mutAted, the protein wAs not secreted into the medium, but wAs retAined within the cells even After 10 h. These results indicAte thAt there is no one specific glycosylAtion site or type of oligosAcchAride (high mAnnose- or complex-type) thAt determines ApicAl sorting, but thAt core N-linked cArbohydrAtes Are required for optimAl enzymAtic Activity And for secretion of Meprin α.
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role of the cooh terminAl domAins of Meprin A in folding secretion And Activity of the metAlloendopeptidAse
Journal of Biological Chemistry, 1998Co-Authors: Takayuki Tsukuba, Judith S BondAbstract:AbstrAct Secreted forms of the α subunit of recombinAnt mouse Meprin A include An NH2-terminAl prosequence, A cAtAlytic domAin, And three COOH-terminAl domAins designAted As MAM (Meprin, A-5 protein, receptor protein-tyrosine phosphAtAse μ), MATH (Meprin AndTRAF homology), And AM (AfterMATH). In this study, the importAnce of these COOH-terminAl domAins for biosynthesis of secreted, ActivAble forms of the proteAse wAs investigAted. TrAnscripts of the Meprin subunit truncAted After the proteAse (α(1–275)), MAM (α(1–452)), And MATH (α(1–528)) domAins or with individuAl domAins deleted (ΔMAM, ΔMATH, And ΔAM), were trAnsfected into humAn embryonic kidney 293 cells. The wild-type subunit, ΔMATH, ΔAM, α(1–452), And α(1–528) were secreted into the mediA, Although the ΔAM mutAnt wAs secreted At very low levels. The ΔMATH And α(1–452) mutAnts were not ActivAble by limited proteolysis. The α(1–528) mutAnt wAs As Active As wild-type Meprin α AgAinst A brAdykinin substrAte, but hAd no Activity AgAinst AzocAsein, And it, As All other mutAnts, wAs more vulnerAble to extensive degrAdAtion by proteAses thAn the wild-type protein. Pulse-chAse experiments reveAled thAt the ΔMAM And α(1–275) mutAnts were rApidly degrAded within cells. TreAtment with lActAcystin, A specific inhibitor of the proteAsome, significAntly decreAsed the degrAdAtion, indicAting thAt the mutAnts lAcking the MAM domAin Are degrAded by the proteAsome As misfolded proteins. These results indicAte thAt the MAM domAin is necessAry for correct folding And trAnsport through the secretory pAthwAy, the MATH domAin is required for folding of An ActivAble zymogen, And the AM domAin is importAnt for Activity AgAinst proteins And efficient secretion of the protein. The work demonstrAtes the interdependence of the domAins for correct folding of An ActivAble, stAble, mAture enzyme.
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Role of the COOH-terminAl domAins of Meprin A in folding, secretion, And Activity of the metAlloendopeptidAse.
The Journal of biological chemistry, 1998Co-Authors: Takayuki Tsukuba, Judith S BondAbstract:Secreted forms of the AlphA subunit of recombinAnt mouse Meprin A include An NH2-terminAl prosequence, A cAtAlytic domAin, And three COOH-terminAl domAins designAted As MAM (Meprin, A-5 protein, receptor protein-tyrosine phosphAtAse mu), MATH (Meprin And TRAF homology), And AM (After MATH). In this study, the importAnce of these COOH-terminAl domAins for biosynthesis of secreted, ActivAble forms of the proteAse wAs investigAted. TrAnscripts of the Meprin subunit truncAted After the proteAse (AlphA(1-275)), MAM (AlphA(1-452)), And MATH (AlphA(1-528)) domAins or with individuAl domAins deleted (DeltAMAM, DeltAMATH, And DeltAAM), were trAnsfected into humAn embryonic kidney 293 cells. The wild-type subunit, DeltAMATH, DeltAAM, AlphA(1-452), And AlphA(1-528) were secreted into the mediA, Although the DeltAAM mutAnt wAs secreted At very low levels. The DeltAMATH And AlphA(1-452) mutAnts were not ActivAble by limited proteolysis. The AlphA(1-528) mutAnt wAs As Active As wild-type Meprin AlphA AgAinst A brAdykinin substrAte, but hAd no Activity AgAinst AzocAsein, And it, As All other mutAnts, wAs more vulnerAble to extensive degrAdAtion by proteAses thAn the wild-type protein. Pulse-chAse experiments reveAled thAt the DeltAMAM And AlphA(1-275) mutAnts were rApidly degrAded within cells. TreAtment with lActAcystin, A specific inhibitor of the proteAsome, significAntly decreAsed the degrAdAtion, indicAting thAt the mutAnts lAcking the MAM domAin Are degrAded by the proteAsome As misfolded proteins. These results indicAte thAt the MAM domAin is necessAry for correct folding And trAnsport through the secretory pAthwAy, the MATH domAin is required for folding of An ActivAble zymogen, And the AM domAin is importAnt for Activity AgAinst proteins And efficient secretion of the protein. The work demonstrAtes the interdependence of the domAins for correct folding of An ActivAble, stAble, mAture enzyme.
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Proteolytic processing And inActivAtion of CCL2/MCP-1 by Meprins.
Biochemistry and biophysics reports, 2016Co-Authors: Christian Herzog, Sudhir V Shah, Randy S Haun, Gur P. KaushalAbstract:Monocyte chemotActic protein 1 (CCL2/MCP-1) is A smAll chemokine involved in the recruitment And trAfficking of mononucleAr immune cells to inflAmmAtion sites. Our studies demonstrAte thAt the metAlloendopeptidAses Meprin A (purified from kidney cortex), recombinAnt Meprin α, And recombinAnt Meprin β cAn All process CCL2/MCP-1. The cleAvAge sites were determined by Amino Acid sequencing And mAss spectrometry AnAlysis of the generAted products, And the biologicAl Activity of the products wAs evAluAted by chemotActic migrAtion AssAy using THP-1 cells. The cleAvAge sites generAted by the Meprin isoforms reveAled thAt Meprin A And Meprin α cleAved the N-terminAl domAin of mouse CCL2/MCP-1 At the Asn6 And AlA7 bond, resulting in significAnt reduction in the chemotActic Activity of the cleAved CCL2/MCP-1. Meprin β wAs unAble to cleAve the N-terminus of mouse CCL2/MCP-1 but cleAved the C-terminAl region between Ser74 And Glu75. HumAn CCL2/MCP-1 thAt lAcks the murine C-terminAl region wAs Also cleAved by Meprin α At the N-terminus resulting in significAnt loss of CCL2/MCP-1 biologicAl Activity, whereAs Meprin β did not Affect the biologicAl Activity. These studies suggest thAt Meprin α And Meprin β mAy plAy importAnt roles in regulAting the CCL2/MCP-1 chemokine Activity during inflAmmAtion.
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Meprin A metAlloproteinAse And its role in Acute kidney injury
American Journal of Physiology-renal Physiology, 2013Co-Authors: Gur P. Kaushal, Randy S Haun, Christian Herzog, Sudhir V ShahAbstract:Meprin A, composed of α- And β-subunits, is A membrAne-AssociAted neutrAl metAlloendoproteAse thAt belongs to the AstAcin fAmily of zinc endopeptidAses. It wAs first discovered As An AzocAsein And ...
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Actinonin A Meprin A inhibitor protects the renAl microcirculAtion during sepsis
Shock, 2011Co-Authors: Zhen Wang, Neriman Gokden, Gur P. Kaushal, Christian Herzog, Philip R MayeuxAbstract:Sepsis-induced Acute kidney injury occurs in 20% to 50% of septic pAtients And neArly doubles the mortAlity rAte of sepsis. BecAuse treAtment in the septic pAtient is usuAlly begun only After the onset of symptoms, therApy thAt is effective even when delAyed would hAve the greAtest impAct on pAtient survivAl. The metAlloproteinAse Meprin A, An oligomeric complex mAde of α- And β-subunits, is highly expressed At the brush-border membrAnes of the kidney And cApAble of degrAding numerous substrAtes including extrAcellulAr mAtrix proteins And cytokines. The goAl of the present study wAs to compAre the therApeutic potentiAl of Actinonin, An inhibitor of Meprin A, when Administered before And After the onset of sepsis. Mice were treAted with Actinonin At 30 min before or 7 h After induction of sepsis by cecAl ligAtion And puncture (CLP). IntrAvitAl videomicroscopy wAs used to imAge renAl peritubulAr cApillAry perfusion And reActive nitrogen species. Actinonin treAtment 30 min before CLP reduced IL-1β levels And prevented the fAll in renAl cApillAry perfusion At 7 And 18 h. Actinonin Also prevented the fAll in renAl cApillAry perfusion even when Administered At 7 h After CLP. In Addition, even lAte AdministrAtion of Actinonin preserved renAl morphology And lowered blood ureA nitrogen And serum creAtinine concentrAtions. These dAtA suggest thAt Agents such As Actinonin should be evAluAted further As possible therApeutic Agents becAuse tArgeting both the eArly systemic And lAter orgAn-dAmAging effects of sepsis should hAve the highest likelihood of success.
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Meprin A And Meprin α generAte biologicAlly functionAl il 1β from pro il 1β
Biochemical and Biophysical Research Communications, 2009Co-Authors: Christian Herzog, Philip R Mayeux, Randy S Haun, Sudhir V Shah, Varsha Kaushal, Gur P. KaushalAbstract:AbstrAct The present study demonstrAtes thAt both oligomeric metAlloendopeptidAse Meprin A purified from kidney cortex And recombinAnt Meprin α Are cApAble of generAting biologicAlly Active IL-1β from its precursor pro-IL-1β. Amino-Acid sequencing AnAlysis reveAls thAt Meprin A And Meprin α cleAve pro-IL-1β At the His 115 -Asp 116 bond, which is one Amino Acid N-terminAl to the cAspAse-1 cleAvAge site And five Amino Acids C-terminAl to the Meprin β site. The biologicAl Activity of the pro-IL-1β cleAved product produced by Meprin A, determined by proliferAtive response of helper T-cells, wAs 3-fold higher to thAt of the IL-1β product produced by Meprin β or cAspAse-1. In A mouse model of sepsis induced by cecAl ligAtion puncture thAt results in elevAted levels of serum IL-1β, Meprin inhibitor Actinonin significAntly reduces levels of serum IL-1β. Meprin A And Meprin α mAy therefore plAy A criticAl role in the production of Active IL-1β during inflAmmAtion And tissue injury.
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role of Meprin A in renAl tubulAr epitheliAl cell injury
Kidney International, 2007Co-Authors: Christian Herzog, Sudhir V Shah, Rohit Seth, Gur P. KaushalAbstract:Meprins Are zinc-dependent metAlloproteinAses thAt Are highly expressed in the brush-border membrAnes of both the kidney And the intestines. Meprins Are cApAble of proteolyticAlly degrAding extrAcellulAr mAtrix proteins, proteolyticAlly processing bioActive proteins, And plAy A role in inflAmmAtory processes. In this study, the function of Meprin A in the Acute kidney injury (AKI) model of cisplAtin nephrotoxicity wAs exAmined. NormAl lineAr locAlizAtion of Meprin A in the brush border membrAnes of proximAl tubules wAs Altered in AKI. The Meprin A α- subunit wAs detected in the urine of both control And cisplAtin-treAted mice. A cleAved product of the Meprin A β- subunit, undetected in the urine of control mice, wAs found to be significAntly increAsed in the urine during the progression of cisplAtin nephrotoxicity. The excretion of this β -frAgment wAs found to be before the rise in serum creAtinine And blood ureA nitrogen (BUN) suggesting usefulness As A biomArker for AKI. PretreAtment of mice with A Meprin A inhibitor Afforded protection from cisplAtin nephrotoxicity As reflected by significAnt decreAses in serum creAtinine, BUN, And the excretion of kidney injury molecule-1. These decreAses in serum And urine biomArkers were AccompAnied by significAnt decreAses in histologic mArkers such As leukocyte infiltrAtion And Apoptosis. Meprin A AppeArs to be An importAnt therApeutic tArget And urinAry excretion AppeArs to be A potentiAl biomArker of AKI.