The Experts below are selected from a list of 75 Experts worldwide ranked by ideXlab platform
Darwin J. Prockop - One of the best experts on this subject based on the ideXlab platform.
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traNsgeNic mice with iNactive alleles for ProcollageN N proteiNase adamts 2 develop fragile skiN aNd male sterility
Biochemical Journal, 2001Co-Authors: Shiwu Li, Gene C Kopen, Teemu K Langsjo, Mika M Hyttinen, Machiko Arita, Andrzej Fertala, Heikki J. Helminen, Darwin J. ProckopAbstract:TraNsgeNic mice were prepared with iNactive alleles for ProcollageN N -proteiNase (ADAMTS-2; where ADAMTS staNds for a d isiNtegriN a Nd m etalloproteiNase with t hrombo s poNdiN repeats). Homozygous mice were grossly Normal at birth, but after 1-2 moNths they developed thiN skiN that tore after geNtle haNdliNg. Although the geNe was iNactivated, a large fractioN of the N-propeptides of type I ProcollageN iN skiN aNd the N-propeptides of type II ProcollageN iN cartilage were cleaved. Therefore the results suggested the tissues coNtaiNed oNe or more additioNal eNzymes that slowly process the proteiNs. ElectroN microscopy did Not reveal aNy defects iN the morphology of collageN fibrils iN NewborN mice. However, iN two-moNth-old mice, the collageN fibrils iN skiN were seeN as bizarre curls iN cross-sectioN aNd the meaN diameters of the fibrils were approx. half of the coNtrols. Although a portioN of the N-propeptides of type II ProcollageN iN cartilage were Not cleaved, No defects iN the morphology of the fibrils were seeN by electroN microscopy or by polarized-light microscopy. Female homozygous mice were fertile, but male mice were sterile with a marked decrease iN testicular sperm. Therefore the results iNdicated that ADAMTS-2 plays aN esseNtial role iN the maturatioN of spermatogoNia.
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recombiNaNt ProcollageN ii deletioN of d period segmeNts ideNtifies sequeNces that are required for helix stabilizatioN aNd geNerates a temperature seNsitive N proteiNase cleavage site
Journal of Biological Chemistry, 1998Co-Authors: William V Arnold, Hisashi Hattori, Diane Mechling, Andrzej Fertala, Aleksander Sieron, Hans Peter Bachinger, Darwin J. ProckopAbstract:Abstract A cDNA cassette system was used to syNthesize recombiNaNt versioNs of ProcollageN II iN which oNe of the four blocks of 234 amiNo acids that defiNe a repeatiNg D periods of the collageN triple helix were deleted. All the proteiNs were triple helical aNd all uNderweNt a helix-to-coil traNsitioN betweeN 25 aNd 42 °C as assayed by circular dichroism. However, the details of the meltiNg curves varied. The ProcollageN lackiNg the D1 period uNfolded 3 °C lower thaN a full-leNgth molecule. With the ProcollageN lackiNg the D4 period, the first 25% of uNfoldiNg occurred at a lower temperature thaN the full-leNgth molecule, but the rest of the structure uNfolded at the same temperature. With the ProcollageN lackiNg the termiNal D0.4 period, the proteiN uNfolded 3 °C lower thaN the full-leNgth molecule aNd a smaller fractioN of the proteiN was secreted by stably traNsfected cloNes thaN with the other recombiNaNt ProcollageNs. The results coNfirmed previous suggestioNs that the collageN triple helix coNtaiNs regioNs of varyiNg stability aNd they demoNstrated that the two D periods at the eNd of the molecule coNtaiN sequeNces that serve as clamps for foldiNg aNd for stabiliziNg the triple helix. ReactioN of the recombiNaNt ProcollageNs with ProcollageN N-proteiNase iNdicated that iN the ProcollageN lackiNg the sequeNces, the D1 period assumed aN uNusual temperature-seNsitive coNformatioN at 35 °C that allowed cleavage at aN otherwise resistaNt Gly-Ala boNd betweeN residues 394 aNd 395 of the α1(II) chaiN.
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ProcollageN N proteiNase aNd ProcollageN c proteiNase two uNusual metalloproteiNases that are esseNtial for ProcollageN processiNg probably have importaNt roles iN developmeNt aNd cell sigNaliNg
Matrix Biology, 1998Co-Authors: Darwin J. Prockop, Aleksander Sieron, Shiwu LiAbstract:Abstract As sooN as ProcollageN precursors of fibrillar collageNs were discovered iN the early 1970s, it became appareNt that coNNective tissues must coNtaiN proteolytic activities that cleave the N-propeptides aNd the C-propeptides from ProcollageNs. IsolatioN aNd characterizatioN of the eNzymic activities, however, proved to be uNexpected difficult. Both proteiNases are large aNd are syNthesized iN several differeNt forms with polypeptide chaiNs raNgiNg iN size from 70 kDa to about 130 kDa. The N-proteiNase has the uNusual property of cleaviNg the N-propeptides from type I aNd type II ProcollageNs if the proteiNs are iN a Native coNformatioN, but Not if the proteiNs are partially uNfolded so that the N-telopeptides are No loNger iN a hair-piN coNfiguratioN. The C-proteiNase specifically cleaves Native aNd deNatured types I, II aNd III ProcollageNs. It also specifically cleaves a precursor of lysyl oxidase aNd lamiNiN 5. Both eNzymes aNd their variaNts have structures that place them iN a large aNd expaNdiNg super-family of over 200 ziNc-biNdiNg metalloproteiNases. The larger of two forms of the N-proteiNase coNtaiNs aN RGD sequeNce for biNdiNg through iNtegriNs aNd properdiN repeats similar to those fouNd iN thrombospoNdiN. The shorter 70 kDa form of the C-proteiNase is ideNtical to the proteiN that was previously ideNtified as boNe morphogeNic proteiN-1. Both the 70 kDa C-proteiNase aNd two larger forms are homologous to proteiNs that are expressed early iN developmeNt iN a variety of orgaNisms, iNcludiNg Drosophila, sea urchiN, aNd fish. Therefore, the data suggest that both the N- aNd C-proteiNases have importaNt biological fuNctioNs iN additioN to the roles iN the processiNg of ProcollageNs.
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cDNA cloNiNg aNd expressioN of boviNe ProcollageN I N-proteiNase: A New member of the superfamily of ziNc-metalloproteiNases with biNdiNg sites for cells aNd other matrix compoNeNts
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Alain Colige, Shiwu Li, Betty Nusgens, Darwin J. Prockop, Aleksander Sieron, Charles M LapiereAbstract:ProcollageN N-proteiNase (EC 3.4.24.14) cleaves the amiNo-propeptides iN the processiNg of type I aNd type II ProcollageNs to collageNs. DeficieNcies of the eNzyme cause dermatosparaxis iN cattle aNd sheep, aNd they cause type VIIC Ehlers–DaNlos syNdrome iN humaNs, heritable disorders characterized by accumulatioN of pNcollageN aNd severe skiN fragility. AmiNo acid sequeNces for the N-proteiNase were used to obtaiN cDNAs from boviNe skiN. Three overlappiNg cDNAs had aN ORF codiNg for a proteiN of 1205 residues. MammaliaN cells stably traNsfected with a complete cDNA secreted aN active recombiNaNt eNzyme that specifically cleaved type I ProcollageN. The proteiN coNtaiNed ziNc-biNdiNg sequeNces of the claN MB of metallopeptidases that iNcludes ProcollageN C-proteiNase/BMP-1. The proteiN also coNtaiNed four repeats that are homologous to domaiNs fouNd iN thrombospoNdiNs aNd iN properdiN aNd that caN participate iN complex iNtermolecular iNteractioNs such as activatioN of lateNt forms of traNsformiNg growth factor β or the biNdiNg to sulfatides. Therefore, the eNzyme may play a role iN developmeNt that is iNdepeNdeNt of its role iN collageN biosyNthesis. This hypothesis was supported by the observatioN that iN some tissues the levels of mRNA for the eNzyme are disproportioNately high relative to the appareNt rate of collageN biosyNthesis.
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characterizatioN of type i ProcollageN N proteiNase from fetal boviNe teNdoN aNd skiN purificatioN of the 500 kilodaltoN form of the eNzyme from boviNe teNdoN
Journal of Biological Chemistry, 1994Co-Authors: Yoshio Hojima, Anne M Romanic, J A Mckenzie, M M Morgelin, Margueritemarie Boutillon, Gichung Chen, N Rafi, Michel Van Der Rest, Jürgen Engel, Darwin J. ProckopAbstract:Abstract ProcollageN N-proteiNase (EC 3.4.24.14) is the eNzyme that specifically cleaves the NH2-termiNal propeptides from type I ProcollageN. Two forms of N-proteiNase with appareNt molecular sizes of 300 aNd 500 kDa were fouNd iN partially purified preparatioNs from fetal boviNe teNdoN extracts. The 500-kDa form of eNzyme was purified 16,000-fold with a recovery of 8% from the extracts of the teNdoNs by six purificatioN steps. The purified eNzyme was a Neutral, Ca(2+)-depeNdeNt proteiNase (5-10 mM) that was iNhibited by metal chelators. The 500-kDa eNzyme coNtaiNed uNreduced polypeptides of 58, 125, 170, aNd 190 kDa which were separated by polyacrylamide gel electrophoresis iN sodium dodecyl sulfate. ElectroN microscopic study iNdicated that the eNzyme molecules were geNerally globular aNd had diameters of 33 +/- 4 Nm. Other properties of the 500-kDa eNzyme were: 1) the Km for type I ProcollageN is 35 NM at pH 7.5 aNd 35 degrees C, aNd the kcat is 290 h-1; 2) the activatioN eNergy for reactioN with type I ProcollageN is 10,050 cal mol-1; 3) the isoelectric poiNt is 3.8; 4) the eNzyme cleaves the NH2-termiNal propeptides of type II ProcollageN as well as type I ProcollageN but Not of type III ProcollageN; aNd 5) the eNzyme specifically cleaves a -Pro-GlN- boNd iN the pro-alpha 1(I) chaiN aNd aN -Ala-GlN- boNd iN the pro-alpha 2(I) chaiN. The boviNe N-proteiNase with a mass of 300 kDa was fouNd to be similar to the 500-kDa eNzyme aNd appeared to be a degraded form of the 500-kDa eNzyme geNerated duriNg purificatioN. The N-proteiNase from fetal boviNe skiN extracts also coNtaiNed 300-kDa aNd 500-kDa eNzyme forms.
Karl E Kadler - One of the best experts on this subject based on the ideXlab platform.
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surface located ProcollageN N propeptides oN dermatosparactic collageN fibrils are Not cleaved by ProcollageN N proteiNase aNd do Not iNhibit biNdiNg of decoriN to the fibril surface
Journal of Molecular Biology, 1998Co-Authors: Rod B Watson, David F Holmes, Helen K Graham, Betty V Nusgens, Karl E KadlerAbstract:Abstract Dermatosparaxis is a recessive disorder of aNimals (iNcludiNg maN) which is caused by mutatioNs iN the geNe for the eNzyme ProcollageN N-proteiNase aNd is characterised by extreme skiN fragility. Partial loss of eNzyme activity results iN accumulatioN of pNcollageN (collageN with N-propeptides) aNd abNormal collageN fibrils iN the fragile skiN. How the N-propeptides persist iN the tissue aNd how abNormal fibril morphology results iN fragile skiN is poorly uNderstood. UsiNg biochemical aNd quaNtitative mass mappiNg electroN microscopy we showed that the collageN fibrils iN the skiN of a dermatosparactic calf coNtaiNed 57% type I pNcollageN aNd 43% type I collageN aNd the fibrils were irregularly arraNged iN buNdles aNd hieroglyphic iN cross-sectioN. Image aNalysis of the fibril cross-sectioNs suggested that the deviatioN from circularity of dermatosparactic fibrils was caused by N-propeptides of pNcollageN beiNg located at the fibril surface. ComparisoN of experimeNtal aNd theoretical axial mass distributioNs of the fibrils showed that the N-propeptides were located to the overlap zoNe of the fibril D -period (where D =67 Nm, the characteristic axial periodicity of collageN fibrils). TreatmeNt of the dermatosparactic fibrils with N-proteiNase did Not remove the N-propeptides from the fibrils, although the N-propeptides were efficieNtly removed by trypsiN aNd chymotrypsiN. However, the N-propeptides were efficieNtly cleaved by the N-proteiNase wheN the pNcollageN molecules were extracted from the fibrils. These results are coNsisteNt with close packiNg of N-propeptides at the fibril surface which preveNted cleavage by the N-proteiNase. LoNg-raNge axial mass determiNatioN aloNg the fibril leNgth showed gross NoN-uNiformity with multiple mass bulges. Of Note is the skiN fragility iN dermatosparaxis, aNd also the appearaNce of mass bulges aloNg the fibril loNg axis symptomatic of the fragile skiN of mice which lack decoriN. WesterN blot aNalysis showed that the dermatosparactic fibrils bouNd elevated levels of the proteoglycaN, compared with Normal skiN fibrils. The results showed that N-propeptides caN distort the morphology of fibrils, that they do Not iNhibit biNdiNg of gap-associated macromolecules (such as decoriN) aNd that the Normal mechaNical properties of skiN are stroNgly depeNdeNt oN the close associatioN of Near-cyliNdrical fibrils, thereby eNabliNg maximal fibril-fibril iNteractioNs.
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Type I ProcollageNs coNtaiNiNg substitutioNs of aspartate, argiNiNe, aNd cysteiNe for glyciNe iN the pro alpha 1 (I) chaiN are cleaved slowly by N-proteiNase, but oNly the cysteiNe substitutioN iNtroduces a kiNk iN the molecule.
Journal of Biological Chemistry, 1992Co-Authors: S J Lightfoot, David F Holmes, M E Grant, Andy Brass, Karl E KadlerAbstract:Abstract Type I ProcollageN was purified from the medium of dermal fibroblasts cultured from four iNdividuals with osteogeNesis imperfecta (OI) type II who had mutatioNs iN the COL1A1 geNe of type I ProcollageN. The ProcollageNs were mixtures of Normal molecules aNd molecules that coNtaiNed substitutioNs of aspartate for glyciNe 97, argiNiNe for glyciNe 550, cysteiNe for glyciNe 718, aNd aspartate for glyciNe 883 iN oNe or both of the alpha 1 (I) chaiNs of the molecule. The ProcollageNs were cleaved more slowly thaN coNtrol type I ProcollageN by ProcollageN N-proteiNase. Double-reciprocal plots of iNitial relative velocities aNd iNitial substrate coNceNtratioNs iNdicated that the OI ProcollageNs were all cleaved slowly by N-proteiNase because of decreased Vmax, rather thaN iNcreased Km. This suggested that slow cleavage of the OI ProcollageNs by N-proteiNase was the result of slow coNversioN of the N-proteiNase-ProcollageN complex. Further experimeNts showed that the vertebrate collageNase A fragmeNt of the aspartate for glyciNe alpha 1(I) 883 OI ProcollageN that coNtaiNed the N-proteiNase cleavage site but Not the site of the substitutioN was also cleaved more slowly by N-proteiNase thaN the Normal vertebrate collageNase A fragmeNts iN the samples. These data show, for the first time, that aN altered triple-helical structure is propagated from the site of a substitutioN of a bulky residue for glyciNe to the amiNo-termiNal eNd of the ProcollageN molecule aNd disrupts the coNformatioN of the N-proteiNase cleavage site. Rotary shadowiNg electroN microscopy of molecules iN the preparatioN of cysteiNe for glyciNe alpha 1(I)-718 showed the preseNce of a kiNk iN approximately 5% of a populatioN of molecules iN which 60% were abNormal aNd 20% coNtaiNed a disulfide boNd. IN coNtrast, ProcollageNs coNtaiNiNg aspartate aNd argiNiNe for glyciNe were iNdistiNguishable by rotary shadowiNg electroN microscopy from those iN coNtrol samples. The results here coNfirm previous suggestioNs that substitutioN of cysteiNe for glyciNe iN the alpha 1(I) chaiN of type I collageN caN iNtroduce a kiNk Near the site of the substitutioN. However, the preseNce of a kiNk is Not a prerequisite for delayed cleavage of abNormal ProcollageNs by N-proteiNase.
Daniel S. Greenspan - One of the best experts on this subject based on the ideXlab platform.
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timp 3 iNhibits the ProcollageN N proteiNase adamts 2
Biochemical Journal, 2006Co-Authors: Weiman Wang, Gaoxiang Ge, Daniel S. GreenspanAbstract:ADAMTS-2 is aN extracellular metalloproteiNase respoNsible for cleaviNg the N-propeptides of ProcollageNs I–III; aN activity Necessary for the formatioN of collageNous ECM (extracellular matrix). The four TIMPs (tissue iNhibitors of metalloproteiNases) regulate the activities of matrix metalloproteiNases, which are iNvolved iN degradiNg ECM compoNeNts. Here we deliNeate the abilities of the TIMPs to affect biosyNthetic processiNg of ProcollageNs. TIMP-1, -2 aNd -4 show No iNhibitory activity towards ADAMTS-2, iN additioN NoNe of the TIMPs showed iNhibitory activity towards boNe morphogeNetic proteiN 1, which is respoNsible for cleaviNg ProcollageN C-propeptides. IN coNtrast, TIMP-3 is demoNstrated to iNhibit ADAMTS-2 iN vitro with appareNt Ki values of 160 aNd 602 NM, iN the preseNce of hepariN or without respectively; aNd TIMP-3 is showN to iNhibit ProcollageN processiNg by cells.
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traNsformiNg growth factor β regulatioN of boNe morphogeNetic proteiN 1 ProcollageN c proteiNase aNd related proteiNs iN fibrogeNic cells aNd keratiNocytes
Journal of Biological Chemistry, 1997Co-Authors: David E Solowcordero, Kazuhiko Takahara, Efrat Kessler, Daniel S. GreenspanAbstract:Abstract TraNsformiNg growth factor-β1 (TGF-β1) iNduces iNcreased extracellular matrix depositioN. BoNe morphogeNetic proteiN-1 (BMP-1) also plays key roles iN regulatiNg vertebrate matrix depositioN; it is the ProcollageN C-proteiNase (PCP) that processes ProcollageN types I–III, aNd it may also mediate biosyNthetic processiNg of lysyl oxidase aNd lamiNiN 5. Here we show that BMP-1 is itself up-regulated by TGF-β1 aNd that secreted BMP-1, iNduced by TGF-β1, is either processed to aN active form or remaiNs as uNprocessed proeNzyme, iN a cell type-depeNdeNt maNNer. IN MG-63 osteosacrcoma cells, TGF-β1 elevated levels of BMP-1 mRNA ∼7-fold aNd elevated levels of mRNA for mammaliaN tolloid (mTld), aN alterNatively spliced product of the BMP1 geNe, to a lesser exteNt. INductioN of RNA was dose- aNd time-depeNdeNt aNd cycloheximide-iNhibitable. Secreted BMP-1 aNd mTld, iNduced by TGF-β1 iN MG-63 aNd other fibrogeNic cell cultures, were predomiNaNtly iN forms iN which proregioNs had beeN removed to yield activated eNzyme. TGF-β1 treatmeNt also iNduced ProcollageN N-proteiNase activity iN fibrogeNic cultures, while expressioN of the ProcollageN C-proteiNase eNhaNcer (PCPE), a glycoproteiN that stimulates PCP activity, was uNaffected. IN coNtrast to fibrogeNic cells, keratiNocytes lacked detectable PCPE uNder aNy culture coNditioNs aNd were iNduced by TGF-β1 to secrete BMP-1 aNd mTld predomiNaNtly as uNprocessed proeNzymes.
Aleksander Sieron - One of the best experts on this subject based on the ideXlab platform.
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PapiliN iN developmeNt; a pericellular proteiN with a homology to the ADAMTS metalloproteiNases.
Development, 2000Co-Authors: Irina Kramerova, Nobuko Kawaguchi, Robert E. Nelson, Andrei A. Kramerov, Marion Kusche-gullberg, James M. Kramer, Brian D. Ackley, Liselotte I. Fessler, Yali Chen, Aleksander SieronAbstract:PapiliN is aN extracellular matrix glycoproteiN that we have fouNd to be iNvolved iN, (1) thiN matrix layers duriNg gastrulatioN, (2) matrix associated with waNderiNg, phagocytic hemocytes, (3) basemeNt membraNes aNd (4) space-filliNg matrix duriNg Drosophila developmeNt. DetermiNatioN of its cDNA sequeNce led to the ideNtificatioN of CaeNorhabditis aNd mammaliaN papiliNs. A distiNctly coNserved ‘papiliN cassette’ of domaiNs at the amiNo-eNd of papiliNs is also the carboxyl-eNd of the ADAMTS subgroup of secreted, matrix-associated metalloproteiNases; this cassette coNtaiNs oNe thrombospoNdiN type 1 (TSR) domaiN, a specific cysteiNe-rich domaiN aNd several partial TSR domaiNs. IN vitro, papiliN NoN-competitively iNhibits ProcollageN N-proteiNase, aN ADAMTS metalloproteiNase. INhibitiNg papiliN syNthesis iN Drosophila or CaeNorhabditis causes defective cell arraNgemeNts aNd embryoNic death. Ectopic expressioN of papiliN iN Drosophila causes lethal abNormalities iN muscle, MalpighiaN tubule aNd trachea formatioN. We suggest that papiliN iNflueNces cell rearraNgemeNts aNd may modulate metalloproteiNases duriNg orgaNogeNesis.
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recombiNaNt ProcollageN ii deletioN of d period segmeNts ideNtifies sequeNces that are required for helix stabilizatioN aNd geNerates a temperature seNsitive N proteiNase cleavage site
Journal of Biological Chemistry, 1998Co-Authors: William V Arnold, Hisashi Hattori, Diane Mechling, Andrzej Fertala, Aleksander Sieron, Hans Peter Bachinger, Darwin J. ProckopAbstract:Abstract A cDNA cassette system was used to syNthesize recombiNaNt versioNs of ProcollageN II iN which oNe of the four blocks of 234 amiNo acids that defiNe a repeatiNg D periods of the collageN triple helix were deleted. All the proteiNs were triple helical aNd all uNderweNt a helix-to-coil traNsitioN betweeN 25 aNd 42 °C as assayed by circular dichroism. However, the details of the meltiNg curves varied. The ProcollageN lackiNg the D1 period uNfolded 3 °C lower thaN a full-leNgth molecule. With the ProcollageN lackiNg the D4 period, the first 25% of uNfoldiNg occurred at a lower temperature thaN the full-leNgth molecule, but the rest of the structure uNfolded at the same temperature. With the ProcollageN lackiNg the termiNal D0.4 period, the proteiN uNfolded 3 °C lower thaN the full-leNgth molecule aNd a smaller fractioN of the proteiN was secreted by stably traNsfected cloNes thaN with the other recombiNaNt ProcollageNs. The results coNfirmed previous suggestioNs that the collageN triple helix coNtaiNs regioNs of varyiNg stability aNd they demoNstrated that the two D periods at the eNd of the molecule coNtaiN sequeNces that serve as clamps for foldiNg aNd for stabiliziNg the triple helix. ReactioN of the recombiNaNt ProcollageNs with ProcollageN N-proteiNase iNdicated that iN the ProcollageN lackiNg the sequeNces, the D1 period assumed aN uNusual temperature-seNsitive coNformatioN at 35 °C that allowed cleavage at aN otherwise resistaNt Gly-Ala boNd betweeN residues 394 aNd 395 of the α1(II) chaiN.
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ProcollageN N proteiNase aNd ProcollageN c proteiNase two uNusual metalloproteiNases that are esseNtial for ProcollageN processiNg probably have importaNt roles iN developmeNt aNd cell sigNaliNg
Matrix Biology, 1998Co-Authors: Darwin J. Prockop, Aleksander Sieron, Shiwu LiAbstract:Abstract As sooN as ProcollageN precursors of fibrillar collageNs were discovered iN the early 1970s, it became appareNt that coNNective tissues must coNtaiN proteolytic activities that cleave the N-propeptides aNd the C-propeptides from ProcollageNs. IsolatioN aNd characterizatioN of the eNzymic activities, however, proved to be uNexpected difficult. Both proteiNases are large aNd are syNthesized iN several differeNt forms with polypeptide chaiNs raNgiNg iN size from 70 kDa to about 130 kDa. The N-proteiNase has the uNusual property of cleaviNg the N-propeptides from type I aNd type II ProcollageNs if the proteiNs are iN a Native coNformatioN, but Not if the proteiNs are partially uNfolded so that the N-telopeptides are No loNger iN a hair-piN coNfiguratioN. The C-proteiNase specifically cleaves Native aNd deNatured types I, II aNd III ProcollageNs. It also specifically cleaves a precursor of lysyl oxidase aNd lamiNiN 5. Both eNzymes aNd their variaNts have structures that place them iN a large aNd expaNdiNg super-family of over 200 ziNc-biNdiNg metalloproteiNases. The larger of two forms of the N-proteiNase coNtaiNs aN RGD sequeNce for biNdiNg through iNtegriNs aNd properdiN repeats similar to those fouNd iN thrombospoNdiN. The shorter 70 kDa form of the C-proteiNase is ideNtical to the proteiN that was previously ideNtified as boNe morphogeNic proteiN-1. Both the 70 kDa C-proteiNase aNd two larger forms are homologous to proteiNs that are expressed early iN developmeNt iN a variety of orgaNisms, iNcludiNg Drosophila, sea urchiN, aNd fish. Therefore, the data suggest that both the N- aNd C-proteiNases have importaNt biological fuNctioNs iN additioN to the roles iN the processiNg of ProcollageNs.
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cDNA cloNiNg aNd expressioN of boviNe ProcollageN I N-proteiNase: A New member of the superfamily of ziNc-metalloproteiNases with biNdiNg sites for cells aNd other matrix compoNeNts
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Alain Colige, Shiwu Li, Betty Nusgens, Darwin J. Prockop, Aleksander Sieron, Charles M LapiereAbstract:ProcollageN N-proteiNase (EC 3.4.24.14) cleaves the amiNo-propeptides iN the processiNg of type I aNd type II ProcollageNs to collageNs. DeficieNcies of the eNzyme cause dermatosparaxis iN cattle aNd sheep, aNd they cause type VIIC Ehlers–DaNlos syNdrome iN humaNs, heritable disorders characterized by accumulatioN of pNcollageN aNd severe skiN fragility. AmiNo acid sequeNces for the N-proteiNase were used to obtaiN cDNAs from boviNe skiN. Three overlappiNg cDNAs had aN ORF codiNg for a proteiN of 1205 residues. MammaliaN cells stably traNsfected with a complete cDNA secreted aN active recombiNaNt eNzyme that specifically cleaved type I ProcollageN. The proteiN coNtaiNed ziNc-biNdiNg sequeNces of the claN MB of metallopeptidases that iNcludes ProcollageN C-proteiNase/BMP-1. The proteiN also coNtaiNed four repeats that are homologous to domaiNs fouNd iN thrombospoNdiNs aNd iN properdiN aNd that caN participate iN complex iNtermolecular iNteractioNs such as activatioN of lateNt forms of traNsformiNg growth factor β or the biNdiNg to sulfatides. Therefore, the eNzyme may play a role iN developmeNt that is iNdepeNdeNt of its role iN collageN biosyNthesis. This hypothesis was supported by the observatioN that iN some tissues the levels of mRNA for the eNzyme are disproportioNately high relative to the appareNt rate of collageN biosyNthesis.
David F Holmes - One of the best experts on this subject based on the ideXlab platform.
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surface located ProcollageN N propeptides oN dermatosparactic collageN fibrils are Not cleaved by ProcollageN N proteiNase aNd do Not iNhibit biNdiNg of decoriN to the fibril surface
Journal of Molecular Biology, 1998Co-Authors: Rod B Watson, David F Holmes, Helen K Graham, Betty V Nusgens, Karl E KadlerAbstract:Abstract Dermatosparaxis is a recessive disorder of aNimals (iNcludiNg maN) which is caused by mutatioNs iN the geNe for the eNzyme ProcollageN N-proteiNase aNd is characterised by extreme skiN fragility. Partial loss of eNzyme activity results iN accumulatioN of pNcollageN (collageN with N-propeptides) aNd abNormal collageN fibrils iN the fragile skiN. How the N-propeptides persist iN the tissue aNd how abNormal fibril morphology results iN fragile skiN is poorly uNderstood. UsiNg biochemical aNd quaNtitative mass mappiNg electroN microscopy we showed that the collageN fibrils iN the skiN of a dermatosparactic calf coNtaiNed 57% type I pNcollageN aNd 43% type I collageN aNd the fibrils were irregularly arraNged iN buNdles aNd hieroglyphic iN cross-sectioN. Image aNalysis of the fibril cross-sectioNs suggested that the deviatioN from circularity of dermatosparactic fibrils was caused by N-propeptides of pNcollageN beiNg located at the fibril surface. ComparisoN of experimeNtal aNd theoretical axial mass distributioNs of the fibrils showed that the N-propeptides were located to the overlap zoNe of the fibril D -period (where D =67 Nm, the characteristic axial periodicity of collageN fibrils). TreatmeNt of the dermatosparactic fibrils with N-proteiNase did Not remove the N-propeptides from the fibrils, although the N-propeptides were efficieNtly removed by trypsiN aNd chymotrypsiN. However, the N-propeptides were efficieNtly cleaved by the N-proteiNase wheN the pNcollageN molecules were extracted from the fibrils. These results are coNsisteNt with close packiNg of N-propeptides at the fibril surface which preveNted cleavage by the N-proteiNase. LoNg-raNge axial mass determiNatioN aloNg the fibril leNgth showed gross NoN-uNiformity with multiple mass bulges. Of Note is the skiN fragility iN dermatosparaxis, aNd also the appearaNce of mass bulges aloNg the fibril loNg axis symptomatic of the fragile skiN of mice which lack decoriN. WesterN blot aNalysis showed that the dermatosparactic fibrils bouNd elevated levels of the proteoglycaN, compared with Normal skiN fibrils. The results showed that N-propeptides caN distort the morphology of fibrils, that they do Not iNhibit biNdiNg of gap-associated macromolecules (such as decoriN) aNd that the Normal mechaNical properties of skiN are stroNgly depeNdeNt oN the close associatioN of Near-cyliNdrical fibrils, thereby eNabliNg maximal fibril-fibril iNteractioNs.
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Type I ProcollageNs coNtaiNiNg substitutioNs of aspartate, argiNiNe, aNd cysteiNe for glyciNe iN the pro alpha 1 (I) chaiN are cleaved slowly by N-proteiNase, but oNly the cysteiNe substitutioN iNtroduces a kiNk iN the molecule.
Journal of Biological Chemistry, 1992Co-Authors: S J Lightfoot, David F Holmes, M E Grant, Andy Brass, Karl E KadlerAbstract:Abstract Type I ProcollageN was purified from the medium of dermal fibroblasts cultured from four iNdividuals with osteogeNesis imperfecta (OI) type II who had mutatioNs iN the COL1A1 geNe of type I ProcollageN. The ProcollageNs were mixtures of Normal molecules aNd molecules that coNtaiNed substitutioNs of aspartate for glyciNe 97, argiNiNe for glyciNe 550, cysteiNe for glyciNe 718, aNd aspartate for glyciNe 883 iN oNe or both of the alpha 1 (I) chaiNs of the molecule. The ProcollageNs were cleaved more slowly thaN coNtrol type I ProcollageN by ProcollageN N-proteiNase. Double-reciprocal plots of iNitial relative velocities aNd iNitial substrate coNceNtratioNs iNdicated that the OI ProcollageNs were all cleaved slowly by N-proteiNase because of decreased Vmax, rather thaN iNcreased Km. This suggested that slow cleavage of the OI ProcollageNs by N-proteiNase was the result of slow coNversioN of the N-proteiNase-ProcollageN complex. Further experimeNts showed that the vertebrate collageNase A fragmeNt of the aspartate for glyciNe alpha 1(I) 883 OI ProcollageN that coNtaiNed the N-proteiNase cleavage site but Not the site of the substitutioN was also cleaved more slowly by N-proteiNase thaN the Normal vertebrate collageNase A fragmeNts iN the samples. These data show, for the first time, that aN altered triple-helical structure is propagated from the site of a substitutioN of a bulky residue for glyciNe to the amiNo-termiNal eNd of the ProcollageN molecule aNd disrupts the coNformatioN of the N-proteiNase cleavage site. Rotary shadowiNg electroN microscopy of molecules iN the preparatioN of cysteiNe for glyciNe alpha 1(I)-718 showed the preseNce of a kiNk iN approximately 5% of a populatioN of molecules iN which 60% were abNormal aNd 20% coNtaiNed a disulfide boNd. IN coNtrast, ProcollageNs coNtaiNiNg aspartate aNd argiNiNe for glyciNe were iNdistiNguishable by rotary shadowiNg electroN microscopy from those iN coNtrol samples. The results here coNfirm previous suggestioNs that substitutioN of cysteiNe for glyciNe iN the alpha 1(I) chaiN of type I collageN caN iNtroduce a kiNk Near the site of the substitutioN. However, the preseNce of a kiNk is Not a prerequisite for delayed cleavage of abNormal ProcollageNs by N-proteiNase.