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Dieter Strack - One of the best experts on this subject based on the ideXlab platform.
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snap shot of Serine Carboxypeptidase like acyltransferase evolution the loss of conserved disulphide bridge is responsible for the completion of neo functionalization
Journal of Phylogenetics & Evolutionary Biology, 2013Co-Authors: Felix Stehle, Dieter Strack, Jurgen Schmidt, Victor Wray, Franziska Gotsch, Wolfgang BrandtAbstract:In this work, it is shown that the At2g23010 gene product encodes 1-O-sinapoyl-β-glucose:1-O-sinapoyl-β-glucose sinapoyltransferase (SST). In contrast to all other functional characterized acyltransferases, the SST protein is highly specific towards this reaction only, and the substrate specificity was correlated to one amino acid substitution. Detailed sequence analysis revealed the lack of the disulphide bond S1 (C78 and C323 in the SMT (sinapoylglucose:malate sinapoyltransferase), that is in SST C80 and D327). The reconstitution of this disulphide bond led to an enzyme accepting many different substrates including disaccharides. Interestingly, the overall changes within the model structures are not very dramatic, but nevertheless, the enzyme models provide some explanations for the broadened substrate specificity: the reconstitution of the disulphide bond provoked more space within the substrate binding pocket simultaneously avoiding electrostatic repulsion. As the SST sequence of A. lyrata also showed the same mutation, the loss of the disulphide bond should has arisen at least 10 mya ago. A Ka/Ks ratio ≤ 1 supports the hypothesis that the loss of this disulphide bond was rather a specification towards a certain reaction than the beginning of a gene death. At the same time, this is also associated with the fixation in the genome.
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heterologous expression of a Serine Carboxypeptidase like acyltransferase and characterization of the kinetic mechanism
FEBS Journal, 2008Co-Authors: Felix Stehle, Dieter Strack, Milton T Stubbs, Carsten MilkowskiAbstract:In plant secondary metabolism, β-acetal ester-dependent acyltransferases, such as the 1-O-sinapoyl-β-glucose:l-malate sinapoyltransferase (SMT; EC 2.3.1.92), are homologous to Serine Carboxypeptidases. Mutant analyses and modeling of Arabidopsis SMT (AtSMT) have predicted amino acid residues involved in substrate recognition and catalysis, confirming the main functional elements conserved within the Serine Carboxypeptidase protein family. However, the functional shift from hydrolytic to acyltransferase activity and structure–function relationship of AtSMT remain obscure. To address these questions, a heterologous expression system for AtSMT has been developed that relies on Saccharomyces cerevisiae and an episomal leu2-d vector. Codon usage adaptation of AtSMT cDNA raised the produced SMT activity by a factor of approximately three. N-terminal fusion to the leader peptide from yeast proteinase A and transfer of this expression cassette to a high copy vector led to further increase in SMT expression by factors of 12 and 42, respectively. Finally, upscaling the biomass production by fermenter cultivation lead to another 90-fold increase, resulting in an overall 3900-fold activity compared to the AtSMT cDNA of plant origin. Detailed kinetic analyses of the recombinant protein indicated a random sequential bi-bi mechanism for the SMT-catalyzed transacylation, in contrast to a double displacement (ping-pong) mechanism, characteristic of Serine Carboxypeptidases.
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structure determinants and substrate recognition of Serine Carboxypeptidase like acyltransferases from plant secondary metabolism
FEBS Letters, 2006Co-Authors: Felix Stehle, Carsten Milkowski, Wolfgang Brandt, Dieter StrackAbstract:Structures of the Serine Carboxypeptidase-like enzymes 1-O-sinapoyl-β-glucose:l-malate sinapoyltransferase (SMT) and 1-O-sinapoyl-β-glucose:choline sinapoyltransferase (SCT) were modeled to gain insight into determinants of specificity and substrate recognition. The structures reveal the α/β-hydrolase fold as scaffold for the catalytic triad Ser-His-Asp. The recombinant mutants of SMT Ser173Ala and His411Ala were inactive, whereas Asp358Ala displayed residual activity of 20%. 1-O-sinapoyl-β-glucose recognition is mediated by a network of hydrogen bonds. The glucose moiety is recognized by a hydrogen bond network including Trp71, Asn73, Glu87 and Asp172. The conserved Asp172 at the sequence position preceding the catalytic Serine meets sterical requirements for the glucose moiety. The mutant Asn73Ala with a residual activity of 13% underscores the importance of the intact hydrogen bond network. Arg322 is of key importance by hydrogen bonding of 1-O-sinapoyl-β-glucose and l-malate. By conformational change, Arg322 transfers l-malate to a position favoring its activation by His411. Accordingly, the mutant Arg322Glu showed 1% residual activity. Glu215 and Arg219 establish hydrogen bonds with the sinapoyl moiety. The backbone amide hydrogens of Gly75 and Tyr174 were shown to form the oxyanion hole, stabilizing the transition state. SCT reveals also the catalytic triad and a hydrogen bond network for 1-O-sinapoyl-β-glucose recognition, but Glu274, Glu447, Thr445 and Cys281 are crucial for positioning of choline.
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formation of a complex pattern of sinapate esters in brassica napus seeds catalyzed by enzymes of a Serine Carboxypeptidase like acyltransferase family
Phytochemistry, 2005Co-Authors: Alfred Baumert, Carsten Milkowski, Jurgen Schmidt, Manfred Nimtz, Victor Wray, Dieter StrackAbstract:Members of the Brassicaceae accumulate complex patterns of sinapate esters, as shown in this communication with seeds of oilseed rape (Brassica napus). Fifteen seed constituents were isolated and identified by a combination of high-field NMR spectroscopy and high resolution electrospray ionisation mass spectrometry. These include glucose, gentiobiose and kaempferol glycoside esters as well as sinapine (sinapoylcholine), sinapoylmalate and an unusual cyclic spermidine amide. One of the glucose esters (1,6-di-Osinapoylglucose), two gentiobiose esters (1-O-caffeoylgentiobiose and 1,2,6 0 -tri-O-sinapoylgentiobiose) and two kaempferol conjugates [4 0 -(6-O-sinapoylglucoside)-3,7-di-O-glucoside and 3-O-sophoroside-7-O-(2-O-sinapoylglucoside)] seem to be new plant products. Serine Carboxypeptidase-like (SCPL) acyltransferases catalyze the formation of sinapine and sinapoylmalate accepting 1-O-b-acetal esters (1-O-b-glucose esters) as acyl donors. To address the question whether the formation of other components of the complex pattern of the sinapate esters in B. napus seeds is catalyzed via 1-O-sinapoyl-b-glucose, we performed a seed-specific dsRNAi-based suppression of the sinapate glucosyltransferase gene (BnSGT1) expression. In seeds of BnSGT1-suppressing plants the amount of sinapoylglucose decreased below the HPLC detection limit resulting in turn in the disappearance or marked decrease of all the other sinapate esters, indicating that formation of the complex pattern of these esters in B. napus seeds is dependent on sinapoylglucose. This gives rise to the assumption that enzymes of an SCPL acyltransferase family catalyze the appropriate transfer reactions to synthesize the accumulating esters. � 2005 Elsevier Ltd. All rights reserved.
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Serine Carboxypeptidase like acyltransferases
Phytochemistry, 2004Co-Authors: Carsten Milkowski, Dieter StrackAbstract:Abstract In plant secondary metabolism, an alternative pathway of ester formation is facilitated by acyltransferases accepting 1- O -β-acetal esters (1- O -β-glucose esters) as acyl donors instead of coenzyme A thioesters. Molecular data indicate homology of these transferases with hydrolases of the Serine Carboxypeptidase type defining them as Serine Carboxypeptidase-like (SCPL) acyltransferases. During evolution, they apparently have been recruited from Serine Carboxypeptidases and adapted to take over acyl transfer function. SCPL acyltransferases belong to the highly divergent class of α/β hydrolases. These enzymes make use of a catalytic triad formed by a nucleophile, an acid and histidine acting as a charge relay system for the nucleophilic attack on amide or ester bonds. In analogy to SCPL acyltransferases, bacterial thioesterase domains are known which favour transferase activity over hydrolysis. Structure elucidation reveals water exclusion and a distortion of the oxyanion hole responsible for the changed activity. In plants, SCPL proteins form a large family. By sequence comparison, a distinguished number of Arabidopsis SCPL proteins cluster with proven SCPL acyltransferases. This indicates the occurrence of a large number of SCPL proteins co-opted to catalyse acyltransfer reactions. SCPL acyltransferases are ideal systems to investigate principles of functional adaptation and molecular evolution of plant genes.
Carsten Milkowski - One of the best experts on this subject based on the ideXlab platform.
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Serine Carboxypeptidase-Like Acyltransferases from Plants
Methods in enzymology, 2012Co-Authors: Sam T. Mugford, Carsten MilkowskiAbstract:Serine Carboxypeptidase-like (SCPL) acyltransferases facilitate transacylation reactions using energy-rich 1-O-β-glucose esters in the synthesis of an array of bioactive compounds and are associated with the diversification of plant natural products. SCPL acyltransferases have evolved from a hydrolytic ancestor by adapting functional elements of the proteases such as the catalytic triad, oxyanion hole, and substrate recognition H-bond network to their new function. As vacuolar proteins, SCPL acyltransferases define an alternative cellular route of transacylation spatially separated from the cytoplasmic enzymes of the BAHD acyltransferase family named according to the first characterized members (BEAT, AHCT, HCBT, and DAT). Recent efforts in cloning and characterization led to the identification of diagnostic peptides for SCPL acyltransferases, enabling the detection of candidate genes in several plant genomes. Detailed biochemical analysis of SCPL acyltransferases is strongly dependent on comprehensive heterologous expression systems, efficient protein purification protocols, and the supply of appropriate substrates. This chapter describes some useful techniques and strategies for identification and characterization of SCPL acyltransferases.
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heterologous expression of a Serine Carboxypeptidase like acyltransferase and characterization of the kinetic mechanism
FEBS Journal, 2008Co-Authors: Felix Stehle, Dieter Strack, Milton T Stubbs, Carsten MilkowskiAbstract:In plant secondary metabolism, β-acetal ester-dependent acyltransferases, such as the 1-O-sinapoyl-β-glucose:l-malate sinapoyltransferase (SMT; EC 2.3.1.92), are homologous to Serine Carboxypeptidases. Mutant analyses and modeling of Arabidopsis SMT (AtSMT) have predicted amino acid residues involved in substrate recognition and catalysis, confirming the main functional elements conserved within the Serine Carboxypeptidase protein family. However, the functional shift from hydrolytic to acyltransferase activity and structure–function relationship of AtSMT remain obscure. To address these questions, a heterologous expression system for AtSMT has been developed that relies on Saccharomyces cerevisiae and an episomal leu2-d vector. Codon usage adaptation of AtSMT cDNA raised the produced SMT activity by a factor of approximately three. N-terminal fusion to the leader peptide from yeast proteinase A and transfer of this expression cassette to a high copy vector led to further increase in SMT expression by factors of 12 and 42, respectively. Finally, upscaling the biomass production by fermenter cultivation lead to another 90-fold increase, resulting in an overall 3900-fold activity compared to the AtSMT cDNA of plant origin. Detailed kinetic analyses of the recombinant protein indicated a random sequential bi-bi mechanism for the SMT-catalyzed transacylation, in contrast to a double displacement (ping-pong) mechanism, characteristic of Serine Carboxypeptidases.
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structure determinants and substrate recognition of Serine Carboxypeptidase like acyltransferases from plant secondary metabolism
FEBS Letters, 2006Co-Authors: Felix Stehle, Carsten Milkowski, Wolfgang Brandt, Dieter StrackAbstract:Structures of the Serine Carboxypeptidase-like enzymes 1-O-sinapoyl-β-glucose:l-malate sinapoyltransferase (SMT) and 1-O-sinapoyl-β-glucose:choline sinapoyltransferase (SCT) were modeled to gain insight into determinants of specificity and substrate recognition. The structures reveal the α/β-hydrolase fold as scaffold for the catalytic triad Ser-His-Asp. The recombinant mutants of SMT Ser173Ala and His411Ala were inactive, whereas Asp358Ala displayed residual activity of 20%. 1-O-sinapoyl-β-glucose recognition is mediated by a network of hydrogen bonds. The glucose moiety is recognized by a hydrogen bond network including Trp71, Asn73, Glu87 and Asp172. The conserved Asp172 at the sequence position preceding the catalytic Serine meets sterical requirements for the glucose moiety. The mutant Asn73Ala with a residual activity of 13% underscores the importance of the intact hydrogen bond network. Arg322 is of key importance by hydrogen bonding of 1-O-sinapoyl-β-glucose and l-malate. By conformational change, Arg322 transfers l-malate to a position favoring its activation by His411. Accordingly, the mutant Arg322Glu showed 1% residual activity. Glu215 and Arg219 establish hydrogen bonds with the sinapoyl moiety. The backbone amide hydrogens of Gly75 and Tyr174 were shown to form the oxyanion hole, stabilizing the transition state. SCT reveals also the catalytic triad and a hydrogen bond network for 1-O-sinapoyl-β-glucose recognition, but Glu274, Glu447, Thr445 and Cys281 are crucial for positioning of choline.
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formation of a complex pattern of sinapate esters in brassica napus seeds catalyzed by enzymes of a Serine Carboxypeptidase like acyltransferase family
Phytochemistry, 2005Co-Authors: Alfred Baumert, Carsten Milkowski, Jurgen Schmidt, Manfred Nimtz, Victor Wray, Dieter StrackAbstract:Members of the Brassicaceae accumulate complex patterns of sinapate esters, as shown in this communication with seeds of oilseed rape (Brassica napus). Fifteen seed constituents were isolated and identified by a combination of high-field NMR spectroscopy and high resolution electrospray ionisation mass spectrometry. These include glucose, gentiobiose and kaempferol glycoside esters as well as sinapine (sinapoylcholine), sinapoylmalate and an unusual cyclic spermidine amide. One of the glucose esters (1,6-di-Osinapoylglucose), two gentiobiose esters (1-O-caffeoylgentiobiose and 1,2,6 0 -tri-O-sinapoylgentiobiose) and two kaempferol conjugates [4 0 -(6-O-sinapoylglucoside)-3,7-di-O-glucoside and 3-O-sophoroside-7-O-(2-O-sinapoylglucoside)] seem to be new plant products. Serine Carboxypeptidase-like (SCPL) acyltransferases catalyze the formation of sinapine and sinapoylmalate accepting 1-O-b-acetal esters (1-O-b-glucose esters) as acyl donors. To address the question whether the formation of other components of the complex pattern of the sinapate esters in B. napus seeds is catalyzed via 1-O-sinapoyl-b-glucose, we performed a seed-specific dsRNAi-based suppression of the sinapate glucosyltransferase gene (BnSGT1) expression. In seeds of BnSGT1-suppressing plants the amount of sinapoylglucose decreased below the HPLC detection limit resulting in turn in the disappearance or marked decrease of all the other sinapate esters, indicating that formation of the complex pattern of these esters in B. napus seeds is dependent on sinapoylglucose. This gives rise to the assumption that enzymes of an SCPL acyltransferase family catalyze the appropriate transfer reactions to synthesize the accumulating esters. � 2005 Elsevier Ltd. All rights reserved.
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Serine Carboxypeptidase like acyltransferases
Phytochemistry, 2004Co-Authors: Carsten Milkowski, Dieter StrackAbstract:Abstract In plant secondary metabolism, an alternative pathway of ester formation is facilitated by acyltransferases accepting 1- O -β-acetal esters (1- O -β-glucose esters) as acyl donors instead of coenzyme A thioesters. Molecular data indicate homology of these transferases with hydrolases of the Serine Carboxypeptidase type defining them as Serine Carboxypeptidase-like (SCPL) acyltransferases. During evolution, they apparently have been recruited from Serine Carboxypeptidases and adapted to take over acyl transfer function. SCPL acyltransferases belong to the highly divergent class of α/β hydrolases. These enzymes make use of a catalytic triad formed by a nucleophile, an acid and histidine acting as a charge relay system for the nucleophilic attack on amide or ester bonds. In analogy to SCPL acyltransferases, bacterial thioesterase domains are known which favour transferase activity over hydrolysis. Structure elucidation reveals water exclusion and a distortion of the oxyanion hole responsible for the changed activity. In plants, SCPL proteins form a large family. By sequence comparison, a distinguished number of Arabidopsis SCPL proteins cluster with proven SCPL acyltransferases. This indicates the occurrence of a large number of SCPL proteins co-opted to catalyse acyltransfer reactions. SCPL acyltransferases are ideal systems to investigate principles of functional adaptation and molecular evolution of plant genes.
Clint Chapple - One of the best experts on this subject based on the ideXlab platform.
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related arabidopsis Serine Carboxypeptidase like sinapoylglucose acyltransferases display distinct but overlapping substrate specificities
Plant Physiology, 2007Co-Authors: Christopher M Fraser, Amber M Shirley, Michael G Thompson, John Ralph, Jessica A Schoenherr, Taksina Sinlapadech, Mark C Hall, Clint ChappleAbstract:The Arabidopsis (Arabidopsis thaliana) genome encodes 51 proteins annotated as Serine Carboxypeptidase-like (SCPL) enzymes. Nineteen of these SCPL proteins are highly similar to one another, and represent a clade that appears to be unique to plants. Two of the most divergent proteins within this group have been characterized to date, sinapoyl-glucose (Glc):malate sinapoyltransferase and sinapoyl-Glc:choline sinapoyltransferase. The fact that two of the least related proteins within this clade are acyltransferases rather than true Serine Carboxypeptidases suggests that some or all of the remaining members of this group may have similar activities. The gene that encodes sinapoyl-Glc:malate sinapoyltransferase (sinapoyl-Glc accumulator1 [SNG1]: At2g22990) is one of five SCPL genes arranged in a cluster on chromosome 2. In this study, an analysis of deletion mutant lines lacking one or more genes in this SCPL gene cluster reveals that three of these genes also encode sinapoyl-Glc-dependent acyltransferases. At2g23000 encodes sinapoyl-Glc:anthocyanin acyltransferase, an enzyme that is required for the synthesis of the sinapoylated anthocyanins in Arabidopsis. At2g23010 encodes an enzyme capable of synthesizing 1,2-disinapoyl-Glc from two molecules of sinapoyl-Glc, an activity shared by SNG1 and At2g22980. Sequence analysis of these SCPL proteins reveals pairwise percent identities that range from 71% to 78%, suggesting that their differing specificities for acyl acceptor substrates are due to changes in a relatively small subset of amino acids. The study of these SCPL proteins provides an opportunity to examine enzyme structure-function relationships and may shed light on the role of evolution of hydroxycinnamate ester metabolism and the SCPL gene family in Arabidopsis and other flowering plants.
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Biochemical characterization of sinapoylglucose:choline sinapoyltransferase, a Serine Carboxypeptidase-like protein that functions as an acyltransferase in plant secondary metabolism.
The Journal of biological chemistry, 2003Co-Authors: Amber M Shirley, Clint ChappleAbstract:Recently, Serine Carboxypeptidase-like (SCPL) proteins that catalyze transacylation reactions in plant secondary metabolism have been identified from wild tomato and Arabidopsis. These include sinapoylglucose: choline sinapoyltransferase (SCT), an enzyme that functions in Arabidopsis sinapate ester synthesis. SCT and the other known SCPL acyltransferases all share the conserved Serine, aspartic acid, and histidine residues employed for catalysis by classical Serine Carboxypeptidases, although the importance of these residues and the mechanism by which this class of SCPL proteins catalyze acyltransferase reactions is unknown. To characterize further SCT and its catalytic mechanism, we have employed the Saccharomyces cerevisiae vacuolar protein localization 1 mutant, which secretes the Serine Carboxypeptidase, Carboxypeptidase Y, and other proteins normally targeted to the vacuole. When expressed in this strain, SCT is similarly secreted. SCT has been purified from the yeast medium and used for kinetic characterization of the protein. Immunological analysis of SCT has revealed that the expected 50-kDa mature protein is proteolytically processed in yeast and in planta, most likely resulting in the production of a heterodimer derived from a 30- and 17-kDa polypeptide.
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cloning of the sng1 gene of arabidopsis reveals a role for a Serine Carboxypeptidase like protein as an acyltransferase in secondary metabolism
The Plant Cell, 2000Co-Authors: Claus Lehfeldt, Amber M Shirley, Knut Meyer, Max O Ruegger, Joanne C Cusumano, Paul V. Viitanen, Dieter Strack, Clint ChappleAbstract:Serine Carboxypeptidases contain a conserved catalytic triad of Serine, histidine, and aspartic acid active-site residues. These enzymes cleave the peptide bond between the penultimate and C-terminal amino acid residues of their protein or peptide substrates. The Arabidopsis Genome Initiative has revealed that the Arabidopsis genome encodes numerous proteins with homology to Serine Carboxypeptidases. Although many of these proteins may be involved in protein turnover or processing, the role of virtually all of these Serine Carboxypeptidase-like (SCPL) proteins in plant metabolism is unknown. We previously identified an Arabidopsis mutant, sng1 (sinapoylglucose accumulator 1), that is defective in synthesis of sinapoylmalate, one of the major phenylpropanoid secondary metabolites accumulated by Arabidopsis and some other members of the Brassicaceae. We have cloned the gene that is defective in sng1 and have found that it encodes a SCPL protein. Expression of SNG1 in Escherichia coli demonstrates that it encodes sinapoylglucose:malate sinapoyltransferase, an enzyme that catalyzes a transesterification instead of functioning like a hydrolase, as do the other Carboxypeptidases. This finding suggests that SCPL proteins have acquired novel functions in plant metabolism and provides an insight into the evolution of secondary metabolic pathways in plants.
Daniel J Klionsky - One of the best experts on this subject based on the ideXlab platform.
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a newly characterized vacuolar Serine Carboxypeptidase atg42 ybr139w is required for normal vacuole function and the terminal steps of autophagy in the yeast saccharomyces cerevisiae
Molecular Biology of the Cell, 2018Co-Authors: Katherine R Parzych, Aileen Ariosa, Muriel Mari, Daniel J KlionskyAbstract:Macroautophagy (hereafter autophagy) is a cellular recycling pathway essential for cell survival during nutrient deprivation that culminates in the degradation of cargo within the vacuole in yeast and the lysosome in mammals, followed by efflux of the resultant macromolecules back into the cytosol. The yeast vacuole is home to many different hydrolytic proteins and while few have established roles in autophagy, the involvement of others remains unclear. The vacuolar Serine Carboxypeptidase Y (Prc1) has not been previously shown to have a role in vacuolar zymogen activation and has not been directly implicated in the terminal degradation steps of autophagy. Through a combination of molecular genetic, cell biological, and biochemical approaches, we have shown that Prc1 has a functional homologue, Ybr139w, and that cells deficient in both Prc1 and Ybr139w have defects in autophagy-dependent protein synthesis, vacuolar zymogen activation, and autophagic body breakdown. Thus, we have demonstrated that Ybr139w and Prc1 have important roles in proteolytic processing in the vacuole and the terminal steps of autophagy.
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A newly characterized vacuolar Serine Carboxypeptidase, Atg42/Ybr139w, is required for normal vacuole function and the terminal steps of autophagy in the yeast Saccharomyces cerevisiae
Molecular biology of the cell, 2018Co-Authors: Katherine R Parzych, Aileen Ariosa, Muriel Mari, Daniel J KlionskyAbstract:Macroautophagy (hereafter autophagy) is a cellular recycling pathway essential for cell survival during nutrient deprivation that culminates in the degradation of cargo within the vacuole in yeast and the lysosome in mammals, followed by efflux of the resultant macromolecules back into the cytosol. The yeast vacuole is home to many different hydrolytic proteins and while few have established roles in autophagy, the involvement of others remains unclear. The vacuolar Serine Carboxypeptidase Y (Prc1) has not been previously shown to have a role in vacuolar zymogen activation and has not been directly implicated in the terminal degradation steps of autophagy. Through a combination of molecular genetic, cell biological, and biochemical approaches, we have shown that Prc1 has a functional homologue, Ybr139w, and that cells deficient in both Prc1 and Ybr139w have defects in autophagy-dependent protein synthesis, vacuolar zymogen activation, and autophagic body breakdown. Thus, we have demonstrated that Ybr139w and Prc1 have important roles in proteolytic processing in the vacuole and the terminal steps of autophagy.
Alessandra Dazzo - One of the best experts on this subject based on the ideXlab platform.
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lack of ppca expression only partially coincides with lysosomal storage in galactosialidosis mice indirect evidence for spatial requirement of the catalytic rather than the protective function of ppca
Human Molecular Genetics, 1998Co-Authors: Robbert J Rottier, Christopher N Hahn, Linda Mann, Richard J Smeyne, Kinuko Suzuki, Maria Del Pilar Martin, Alessandra DazzoAbstract:Protective protein/cathepsin A (PPCA) is a pleiotropic lysosomal enzyme that complexes with beta-galactosidase and neuraminidase, and possesses Serine Carboxypeptidase activity. Its deficiency in man results in the neurodegenerative lysosomal storage disorder galactosialidosis (GS). The mouse model of this disease resembles the human early onset phenotype and results in severe nephropathy and ataxia. To understand better the pathophysiology of the disease, we compared the occurrence of lysosomal PPCA mRNA and protein in normal adult mouse tissues with the incidence of lysosomal storage in PPCA(-/-) mice. PPCA expression was markedly variable among different tissues. Most sites that produced both mRNA and protein at high levels in normal mice showed extensive and overt storage in the knockout mice. However, this correlation was not consistent as some cells that normally expressed high levels of PPCA were unaffected in their storage capability in the PPCA(-/-) mice. In addition, some normally low expressing cells accumulated large amounts of undegraded products in the GS mouse. This apparent discrepancy may reflect a requirement for the catalytic rather than the protective function of PPCA and/or the presence of cell-specific substrates in certain cell types. A detailed map showing the cellular distribution of PPCA in nomal mouse tissues as well as the sites of lysosomal storage in deficient mice is critical for accurate assessment of the effects of therapeutic interventions.
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lack of ppca expression only partially coincides with lysosomal storage in galactosialidosis mice indirect evidence for spatial requirement of the catalytic rather than the protective function of ppca
Human Molecular Genetics, 1998Co-Authors: Robbert J Rottier, Christopher N Hahn, Linda Mann, Maria Del Pilar Martin, Richard J Smeyne, Kinuko Suzuki, Alessandra DazzoAbstract:Protective protein/cathepsin A (PPCA) is a pleiotropic lysosomal enzyme that complexes with β-galactosidase and neuraminidase, and possesses Serine Carboxypeptidase activity. Its deficiency in man results in the neurodegenerative lysosomal storage disorder galactosialidosis (GS). The mouse model of this disease resembles the human early onset phenotype and results in severe nephropathy and ataxia. To understand better the pathophysiology of the disease, we compared the occurrence of lysosomal PPCA mRNA and protein in normal adult mouse tissues with the incidence of lysosomal storage in PPCA(-/-) mice. PPCA expression was markedly variable among different tissues. Most sites that produced both mRNA and protein at high levels in normal mice showed extensive and overt storage in the knockout mice. However, this correlation was not consistent as some cells that normally expressed high levels of PPCA were unaffected in their storage capability in the PPCA(-/-) mice. In addition, some normally low expressing cells accumulated large amounts of undegraded products in the GS mouse. This apparent discrepancy may reflect a requirement for the catalytic rather than the protective function of PPCA and/or the presence of cell-specific substrates in certain cell types. A detailed map showing the cellular distribution of PPCA in nomal mouse tissues as well as the sites of lysosomal storage in deficient mice is critical for accurate assessment of the effects of therapeutic interventions.
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a point mutation in the neu 1 locus causes the neuraminidase defect in the sm j mouse
Human Molecular Genetics, 1998Co-Authors: Robbert J Rottier, Erik Bonten, Alessandra DazzoAbstract:Lysosomal neuraminidase (sialidase) occurs in a high molecular weight complex with the glycosidase beta-galactosidase and the Serine Carboxypeptidase protective protein/cathepsin A (PPCA). Association of the enzyme with PPCA is crucial for its correct targeting and lysosomal activation. In man two genetically distinct storage disorders are associated with either a primary or a secondary deficiency of lysosomal neuraminidase: sialidosis and galactosialidosis. In the mouse the naturally occurring inbred strain SM/J presents with a number of phenotypic abnormalities that have been attributed to reduced neuraminidase activity. SM/J mice were originally characterized by their altered sialylation of several lysosomal glycoproteins. This defect was linked to a single gene, neu-1 , on chromosome 17, which was mapped by linkage analysis to the H-2 locus. In addition, these mice have an altered immune response that has also been coupled to a deficiency of the Neu-1 neuraminidase. Here we report the identification in SM/J mice of a single amino acid substitution (L209I) in the Neu-1 protein which is responsible for the partial deficiency of lysosomal neuraminidase. We propose that the reduced activity is caused by the enzyme's altered affinity for its substrate, rather than a change in substrate specificity or turnover rate. The mutant enzyme is correctly compartmentalized in lysosomes and maintains the ability to associate with its activating protein, PPCA. We propose that it is this mutation that is responsible for the SM/J phenotype.
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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.
