Cloning, expression, and characterization of a soluble calcium-activated nucleotidase, a human enzyme belonging to a new family of extracellular nucleotidases

Smith, Thomas M.; Hicks-Berger, Carrie A.; Kim, Sunkyu; Kirley, Terence L.

doi:10.1016/s0003-9861(02)00420-4

Cited by 71 publications

(116 citation statements)

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“…We have identified and named APY-1, a C. elegans homologue of the apyrase first found in the saliva of bloodsucking insects (Valenzuela et al, 1998;Charlab et al, 1999; Valenzuela et al, 2001); closely related enzymes have now been described in humans (Smith et al, 2002) and rats (Failer et al, 2002). The substrate specificity of APY-1 is very similar to that of the purified E-NDPase SCAN-1 of human and of the rat Ca 2ϩ -NDPase.…”

Section: Discussionmentioning

confidence: 79%

“…The presence in C. elegans ORFeome Database of the ORF F08C6.6 (hereinafter referred to as C. elegans apy-1), similar to a rat brain ER Ca 2ϩ -NDPase (Failer et al, 2002) and to human UDPase SCAN-1 (Smith et al, 2002), prompted us to ask whether APY-1 was an NDPase too. To answer this point, we carried out a nucleotidase activity assay on membrane fractions from apy-1(RNAi) mixed-stage worms.…”

Section: The Orf F08c66 Encodes For a Uridine Diphosphatase Up-regulmentioning

confidence: 99%

“…Although analogous to the E-NTPDases in their enzymatic action, these enzymes are not similar to the E-NTPDase family with respect to their amino acid sequences, and it appears that these two families of apyrases are evolutionarily unrelated (Valenzuela et al, 2001). Sequences related to the Ca 2ϩ -dependent secreted apyrases from bloodsucking arthropods were also found in other metazoans from Xenopus to mammalian species (Failer et al, 2002;Smith et al, 2002;Devader et al, 2006). The cDNA isolated from rat brain encodes a membrane-bound Ca 2ϩ -dependent NDPase (Ca 2ϩ -NDPase) that is targeted to the ER after heterologous expression.…”

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confidence: 97%

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APY-1, a NovelCaenorhabditis elegansApyrase Involved in Unfolded Protein Response Signalling and Stress Responses

Uccelletti

Pascoli

Farina

et al. 2008

MBoC

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Protein glycosylation modulates a wide variety of intracellular events and dysfunction of the glycosylation pathway has been reported in a variety of human pathologies. Endo-apyrases have been suggested to have critical roles in protein glycosylation and sugar metabolism. However, deciphering the physiological relevance of Endo-apyrases activity has actually proved difficult, owing to their complexity and the functional redundancy within the family. We report here that a UDP/GDPase, homologous to the human apyrase Scan-1, is present in the membranes of Caenorhabditis elegans, encoded by the ORF F08C6.6 and hereinafter-named APY-1. We showed that ER stress induced by tunicamycin or high temperature resulted in increased transcription of apy-1. This increase was not observed in C. elegans mutants defective in ire-1 or atf-6, demonstrating the requirement of both ER stress sensors for up-regulation of apy-1. Depletion of APY-1 resulted in constitutively activated unfolded protein response. Defects in the pharynx and impaired organization of thin fibers in muscle cells were observed in adult worms depleted of APY-1. Some of the apy-1(RNAi) phenotypes are suggestive of premature aging, because these animals also showed accumulation of lipofuscin and reduced lifespan that was not dependent on the functioning of DAF-2, the receptor of the insulin/IGF-1 signaling pathway. INTRODUCTIONFrom observations on human diseases and mutant mice, it has become clear that glycosylation plays a major role in metazoan development (Schachter, 2004). Given the numerous physiological roles of glycoproteins, which encompass many aspects of normal cellular function and survival, the structures of glycan moieties vary markedly providing the chemical diversity required to fulfill the vast range of their biological tasks. The diversity of natural sugar structures is ultimately derived from the activities of the enzymes responsible for sugar attachment, namely glycosyltransferases, which determine the feasibility of natural product enzymatic glycodiversification. Lumenal ecto-nucleoside triand diphosphohydrolases (E-NTPDases) of the secretory pathway of eukaryotes hydrolyzes the nucleoside diphosphates (NDPs) generated by glycosyltransferases. The resulting nucleoside monophosphates (NMPs) are weaker inhibitors of glycosyltransferases than NDPs. NMPs also serve as antiporters in the transport of nucleotide-sugars from the cytosol to the lumen of the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylation plays an important role in the biogenesis of secreted and transmembrane proteins and an elaborate mechanism ensure that only properly folded and assembled proteins exit the ER, a process termed "quality control."However, The ER's capacity to process proteins is relatively limited and the stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to a number of important human diseases (Zhang and Kaufman, 2006). Cells respond to ER stress by activating the unfolded protein response (UPR), which limits new pr...

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Section: Discussionmentioning

confidence: 79%

Section: The Orf F08c66 Encodes For a Uridine Diphosphatase Up-regulmentioning

confidence: 99%

mentioning

confidence: 97%

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APY-1, a NovelCaenorhabditis elegansApyrase Involved in Unfolded Protein Response Signalling and Stress Responses

Uccelletti

Pascoli

Farina

et al. 2008

MBoC

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“…In insects, these soluble proteins hydrolyze ADP at the site of host skin puncture, inhibiting the host's blood from clotting and allowing the insect to feed for extended times [5][6][7]. The homologous mammalian enzymes are termed calcium-activated nucleotidases (CANs, sometimes abbreviated as CANTs), and they exist as both intracellular membranebound forms in the ER and pre-Golgi membranes [8], as well as secreted, soluble forms (SCAN, [9]). Unlike the insect members of this nucleotidase family, the mammalian enzymes do not hydrolyze ADP effectively [8][9][10].…”

Section: Conflict Of Interest Statementmentioning

confidence: 99%

“…The homologous mammalian enzymes are termed calcium-activated nucleotidases (CANs, sometimes abbreviated as CANTs), and they exist as both intracellular membranebound forms in the ER and pre-Golgi membranes [8], as well as secreted, soluble forms (SCAN, [9]). Unlike the insect members of this nucleotidase family, the mammalian enzymes do not hydrolyze ADP effectively [8][9][10]. However, Dai et al were able to engineer the soluble human enzyme to hydrolyze ADP efficiently, by combining 5 point mutations in the active site [11], thus making the human enzyme functionally similar to the insect homologues regarding the ability to inhibit platelet aggregation.…”

Section: Conflict Of Interest Statementmentioning

confidence: 99%

Engineered human soluble calcium-activated nucleotidase inhibits coagulation in vitro and thrombosis in vivo

Yang

Kirley

2008

Thrombosis Research

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Human soluble calcium-activated nucleotidase (human SCAN) is a homologue of the salivary anticoagulant apyrases injected by insects into their hosts to allow blood feeding. However, the human enzyme, unlike its insect counterparts, does not efficiently hydrolyze the platelet agonist, ADP. By site-directed mutagenesis, two mutant human SCANs were constructed and expressed in bacteria. Following refolding from inclusion bodies and purification, these enzymes were assessed for anticoagulant and anti-thrombotic efficacy. These engineered proteins include both active site mutations and a dimer interface mutation to increase the stability and ADPase activity of the modified human nucleotidase. The ADPase activity of these mutants increased more than ten fold. The E130Y/ K201M/E216M SCAN mutant efficiently inhibited platelet aggregation in vitro. In addition, the E130Y/K201M/T206K/T207E/E216M mutant inhibited jugular vein thrombosis in the murine ferric chloride-induced model of thrombosis, as assessed by laser Doppler blood flow measurements. The bed bug insect homologue of human SCAN was also expressed and purified, and used in these in vivo experiments as a benchmark to assess the therapeutic potential of the engineered human enzymes. The most active modified human enzyme was able to completely inhibit the thrombosis induced by ferric chloride at roughly double the protein dose used for the bed bug enzyme. Thus, for the first time, we show that an engineered form of this human protein is efficacious in an in vivo model of thrombosis, demonstrating that suitably modified human SCAN enzymes have therapeutic potential as anti-coagulant and anti-thrombotic therapeutic agents. This suggests their utility in future treatment strategies for thrombotic cardiovascular diseases, including myocardial infarctions and ischemic strokes. Keywordscalcium-activated nucleotidase; ferric chloride-induced thrombosis; aggregometry; platelets; anticoagulant proteins; laser Doppler blood flow Cardiovascular and cerebrovascular diseases continue to be the leading causes of death in the developed world, with more than 450,000 patients in the United States afflicted by stroke annually [1]. The platelet is a major contributor to occlusive thrombus formation in acute Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of Interest StatementThere are no conflicts of interest to declare. The sole funding for this study was obtained via NIH grant HL72882 to T.L.K. Mammals express a protein homologous to the nucleotidases found in the saliva of bloodsucking insects. In insects, these soluble pro...

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CANT1 deficiency in a mouse model of Desbuquois dysplasia impairs glycosaminoglycan synthesis and chondrocyte differentiation in growth plate cartilage

et al. 2020

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Desbuquois dysplasia (DD) type 1 is a rare skeletal dysplasia characterized by a short stature, round face, progressive scoliosis, and joint laxity. The causative gene has been identified as calcium‐activated nucleotidase 1 (CANT1), which encodes a nucleotidase that preferentially hydrolyzes UDP to UMP and phosphate. In this study, we generated Cant1 KO mice using CRISPR/Cas9‐mediated genome editing. All F0 mice possessing frameshift deletions at both Cant1 alleles exhibited a dwarf phenotype. Germline transmission of the edited allele was confirmed in an F0 heterozygous mouse, and KO mice were generated by crossing of the heterozygous breeding pairs. Cant1 KO mice exhibited skeletal defects, including short stature, thoracic kyphosis, and delta phalanx, all of which are observed in DD type 1 patients. The glycosaminoglycan (GAG) content and extracellular matrix space were reduced in the growth plate cartilage of mutants, and proliferating chondrocytes lost their typical flat shape and became round. Chondrocyte differentiation, especially terminal differentiation to hypertrophic chondrocytes, was impaired in Cant1 KO mice. These findings indicate that CANT1 is involved in the synthesis of GAG and regulation of chondrocyte differentiation in the cartilage and contribute to a better understanding of the pathogenesis of DD type 1.

show abstract

Cloning, expression, and characterization of a soluble calcium-activated nucleotidase, a human enzyme belonging to a new family of extracellular nucleotidases

Cited by 71 publications

References 53 publications

APY-1, a NovelCaenorhabditis elegansApyrase Involved in Unfolded Protein Response Signalling and Stress Responses

APY-1, a NovelCaenorhabditis elegansApyrase Involved in Unfolded Protein Response Signalling and Stress Responses

Engineered human soluble calcium-activated nucleotidase inhibits coagulation in vitro and thrombosis in vivo

CANT1 deficiency in a mouse model of Desbuquois dysplasia impairs glycosaminoglycan synthesis and chondrocyte differentiation in growth plate cartilage

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