Dataset Information

Various N-glycoforms differentially upregulate E-NTPDase activity of the NTPDase3/CD39L3 ecto-enzymatic domain.

ABSTRACT: The GDA1/CD39 ecto-nucleoside triphosphate diphosphosphohydrolase (E-NTPDase) superfamily is a group of eight heavily glycosylated ecto-enzymes that hydrolyze extracellular nucleosides di- and tri-phosphates in the presence of divalent cations, to generate the monophosphate derivatives. This catalytic process differentially regulates a complex array of purinergic signaling responses. NTPDase3/CD39L3is dominantly expressed in pancreatic islet cells, where it may regulate insulin secretion, and has seven N-linked glycosylation sites with four close to five highly conserved domains called "apyrase conserved regions" (ACRs). In a manner similar to CD39, NTPDase3/CD39L3 uses ATP as its preferential substrate and also possesses significant activities toward other triphosphate and diphosphate nucleosides. To understand the mechanism of the ecto-NTPDase activity and substrate specificity, potentially impacted by N-glycans, we have generated soluble enzymatic domains of NTPDase3/CD39L3 in human embryotic kidney cells with four different glycan modifications. These include mannose5-9 glycans with kifunesine treatment, single GlcNAc-Asn by treatment with EndoH, de-glycosylated form by treatment with PNGaseF, and wild-type glycans. Our functional data indicate that the non-glycosylated NTPDase3/CD39L3 ecto-enzymatic domain retains activity, but that N-glycan attachments, such as the GlcNAc-Asn, substantially upregulate specific NTPDase activity by 2-20 fold. Both the Vmax and the Km on di- or tri-phosphate nucleosides are substantially and differentially altered by the glycan attachments. Structural modeling analysis based on putative structures derived from bacterial-originated CD39 domain proteins suggests that N-glycan modifications at Asn149 next to ACR2 and/or Asn454, N-terminal to ACR5 have critical roles in regulating the catalytic pocket of NTPDase3/CD39L3. Our data provide both new insights into the enzymatic mechanisms of NTPDase family members and further evidence that N-glycans directly modulate functional ectonucleotidase activities.

SUBMITTER: Zhong AH

PROVIDER: S-EPMC5714850 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Various N-glycoforms differentially upregulate E-NTPDase activity of the NTPDase3/CD39L3 ecto-enzymatic domain.

Zhong Alexander H AH Gordon Jiang Z Z Cummings Richard D RD Robson Simon C SC

Purinergic signalling 20170927 4

The GDA1/CD39 ecto-nucleoside triphosphate diphosphosphohydrolase (E-NTPDase) superfamily is a group of eight heavily glycosylated ecto-enzymes that hydrolyze extracellular nucleosides di- and tri-phosphates in the presence of divalent cations, to generate the monophosphate derivatives. This catalytic process differentially regulates a complex array of purinergic signaling responses. NTPDase3/CD39L3is dominantly expressed in pancreatic islet cells, where it may regulate insulin secretion, and ha ...[more]

PMID: 28956227

Similar Datasets

Project description:Macrophages are key elements in the inflammatory process, whereas depending on the micro-environmental stimulation they exhibit a pro-inflammatory (classical/M1) or an anti-inflammatory/reparatory (alternative/M2) phenotype. Extracellular ATP can act as a danger signal whereas adenosine generally serves as a negative feedback mechanism to limit inflammation. The local increase in nucleotides communication is controlled by ectonucleotidases, such as members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family and ecto-5'-nucleotidase/CD73 (ecto-5'-NT). In the present work we evaluated the presence of these enzymes in resident mice M1 (macrophages stimulated with LPS), and M2 (macrophages stimulated with IL-4) macrophages. Macrophages were collected by a lavage of the mice (6-8 weeks) peritoneal cavity and treated for 24 h with IL-4 (10 ng/mL) or LPS (10 ng/mL). Nitrite concentrations were measured using the Greiss reaction. Supernatants were harvested to determine cytokines and the ATPase, ADPase and AMPase activities were determined by the malachite green method and HPLC analysis. The expression of selected surface proteins was evaluated by flow cytometry. The results reveal that M1 macrophages presented a decreased ATP and AMP hydrolysis in agreement with a decrease in NTPDase1, -3 and ecto-5'-nucleotidase expression compared to M2. In contrast, M2 macrophages showed a higher ATP and AMP hydrolysis and increased NTPDase1, -3 and ecto-5'-nucleotidase expression compared to M1 macrophages. Therefore, macrophages of the M1 phenotype lead to an accumulation of ATP while macrophages of the M2 phenotype may rapidly convert ATP to adenosine. The results also showed that P1 and P2 purinoreceptors present the same mRNA profile in both phenotypes. In addition, M2 macrophages, which have a higher ATPase activity, were less sensitive to cell death. In conclusion, these changes in ectoenzyme activities might allow macrophages to adjust the outcome of the extracellular purinergic cascade in order to fine-tune their functions during the inflammatory set.

Dataset Information

Various N-glycoforms differentially upregulate E-NTPDase activity of the NTPDase3/CD39L3 ecto-enzymatic domain.

Publications

Various N-glycoforms differentially upregulate E-NTPDase activity of the NTPDase3/CD39L3 ecto-enzymatic domain.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets