Molecular cloning and functional expression of bovine spleen ecto-NAD+ glycohydrolase: structural identity with human CD38

Bovine spleen ecto-NAD+ glycohydrolase, an archetypal member of the mammalian membrane-associated NAD(P)+ glycohydrolase enzyme family (EC 3.2.2.6), displays catalytic features similar to those of CD38, i.e. a protein originally described as a lymphocyte differentiation marker involved in the metabolism of cyclic ADP-ribose and signal transduction. Using amino acid sequence information obtained from NAD+ glycohydrolase and from a truncated and hydrosoluble form of the enzyme (hNADase) purified to homogeneity, a full-length cDNA clone was obtained. The deduced sequence indicates a protein of 278 residues with a molecular mass of 31.5 kDa. It predicts that bovine ecto-NAD+ glycohydrolase is a type II transmembrane protein, with a very short intracellular tail. The bulk of the enzyme, which is extracellular and contains two potential N-glycosylation sites, yields the fully catalytically active hNADase which is truncated by 71 residues. Transfection of HeLa cells with the full-length cDNA resulted in the expression of the expected NAD+ glycohydrolase, ADP-ribosyl cyclase and GDP-ribosyl cyclase activities at the surface of the cells. The bovine enzyme, which is the first ‘classical’ NAD(P)+ glycohydrolase whose structure has been established, presents a particularly high sequence identity with CD38, including the presence of 10 strictly conserved cysteine residues in the ectodomain and putative catalytic residues. However, it lacks two otherwise conserved cysteine residues near its C-terminus. Thus hNADase, the truncated protein of 207 amino acids, represents the smallest functional domain endowed with all the catalytic activities of CD38/NAD+ glycohydrolases so far identified. Altogether, our data strongly suggest that the cloned bovine spleen ecto-NAD+ glycohydrolase is the bovine equivalent of CD38.

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Identification of differentially expressed genes during early development of tomato fruit. Characterisation of a novel cDNA coding for a RAD23 protein.

Functional Plant Biology ◽

10.1071/pp00047 ◽

2000 ◽

Vol 27 (10) ◽

pp. 911

Author(s):

Martine Lemaire-Chamley ◽

Johann Petit ◽

Mathilde Causse ◽

Philippe Raymond ◽

Christian Chevalier

Keyword(s):

Fruit Development ◽

Sequence Data ◽

Excision Repair ◽

Tomato Fruit ◽

Full Length ◽

Nucleotide Sequencing ◽

Sequence Information ◽

Repair System ◽

Full Length Cdna ◽

A Cell

Before the onset of ripening, tomato fruit development comprises three distinct phases: fruit set, a cell division phase and a cell expansion phase. In this study, we used the method of mRNA differential display in order to isolate tomato genes specifically expressed during these early phases of fruit development. Among 40 differen-tial bands, nine cDNAs were selected for further investigations based on their identification after nucleotide sequencing. We isolated the full-length cDNA corresponding to one of these fragments, coding for RAD23, a protein involved in the excision repair system, thus providing new sequence information on a poorly characterised protein in plants. All the isolated cDNAs were mapped on the tomato genome and their expression studied by northern blot and semi-quantitative RT–PCR during early fruit development and in vegetative organs of tomato plants. The sequence data are deposited in the GenBank under the accession numbers: AJ270956 (mo5-3C11/1), AJ270957 (mo5-3G12/4), AJ270958 (mo5-3G17), AJ270959 (mo5-3T12), AJ270960 (mo1-6A1), AJ270961 (mo1-6T1), AJ270962 (mo5-10G1), AJ270963 (mo6-20G1), AJ270964 (mo6-MGT2) and AJ243875 (LeRAD23-8 full-length cDNA).

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Multiple epithelial Na+ channel domains participate in subunit assembly

AJP Renal Physiology ◽

10.1152/ajprenal.00095.2003 ◽

2003 ◽

Vol 285 (4) ◽

pp. F600-F609 ◽

Cited By ~ 13

Author(s):

James B. Bruns ◽

Baofeng Hu ◽

Yoon J. Ahn ◽

Shaohu Sheng ◽

Rebecca P. Hughey ◽

...

Keyword(s):

Transmembrane Protein ◽

Expression System ◽

Functional Expression ◽

Full Length ◽

Na Channel ◽

Xenopus Laevis Oocyte ◽

Subunit Interactions ◽

Α Subunit ◽

Hydrophobic Domain ◽

Membrane Spanning

Epithelial sodium channels (ENaCs) are composed of three structurally related subunits that form a tetrameric channel. The Xenopus laevis oocyte expression system was used to identify regions within the ENaC α-subunit that confer a dominant negative phenotype on functional expression of αβγ-ENaC to define domains that have a role in subunit-subunit interactions. Coexpression of full-length mouse αβγ-ENaC with either 1) the α-subunit first membrane-spanning domain and short downstream hydrophobic domain (α-M1H1); 2) α-M1H1 and its downstream hydrophilic extracellular loop (α-M1H1-ECL); 3) the membrane-spanning domain of a control type 2 transmembrane protein (glutamyl transpeptidase; γ-GT) fused to the α-ECL (γ-GT-α-ECL); 4) the extracellular domain of a control type 1 transmembrane protein (Tac) fused to the α-subunit second membrane-spanning domain and short upstream hydrophobic domain (Tac-α-H2M2); or 5) the α-subunit cytoplasmic COOH terminus (α-Ct) significantly reduced amiloride-sensitive Na+ currents in X. laevis oocytes. Functional expression of Na+ channels was not inhibited when full-length αβγ-ENaC was coexpressed with either 1) the α-ECL lacking a signal-anchor sequence, 2) α-M1H1 and α-Ct expressed as a fusion protein, 3) full-length γ-GT, or 4) full-length Tac. Furthermore, the expression of ROMK channels was not inhibited when full-length ROMK was coexpressed with either α-M1H1-ECL or α-Ct. Full-length FLAG-tagged α-, β-, or γ-ENaC coimmunoprecipitated with myc-tagged α-M1H1-ECL, whereas wild-type γ-GT did not. These data suggest that multiple sites within the α-subunit participate in subunit-subunit interactions that are required for proper assembly of the heterooligomeric ENaC complex.

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Biosynthesis of surfactant protein C: characterization of aggresome formation by EGFP chimeras containing propeptide mutants lacking conserved cysteine residues

Journal of Cell Science ◽

10.1242/jcs.114.2.293 ◽

2001 ◽

Vol 114 (2) ◽

pp. 293-302

Author(s):

A.F. Kabore ◽

W.J. Wang ◽

S.J. Russo ◽

M.F. Beers

Keyword(s):

Protein C ◽

Transmembrane Protein ◽

A549 Cells ◽

Surfactant Protein ◽

Mutant Protein ◽

Cysteine Residues ◽

Western Blots ◽

Surfactant Protein C ◽

C Terminus ◽

Aggresome Formation

Surfactant protein C (SP-C) is a lung-specific secreted protein, which is synthesized as a 21-kDa propeptide (SP-C(21)) and then proteolytically processed as a bitopic transmembrane protein in subcellular compartments distal to the medial Golgi to produce a 3.7 kDa mature form. We have shown that initial processing of SP-C(21) involves two endoproteolytic cleavages of the C terminus and that truncation of nine amino acids from the C-flanking peptide resulted in retention of mutant protein in proximal compartments. Because these truncations involved removal of a conserved cysteine residue (Cys(186)), we hypothesized that intralumenal disulfide-mediated folding of the C terminus of SP-C(21) is required for intracellular trafficking. To test this, cDNA constructs encoding heterologous fusion proteins consisting of enhanced green fluorescent protein (EGFP) attached to the N terminus of wild-type rat proSP-C (EGFP/SP-C(1–194)), C-terminally deleted proSP-C (EGFP/SP-C(1–185); EGFP/SP-C(1–191)) or point mutations of conserved cysteine residues (EGFP/SP-C(C122G); EGFP/SP-C(C186G); or EGFP/SP-C(C122/186G)) were transfected into A549 cells. Fluorescence microscopy revealed that transfected EGFP/SP-C(1–194) and EGFP/SP-C(1–191)were expressed in a punctate pattern within CD-63 positive, EEA-1 negative cytoplasmic vesicles. In contrast, EGFP/SP-C(1–185), EGFP/SP-C(C122G), EGFP/SP-C(C186G) and EGFP/SP-C(C122/186G) were expressed but retained in a juxtanuclear compartment that stained for ubiquitin and that contained (γ)-tubulin and vimentin, consistent with expression in aggresomes. Treatment of cells transfected with mutant proSP-C with the proteasome inhibitor lactacysteine enhanced aggresome formation, which could be blocked by coincubation with nocodazole. Western blots using a GFP antibody detected a single form in lysates of cells transfected with EGFP/SP-C cysteine mutants, without evidence of smaller degradation fragments. We conclude that residues Cys(122) and Cys(186) of proSP-C are required for proper post-translational trafficking. Mutation or deletion of one or both of these residues results in misfolding with mistargeting of unprocessed mutant protein, leading to formation of stable aggregates within aggresomes.

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Trimerization of collagen IX α-chains does not require the presence of the COL1 and NC1 domains

Biochemical Journal ◽

10.1042/bj20070984 ◽

2007 ◽

Vol 409 (2) ◽

pp. 545-554 ◽

Cited By ~ 6

Author(s):

Juha Jäälinoja ◽

Joni Ylöstalo ◽

William Beckett ◽

David J. S. Hulmes ◽

Leena Ala-Kokko

Keyword(s):

Triple Helix ◽

Full Length ◽

Cysteine Residues ◽

C Terminus ◽

Study Association ◽

Collagen Ix ◽

Selection Of

Collagen IX is a heterotrimer of three α-chains, which consists of three COL domains (collagenous domains) (COL1–COL3) and four NC domains (non-collagenous domains) (NC1–NC4), numbered from the C-terminus. Although collagen IX chains have been shown to associate via their C-terminal NC1 domains and form a triple helix starting from the COL1 domain, it is not known whether chain association can occur at other sites and whether other collagenous and non-collagenous regions are involved. To address this question, we prepared five constructs, two long variants (beginning at the NC4 domain) and three short variants (beginning at the COL2 domain), all ending at the NC2 domain (or NC2 replaced by NC1), to study association and selection of collagen IX α-chains. Both long variants were able to associate with NC1 or NC2 at the C-terminus and form various disulfide-bonded trimers, but the specificity of chain selection was diminished compared with full-length chains. Trimers of the long variant ending at NC2 were shown to be triple helical by CD. Short variants were not able to assemble into disulfide-bonded trimers even in the presence of both conserved cysteine residues from the COL1–NC1 junction. Our results demonstrate that collagen IX α-chains can associate in the absence of COL1 and NC1 domains to form a triple helix, but the COL2–NC2 region alone is not sufficient for trimerization. The results suggest that folding of collagen IX is a co-operative process involving multiple COL and NC domains and that the COL1–NC1 region is important for chain specificity.

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Pharmacokinetics of Full Length and Two-Domain Tissue Factor Pathway Inhibitor in Combination with Heparin in Rabbits

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649604 ◽

1993 ◽

Vol 70 (03) ◽

pp. 454-457 ◽

Cited By ~ 14

Author(s):

Claus Bregengaard ◽

Ole Nordfang ◽

Per Østergaard ◽

Jens G L Petersen ◽

Giorgio Meyn ◽

...

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Anticoagulant Activity ◽

Fold Increase ◽

Volume Of Distribution ◽

Full Length ◽

Heparin Binding ◽

Extrinsic Pathway ◽

C Terminus ◽

Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a feed back inhibitor of the initial activation of the extrinsic pathway of coagulation. In humans, injection of heparin results in a 2-6 fold increase in plasma TFPI and recent studies suggest that TFPI may be important for the anticoagulant activity of heparin. Full length (FL) TFPI, but not recombinant two-domain (2D) TFPI, has a poly cationic C-terminus showing very strong heparin binding. Therefore, we have investigated if heparin affects the pharmacokinetics of TFPI with and without this C-terminus.FL-TFPI (608 U/kg) and 2D-TFPI (337 U/kg) were injected intravenously in rabbits with and without simultaneous intravenous injections of low molecular weight heparin (450 anti-XaU/kg).Heparin decreased the volume of distribution and the clearance of FL-TFPI by a factor 10-15, whereas the pharmacokinetics of 2D-TFPI were unaffected by heparin. When heparin was administered 2 h following TFPI the recovery of FL-TFPI was similar to that found in the group receiving the two compounds simultaneously, suggesting that the releasable pool of FL-TFPI is removed very slowly in the absence of circulating heparin.

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