Teladorsagia circumcincta 1,6-Bisphosphate Aldolase: Molecular and Biochemical Characterisation, Structure Analysis and Recognition by Immune Hosts

A 1095 bp full length cDNA encoding Teladorsagia circumcincta aldolase (TciALDO-1) was cloned and expressed in Escherichia coli. Recombinant TciALDO-1 was purified, and its kinetic properties determined. The predicted protein consisted of 365 amino acids, and was present as a single band of about 44 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TciALDO-1 with homologues from other helminths showed the greatest similarity (93%) to the aldolases of Haemonchus contortus and Dictyocaulus viviparus, 82–86% similarity to the other nematode sequences, and 68–71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified, and were completely conserved in other homologues. At 30 °C, the optimum pH for TciALDO-1 activity was pH 7.5, the Vmax was 432 ± 23 nmol × min−1 × mg−1 protein, and the apparent Km for the substrate fructose 1,6-bisphosphate was 0.24 ± 0.01 µM (mean ± SEM, n = 3). Recombinant TciALDO-1 was recognized by antibodies in both serum and saliva from field-immune sheep in ELISA, however, that was not the case with nematode-naïve sheep. Teladorsagia circumcincta fructose 1,6-bisphosphate aldolase appears to have potential as a vaccine candidate to control this common sheep parasite.

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Teladorsagia circumcincta 1,6 bisphosphate aldolase: molecular and biochemical characterisation, structure analysis and recognition by immune hosts

10.1101/2020.07.13.201335 ◽

2020 ◽

Author(s):

S. Umair ◽

C.L.G. Bouchet ◽

N. Palevich ◽

J.S. Knight ◽

H.V. Simpson

Keyword(s):

Recombinant Protein ◽

Kinetic Properties ◽

Dictyocaulus Viviparus ◽

Teladorsagia Circumcincta ◽

Multiple Alignments ◽

Sds Page ◽

Biochemical Characterisation ◽

Optimum Ph ◽

Fructose 1,6 Bisphosphate ◽

Substrate Binding Sites

ABSTRACTA 1095 bp full length cDNA encoding Teladorsagia circumcincta aldolase (TciALDO) was cloned, expressed in Escherichia coli, the recombinant protein purified and its kinetic properties determined. A phylogenetic tree was constructed using helminth aldolase sequences. The predicted protein consisted of 365 amino acids and was present as a single band of about 44 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TciALDO with homologues from other helminths showed that the greatest similarity (93%) to the aldolases of Haemonchus contortus and Dictyocaulus viviparus, 82-86% similarity to the other nematode sequences and 68-71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. At 25 °C, the optimum pH for TciALDO activity was pH 7.5, the Vmax was 432 ± 23 nmoles.min−1.mg−1 protein and the apparent Km for the substrate fructose 1,6-bisphosphate was 0.24 ± 0.01 μM (mean ± SEM, n = 3). Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciALDO in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native aldolase indicates similar antigenicity of the two proteins.

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Characterisation of a Teladorsagia circumcincta glutathione transferase

10.1101/2020.07.13.201368 ◽

2020 ◽

Author(s):

Saleh Umair ◽

Charlotte L.G. Bouchet ◽

Qing Deng ◽

Nikola Palevich ◽

Heather V. Simpson

Keyword(s):

Recombinant Protein ◽

Glutathione Transferase ◽

Kinetic Properties ◽

3 Dimensional ◽

Teladorsagia Circumcincta ◽

Multiple Alignments ◽

Novel Drugs ◽

Sds Page ◽

Vaccine Antigens ◽

Substrate Binding Sites

ABSTRACTA 615 bp full length cDNA encoding a Teladorsagia circumcincta glutathione transferase (TcGST) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. The predicted protein consisted of 205 amino acids and was present as a single band of about 24 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TcGST with homologues from other helminths showed that the highest identity of 53-68% with haem-binding nematode proteins designated as members of the nu class of GSTs. Substrate binding sites and conserved regions were identified and were generally conserved. The predicted 3-dimensional structures of TcGST and HcGST revealed highly open binding cavities typical of this class of GST, considered to allow greater accessibility to diverse ligands compared with other classes of GST. At 25 °C, the optimum pH for TcGST activity was pH 7, the Vmax was 1535 ± 33 nmoles.min-1.mg-1 protein and the apparent Km for the substrate 1-chloro-2,4-dinitrobenzene (CDNB) was 0.22 ± 0.01 mM (mean ± SD, n = 2). Antibodies in both serum and saliva from field-immune, but not nematode-naÏve, sheep, recognised recombinant TcGST in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins. These findings could aid in the design of novel drugs and vaccine antigens for economically important parasites of livestock.

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Faculty Opinions recommendation of Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1046453.496422 ◽

2006 ◽

Author(s):

Amnon Horovitz

Keyword(s):

Binding Sites ◽

Substrate Binding ◽

Substrate Binding Sites

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Faculty Opinions recommendation of Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporter.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718076922.793482351 ◽

2013 ◽

Author(s):

Robert Vandenberg

Keyword(s):

Dopamine Transporter ◽

Binding Sites ◽

Substrate Binding ◽

Multiple Substrate ◽

Substrate Binding Sites

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Covalent modification of substrate-binding sites of Escherichia coli ADP-glucose synthetase. Isolation and structural characterization of 8-azido-ADP-glucose-incorporated peptides.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)36052-0 ◽

1986 ◽

Vol 261 (3) ◽

pp. 1058-1064

Author(s):

Y M Lee ◽

J Preiss

Keyword(s):

Escherichia Coli ◽

Structural Characterization ◽

Binding Sites ◽

Substrate Binding ◽

Covalent Modification ◽

Substrate Binding Sites

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Substrate-binding sites in acetylcholinesterase

Trends in Pharmacological Sciences ◽

10.1016/0165-6147(91)90621-x ◽

1991 ◽

Vol 12 ◽

pp. 422-426 ◽

Cited By ~ 26

Author(s):

Ferdinand Hucko ◽

Jaak Järv ◽

Christoph Weise

Keyword(s):

Binding Sites ◽

Substrate Binding ◽

Substrate Binding Sites

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Opposite stereospecificity of two tropinone reductases is conferred by the substrate-binding sites.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)32627-3 ◽

1994 ◽

Vol 269 (16) ◽

pp. 11695-11698 ◽

Cited By ~ 1

Author(s):

K. Nakajima ◽

T. Hashimoto ◽

Y. Yamada

Keyword(s):

Binding Sites ◽

Substrate Binding ◽

Substrate Binding Sites

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Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites

AJP Renal Physiology ◽

10.1152/ajprenal.00133.2004 ◽

2005 ◽

Vol 288 (2) ◽

pp. F327-F333 ◽

Cited By ~ 147

Author(s):

Rémon A. M. H. Van Aubel ◽

Pascal H. E. Smeets ◽

Jeroen J. M. W. van den Heuvel ◽

Frans G. M. Russel

Keyword(s):

Binding Sites ◽

Efflux Pump ◽

Substrate Binding ◽

Organic Anion ◽

Apical Cell ◽

Cell Uptake ◽

Hek293 Cells ◽

Hill Coefficient ◽

Anion Transporter ◽

Substrate Binding Sites

The end product of human purine metabolism is urate, which is produced primarily in the liver and excreted by the kidney through a well-defined basolateral blood-to-cell uptake step. However, the apical cell-to-urine efflux mechanism is as yet unidentified. Here, we show that the renal apical organic anion efflux transporter human multidrug resistance protein 4 (MRP4), but not apical MRP2, mediates ATP-dependent urate transport via a positive cooperative mechanism ( Km of 1.5 ± 0.3 mM, Vmax of 47 ± 7 pmol·mg−1·min−1, and Hill coefficient of 1.7 ± 0.2). In HEK293 cells overexpressing MRP4, intracellular urate levels were lower than in control cells. Urate inhibited methotrexate transport (IC50 of 235 ± 8 μM) by MRP4, did not affect cAMP transport, whereas cGMP transport was stimulated. Urate shifted cGMP transport by MRP4 from positive cooperativity ( Km and Vmax value of 180 ± 20 μM and 58 ± 4 pmol·mg−1·min−1, respectively, Hill coefficient of 1.4 ± 0.1) to single binding site kinetics ( Km and Vmax value of 2.2 ± 0.9 mM and 280 ± 50 pmol·mg−1·min−1, respectively). Finally, MRP4 could transport urate simultaneously with cAMP or cGMP. We conclude that human MRP4 is a unidirectional efflux pump for urate with multiple allosteric substrate binding sites. We propose MRP4 as a candidate transporter for urinary urate excretion and suggest that MRP4 may also mediate hepatic export of urate into the circulation, because of its basolateral expression in the liver.

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