Characterization of Dog Glutathione Transferase P1-1, an Enzyme Relevant to Veterinary Medicine

Ismail, Aram; Lewis, Elizabeth; Sjödin, B.; Mannervik, Bengt

doi:10.3390/ijms22084079

Cited by 3 publications

(8 citation statements)

References 43 publications

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“…The third class of inhibitors of compounds referred to as non-substrate ligands which are noncompetitive inhibitors of GSTs [ 36 ]. In the present investigation, hematin and CB were the most potent inhibitors for BaGST2 and BaGST3, with IC 50 approximately of 0.5 μM, whereas in IC 50 values of S-hexyl-GSH, S-butyl-GSH, and S-P-bromobenzyl-GSH, BSP are approximatly100-fold higher but remain within the range reported for other GSTs [ 35 , 37 ]. The type of inhibition of CB is competitive for BaGST2 and BaGST3.…”

Section: Discussionsupporting

confidence: 71%

“…In addition to being inhibitor of GSTs, EA interacts with GSTs as a substrate for conjugation with GSH to yield an EA-GSH conjugate. BaGST2 and BaGST3 showed a comparatively high specific activity values with EA as that reported for the pi-class GSTs [ 35 ]. Compounds well-established as GST inhibitors were classified into three classes, determined by their binding site on the protein and mechanism of inhibition.…”

Section: Discussionsupporting

confidence: 60%

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Biochemical characterization and peptide mass fingerprinting of two glutathione transferases from Biomphalaria alexandrina snails (Gastropoda: Planorbidae)

Abdalla

Karim

2022

Journal of Genetic Engineering and Biotechnology

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Background The freshwater snails Biomphalaria alexandrina (Gastropoda: Planorbidae) has public health importance of being an intermediate host of Schistosoma mansoni, the parasite species that causes intestinal schistosomiasis in humans. Glutathione transferases (GSTs) play an important role in detoxification of a broad range of compounds including secondary metabolites and exogenous compounds. Studying GSTs in snails may clarify their role in detoxification of molluscicides. Results Two glutathione transferases (BaGST2 and BaGST3) were purified and characterized from B. alexandrina snails. BaGST2 and BaGST3 were electrophoretically homogeneous preparations with subunit molecular weight of 23.6 kDa and molecular weight of 45 kDa. Isoelectric focusing of BaGST2 revealed the presence of two components at pI 4.47 and 4.67, while BaGST3 showed one band at pI 4.17. The specific activity of BaGST2 and BaGST3 toward 1-chloro-2,4-dinitrobenzene (CDNB) was 19.0 and 45.2 μmol/min/mg protein following 146- and 346-fold purification, respectively. The catalytic pH optima, km values, and the activation energies for BaGST2 and BaGST3 were determined. BaGST2 and BaGST3 were significantly inhibited by hematin and Cibacron Blue and to a less extent by bromosulfophthalein, S-butyl-GSH, S-hexyl-GSH, and S-P-bromobenzyl-GSH. BaGST2 and BaGST3 showed high activity against ethacrynic acid as substrate, and they also exhibited peroxidase activity on cumene hydroperoxide. The two enzymes showed identical patterns of lysine-C digestion after high-performance liquid chromatography. The amino acid sequences of three peptide fragments and peptide mass fingerprinting of fourteen peptides were used to predict the primary structure of BaGST2. A polypeptide of 206 amino acids (with 7 gaps, 3 of which could not identified) was predicted for BaGST2. The theoretical subunit molecular weight of BaGST2 is 22.6 kDa, with pI of 8.58. BaGST2 has 65% sequence identity and 78% positive with Biomphalaria glabrata GST7. The overall structure of BaGST2 at the N-terminal domain is identical to the canonical GST N-terminal domain, having the typical thioredoxin-like fold with a βαβ-α-ββα motif, whereas the C-terminal domain is made from 6 α-helices. A conservative GST-N-domain includes glutathione binding sites Y11, L17, Q53, M54, Q65, and S66, while a variable GST-C domain contains electrophilic substrate binding site H99, R102, A103, F106, K107, L161, and Y167. Phylogenetic tree showed that BaGST2 was clustered in the sigma group with GSTs sigma class from invertebrates and vertebrates. Conclusions We have purified and characterized two GSTs from B. alexandrina snails. Our study broadens the biochemical information on freshwater snail GSTs by demonstrating the role of BaGSTs in defense mechanisms against structurally different electrophilic compounds. BaGST2 and BaGST3 have Se-independent peroxidase activity, which indicates their role in cellular antioxidant defense by reducing organic hydroperoxides in B. alexandrina. A polypeptide chain of 206 amino acids was predicted. The primary structure of BaGST2 showed 65% sequence identity with Biomphalaria glabrata GST7. Sequence analysis indicates that BaGST2 is a GST-N-sigma-like with a thioredoxin-like superfamily. Phylogenetic tree confirms that BaGST2 belongs to the sigma class of GSTs superfamily.

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Section: Discussionsupporting

confidence: 71%

Section: Discussionsupporting

confidence: 60%

Biochemical characterization and peptide mass fingerprinting of two glutathione transferases from Biomphalaria alexandrina snails (Gastropoda: Planorbidae)

Abdalla

Karim

2022

Journal of Genetic Engineering and Biotechnology

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“…All the human GST P1-1 variants were based on the same wildtype sequence. Expression clones for mouse and rat GST P1-1, as well as mouse GST P2-2, were synthesized by ATUM (Newark, CA, USA) as previously described for dog GST P1-1 [20].…”

Section: Methodsmentioning

confidence: 99%

Human GST P1-1 Redesigned for Enhanced Catalytic Activity with the Anticancer Prodrug Telcyta and Improved Thermostability

Ismail,

Govindarajan,

Mannervik

2024

Cancers

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Protein engineering can be used to tailor enzymes for medical purposes, including antibody-directed enzyme prodrug therapy (ADEPT), which can act as a tumor-targeted alternative to conventional chemotherapy for cancer. In ADEPT, the antibody serves as a vector, delivering a drug-activating enzyme selectively to the tumor site. Glutathione transferases (GSTs) are a family of naturally occurring detoxication enzymes, and the finding that some of them are overexpressed in tumors has been exploited to develop GST-activated prodrugs. The prodrug Telcyta is activated by GST P1-1, which is the GST most commonly elevated in cancer cells, implying that tumors overexpressing GST P1-1 should be particularly vulnerable to Telcyta. Promising antitumor activity has been noted in clinical trials, but the wildtype enzyme has modest activity with Telcyta, and further functional improvement would enhance its usefulness for ADEPT. We utilized protein engineering to construct human GST P1-1 gene variants in the search for enzymes with enhanced activity with Telcyta. The variant Y109H displayed a 2.9-fold higher enzyme activity compared to the wild-type GST P1-1. However, increased catalytic potency was accompanied by decreased thermal stability of the Y109H enzyme, losing 99% of its activity in 8 min at 50 °C. Thermal stability was restored by four additional mutations simultaneously introduced without loss of the enhanced activity with Telcyta. The mutation Q85R was identified as an important contributor to the regained thermostability. These results represent a first step towards a functional ADEPT application for Telcyta.

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“…The molecular mass of dog GSTP1‐1 is 23 398 Da, isoelectric point is 6.39. The highest specific activities are with benzyl isothiocyanate and 1‐chloro‐2,4‐dinitrobenzene, the lowest – with ∆ 5 ‐androstene‐3,17‐dione, cumene hydroperoxide, and the trans‐2‐alkenals 18 …”

Section: Introductionmentioning

confidence: 99%

“…The highest specific activities are with benzyl isothiocyanate and 1-chloro-2,4-dinitrobenzene, the lowestwith Δ 5 -androstene-3,17-dione, cumene hydroperoxide, and the trans-2-alkenals. 18 Unfortunately, there is little information available about GSTs in canine cancers.…”

Section: Introductionmentioning

confidence: 99%

Polymorphism of glutathione S‐transferase P1 of dogs with mammary tumours

Fedets

Dmytruk

Adaszek

et al. 2023

Vet Comparative Oncology

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Mammary tumours constitute more than half of neoplasms in female dogs from different countries. Genome sequences are associated with cancer susceptibility but there is little information available about genetic polymorphisms of glutathione S‐transferase P1 (GSTP1) in canine cancers. The aim of this study was to find single nucleotide polymorphisms (SNPs) in GSTP1 of dogs (Canis lupus familiaris) with mammary tumours compared to healthy dogs and to determine the association between GSTP1 polymorphisms and the occurrence of these tumours. The study population included 36 client‐owned female dogs with mammary tumours and 12 healthy female dogs, with no previous diagnosis of cancer. DNA was extracted from blood and amplified by PCR assay. PCR‐products were sequenced by Sanger method and analysed manually. The 33 polymorphisms were found in GSTP1: 1 coding SNP (exon 4), 24 non‐coding SNPs (9 in exon 1), 7 deletions and 1 insertion. The 17 polymorphisms have been found in introns 1, 4, 5 and 6. The dogs with mammary tumours have significant difference from healthy in SNPs I4 c.1018 + 123 T > C (OR 13.412, 95%CI 1.574–114.267, P = .001), I5 c.1487 + 27 T > C (OR 10.737, 95%CI 1.260–91.477, P = .004), I5 c.1487 + 842 G > C (OR 4.714, 95% CI 1.086–20.472, P = .046) and I6 c.2481 + 50 A > G (OR 12.000, 95% CI 1.409–102.207, P = .002). SNP E5 c.1487 T > C and I5 c.1487 + 829 delG also differed significantly (P = .03) but not to the confidence interval. The study, for the first time, showed a positive association of SNPs in GSTP1 with mammary tumours of dogs, that can possibly be used to predict the occurrence of this pathology.

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Characterization of Dog Glutathione Transferase P1-1, an Enzyme Relevant to Veterinary Medicine

Cited by 3 publications

References 43 publications

Biochemical characterization and peptide mass fingerprinting of two glutathione transferases from Biomphalaria alexandrina snails (Gastropoda: Planorbidae)

Biochemical characterization and peptide mass fingerprinting of two glutathione transferases from Biomphalaria alexandrina snails (Gastropoda: Planorbidae)

Human GST P1-1 Redesigned for Enhanced Catalytic Activity with the Anticancer Prodrug Telcyta and Improved Thermostability

Polymorphism of glutathione S‐transferase P1 of dogs with mammary tumours

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