Expression of the theta class GST isozyme, YdfYdf, in low GST dogs

Abstract

We have reported the existence of low glutathione S-transferase (GST) dogs whose GST activity to 1,2-dichloro-4-nitrobenzene (DCNB) as a substrate (GST-D activity) is quite low, and have also reported significant individual differences in dog liver GST-D activity. The dogs were classified as "low", "middle", or "high" GST dogs based on their GST-D activity. In the present study, in order to investigate the causes of quite low GST-D activity in low GST dogs and the individual differences in dog GST-D activity, glutathione (GSH) conjugation of DCNB was kinetically analyzed. Moreover, liver cytosolic proteins whose expression levels were significantly lower in low GST dogs than in high GST dogs were identified by two-dimensional difference gel electrophoresis (2D-DIGE) and LC tandem mass spectrometry (LC/MS/MS). Interestingly, Vmax values for this reaction well reflected their GST-D activities in all groups, i.e. they were 3.8, 80.6, and 169.2 nmol/min/mg protein in the low, middle, and high GST dogs, respectively. However, Km values were almost the same (260.0-283.7 microM) among these groups. These suggest that GSH conjugation of DCNB should be catalyzed by the same enzyme in all the dogs, and individual differences in the GST-D activity should be the result of individual differences in the expression level of the GST isozyme, which catalyzes conjugation of DCNB. In 2D-DIGE, the expression levels of the two protein spots were significantly lower in the low GST dogs than in the high GST dogs. Positive good correlation (r > 0.800) was observed between GST-D activity and expression levels of these two protein spots. Moreover, expression levels were quite low in low GST dogs. These two proteins were both identified as the theta class GST isozyme, YdfYdf, which specifically catalyzes GSH conjugation of DCNB in dog livers. In the present study, we present two novel findings based on an enzyme kinetic study and protein-expression analysis: (1) GSTYdfYdf is expressed at quite a low level in the liver of low GST dogs, and (2) individual differences in dog liver GST-D activity would be due to individual differences in the expression level of GSTYdfYdf. Considering these findings, low GST dogs might have high susceptibility, including an unexpected toxicity at abnormal exposure to chemicals metabolized by GSTYdfYdf.

A 6‐bp Deletion Variant in a Novel Canine Glutathione‐S‐Transferase Gene (GSTT5) Leads to Loss of Enzyme Function

“…The overall MAF for GSTT5 385_390delGACCAG was 0.144 in our sample of 45 livers, and was significantly higher (0.444) in a small subpopulation of purpose‐bred laboratory beagles, with 2 of 9 (22%) beagles being homozygous. This observation is consistent with the previous finding of low DCNB activities in 12% of research colony beagles . Our study was limited by a relatively low number of individual dog livers available, many of which were from purpose‐bred or mixed‐breed dogs.…”

“…Polymorphisms in genes that encode GST enzymes are important in the epidemiology of cancer risk in humans, but little is known about GST variants in dogs. Previous studies had found polymorphic activity for the GST‐theta substrate, DCNB, in beagle dog livers, with some dogs having virtually no activity . These latter dogs were reportedly missing a GST‐theta protein with an N‐terminal amino acid sequence, MPPELFLDLYSPPPRAIYIFALKNGIP, which we matched to a novel canine GST gene on chromosome 26.…”

“…Furthermore, we found a single nucleotide polymorphism in canine GSTT1 that was significantly associated with lymphoma risk in dogs . However, the predicted amino acid sequence of reference canine GSTT1 was different from that of the GST‐theta protein that was absent in 12% of beagle dogs . We hypothesized that a second GST‐theta enzyme was expressed in canine liver, and that this isozyme was responsible for the low activity GST‐theta variation previously documented in research colony dogs.…”

Positive association between a glutathione‐S‐transferase polymorphism and lymphoma in dogs

“…Epidemiologic studies have found significant associations between canine lymphoma and exposure to industrial pollutants, solvents and herbicides, but the role of specific environmental carcinogens in the development of lymphoma in dogs is not clear . GSTT1 activities have been characterized in livers from research beagles, and 12% of these dogs had virtually no GSTT1 protein expression or activity . However, the genetic basis for this variability, and its toxicological consequences, has not been explored.…”

Glutathione‐S‐transferase‐theta genotypes and the risk of cyclophosphamide toxicity in dogs

“…We chose GST‐theta variants for this study because of marked variability in GST‐theta activities in dogs and functionally defective canine GST‐theta alleles . However, CP response in humans can be influenced by other GST gene variants, alone or in combination .…”