Phase II detoxification: Glycine conjugation

                                                                   

                                                           


Research Focus:

We have determined that the major role of glycine conjugation is to dispose of the end products formed by the gut microbiome through the metabolism of dietary polyphenols. Glycine conjugation facilitates the metabolism of toxic aromatic acids, capable of disrupting mitochondrial integrity. The importance of functional hepatic glycine conjugation has been underestimated in the last few decades. The glycine conjugation capacity of individuals vary significantly. Glycine conjugation is a two-step process and the overall rate of glycine conjugation can be influenced by several factors, including the availability of ATP, Coenzyme A, and glycine; genetic variation in the ATP dependent acid: CoA ligase (ACSM2B) (encoding HXMA) and glycine N-acyltransferase (GLYAT) (encoding GLYAT) genes as well as variable expression of HXMA and GLYAT. We have previously established that the observed variation is not due to genetic variation in GLYAT as the GLYAT gene is highly conserved among humans and polymorphisms with deleterious effects are rare. Due to the high exposure to toxic substrates such as benzoate and aspirin, characterisation of the factors that contribute to the variation in individual glycine conjugation capacity needs to be further investigated.   


Research Leaders:

Dr Rencia van der Sluis

 

Prof Albie van Dijk


Active Researchers (Technicians and Students):

Staff:

  • Mr Lardus Erasmus

MSc:

  • Mr Phillip Venter

  • Mrs Chantalle Schutte

Hons:

  • Mr Francois Boshoff

  • Ms Dane Vermeulen


Research Background:

The glycine conjugation pathway was one of the first metabolic pathways to be discovered, in 1841. The biggest challenge regarding the investigation of the glycine conjugation pathway remains the fact that this pathway is still very poorly characterised when compared to the cytochrome P450 (CYP450) phase I detoxification enzymes. This may be because of the small number of pharmaceutical drugs that are metabolised to glycine conjugates and the difficulty in obtaining human liver samples and xenobiotic acyl-CoA substrates for research. There is now a growing realisation that glycine conjugation is one of the fundamentally important homeostatic mechanisms in animal physiology.  The glycine conjugation pathway is important in preventing Coenzyme A sequestration, which would result from the accumulation of acyl-CoA metabolites such as benzoyl-CoA. Glycine conjugation also decreases the toxicity of benzoate and other aromatic acids by forming less lipophilic conjugates rather than more water soluble conjugates. In several organic acidemias an acyl-CoA accumulates to toxic levels because of a defect of the enzyme acting on it. Because some of the acyl-CoAs that accumulate in organic acidemias are substrates for GLYAT, glycine conjugation impacts on the biochemical profiles and clinical outcomes of some of these metabolic defects. Recent studies have shown that the metabolic demand for glycine often exceeds the capacity for glycine synthesis. This glycine deficiency can influence the metabolism of collagen, glutathione, creatine, nucleic acids, and porphyrins. Glycine shortage can be exacerbated by the presence of benzoate and other substrates for glycine conjugation. In addition to natural sources of benzoate, humans are nowadays exposed to ever increasing amounts of this compound, since it is used as a preservative in food and pharmaceuticals. The GLYAT gene has also been linked to the development of liver cancer and musculoskeletal development, although the mechanism behind this is still unclear at present.


Research Objectives and Technologies used:

The project has four main objectives:

  1. To characterise the kinetic parameters and substrate specificity of five of the 14 haplotypes identified in GLYAT (S156; T17S156; L61S156; wild-type and S156C199). S156 and T17S156 have the highest haplotype frequencies identified in all populations studied (70% and 20% respectively) while L61S156 is a novel haplotype identified in the Afrikaner Caucasian population. S156 has the highest enzyme activity while S156C199 only has 5% enzyme activity when compared to the wild-type enzyme.

  2. To characterise the genetic variation in the ACSM2B gene by analysing world-wide population data as well as sequence data from a South African co-hort.

  3. To determine whether a functional protein is encoded by the ACSM2A gene and compare the substrate specificity of the enzyme to the substrate specificity of HXMA (encoded by ASCM2B) reported in the literature [25].

  4. To establish a cell culture model system in which variants of HXMA and GLYAT can be co-expressed to investigate the effect of genetic variation on the detoxification ability of the pathway.

 

Students involved in these projects will get experience in the following molecular genetic techniques:

  • Basic molecular techniques: PCR; qPCR; cloning

  • Recombinant protein expression and purification: Optimisation of conditions to express soluble enzymatically active protein in bacterial, insect and mammalian expression systems.

  • Determining enzyme kinetic parameters

  • Cell culture: Insect cells; Mammalian cells.

  • Cytotoxicity assays; confocal microscopy and immunofluorescence and subcellular fractionation assays.

  • Single nucleotide polymorphism (SNP) detoxification genotyping: Data analysis and interpretation.

  • Next generation sequence data analysis– specifically identifying and validating single nucleotide polymorphisms.

  • Basic population genetic analyses of worldwide genetic variation data obtained from public available databases such as the 1000 Genome Project database e.g. haplotype determination and phylogenetic analyses.


Publications:

  1. Badenhorst C.P.S., Jooste M., van Dijk A.A. 2012. Enzymatic characterization and elucidation of the catalytic mechanism of a recombinant bovine glycine N-acyltransferase. Drug Metababolism and Disposition. 40(2):346-52.

  2. van der Sluis R., Badenhorst C.P.S., van der Westhuizen F.H., and van Dijk A.A. 2013. Characterisation of the influence of genetic variations on the enzyme activity of a recombinant human glycine N-acyltransferase. Gene (2013) 515: 447-453.

  3. Badenhorst C.P.S., van der Sluis R., Erasmus E., and van Dijk A.A. 2013. Glycine conjugation: Importance in metabolism, the role of glycine N-acyltransferase, and the factors that influence interindividual variation. Expert Opinion on Drug Metabolism and Toxicology 9: 1139-1153.

  4. Badenhorst C.P.S., Erasmus E., van der Sluis R., Nortje C., and van Dijk A.A. 2014. A new perspective on the importance of glycine conjugation in the metabolism of aromatic acids. Drug Metabolism Reviews 46 (3): 343-361.

  5. van der Sluis R., Badenhorst C.P.S., Erasmus, E. van Dyk, E., van der Westhuizen F.H., and van Dijk A.A. 2015. Conservation of the coding regions of the glycine N-acyltransferase gene further suggests that glycine conjugation is an essential detoxification pathway. Gene. 571: 126 – 134.

  6. Nortje C., van der Sluis R., van Dijk A.A., Erasmus E. 2016. The Use of p-aminobenzoic acid as a probe substance for the targeted profiling of glycine conjugation. Journal of Biochemical and Molecular Toxicology. 30(3):136-47

  7.  van der Sluis R. and Erasmus E. 2016. Xenobiotic/medium chain fatty acid: CoA ligase - a critical review on its role in fatty acid metabolism and the detoxification of benzoic acid and aspirin. Expert Opinion on Drug Metabolism and Toxicology. 12 (10): 1169–1179.