AKSM2A en AKSM2B ensiemtoets optimalisering en rekombinante uitdrukking

Abstract

Glisienkonjugasie is ʼn Fase 2-biotransformasieweg wat verantwoordelik is vir die detoksifisering van xenobiotika soos bensoaat en salisilaat. Hierdie biotransformasieweg is gekonserveerd en speel ʼn baie belangrike rol in die lewer se metabolisme. Dit, tesame met die feit dat daar nog geen defek in die glisienkonjugasieweg beskryf is nie, dui daarop dat hierdie weg noodsaaklik is vir oorlewing. Hierdie biotransformasieweg is in die verlede as ʼn een-stap proses bestudeer, maar onlangse studies het die kritiese tekort ensimatiese inligting aangaande beide ensieme betrokke in die weg, naamlik die asiel-KoA-sintetase mediumketting 2B (AKSM2B) en glisienasieltransferase (GLIAT), uitgewys. Dit is belangrik om hierdie weg beter te karakteriseer veral in die lig van die groot hoeveelhede xenobiotika soos bensoaat en salisilaat wat in die moderne omgewing ingeneem word en die newe-effekte wat ervaar word as gevolg van hierdie toksiese stowwe. Die vermoë van ʼn biotransformasieweg om effektief van xenobiotika ontslae te raak, is ʼn som totaal van elke individuele ensiem wat betrokke is in die weg, se vermoë om optimaal te funksioneer. Die limiet van die hoeveelheid xenobiotika wat ingeneem kan word, sonder om die newe-effekte te ervaar, word deur hierdie vermoë aangedui. Indien die ensieme in hierdie weë nie gekarakteriseer is nie, soos AKSM2B, is dit nie moontlik om te bepaal of die hoeveelheid xenobiotika wat ingeneem word, veilig gedetoksifiseer word nie. Bensoaat en salisilaat word ten spyte hiervan steeds algemeen en in groot hoeveelhede gebruik. Dit word groot en deels toegelaat as gevolg van die feit dat toksisiteit studies die effek van blootstelling van hierdie stowwe slegs oor ʼn kort termyn in diere modelle bestudeer. Om toekomstige navorsing in staat te stel om die AKSM2B-ensiem te karakteriseer word ʼn ensiemtoets vir die AKSM2B-ensiem benodig en die AKSM2B-ensiem self word benodig. Die eerste hoof doelstelling van hierdie studie was dus om ʼn ensiemtoets vir die AKSM2B-ensiem te optimaliseer. Die AKSM2B-ensiem word geproduseer in die mens se lewer en word nie uitgedruk in menslewer kanker sellyne nie. Dit is dus onprakties om die ensiem te probeer isoleer vanuit natuurlike bronne. Die tweede hoof doelstelling van hierdie studie was dus om die AKSM2B-proteïen rekombinant te produseer. Die AKSM2B-geen is gesintetiseer en in verskillende ekspressieplasmiede gekloneer. Die AKSM2B-geen is 98.8% identies aan die AKSM2A-geen. Hierdie twee gene het ontstaan as gevolg van ʼn chromosomale verdubbeling gebeurtenis van die AKSM2-geen. Die biologiese rol van die AKSM2A-ensiem is onbekend. Die hoë ooreenkoms tussen die twee ensieme laat die vraag ontstaan of hierdie twee ensieme by dieselfde weg betrokke is en of die ensieme se substraatspesifisiteit oorvleuel. Die karakterisering van die AKSM2A-ensiem is dus ook belangrik omdat dit ook ʼn effek kan hê in glisienkonjugasie. Die AKSM2A-geen is dus saam met die AKSM2B-geen gekloneer en beide ensiem is rekombinant uitgedruk. ʼn Ensiemtoets vir die AKSM2-ensieme was geïdentifiseer, afgeskaal en geoptimaliseer om op ʼn plaatleser te geskied. Verskillende bakteriële ekspressiemetodes is getoets vir oplosbare uitdrukking van AKSM2A- en AKSM2B-ensieme en ʼn metode wat oplosbare AKSM2-ensieme produseer is geïdentifiseer en gedeeltelik geoptimaliseer

Glycine conjugation is a Phase 2 biotransformation pathway that is responsible for the detoxification of xenobiotics such as benzoate and salicylate. This biotransformation pathway is conserved and plays an important role in the liver’s metabolism. This alongside the fact that no defect in the glycine conjugation pathway has been described, indicates that this pathway is essential for survival. This biotransformation pathway has been studied as a one-step proses in the past, but recent studies indicate the lack of enzymatic information on both enzymes in the pathway, namely the acyl-CoA synthetase medium chain 2B (ACSM2B) and the glycine acyl transferase (GLYAT). It is important to better characterise this pathway especially in light of the high volumes of xenobiotics such as benzoate and salicylate that are consumed in modern society and the side effects, which are experienced due to these toxic materials. The ability of a biotransformation pathway to eliminate xenobiotics is the sum of the individual participating enzymes’ ability to function optimally. The amount of xenobiotic that can be ingested without experiencing side effects is indicated by this ability. If the enzymes in these pathways are not characterized, as it is with ACSM2B, then it is not possible to determine if the amount of xenobiotic that is consumed, is safely detoxified. Benzoate and salicylate are generally still consumed in high amounts despite this. This consumption is permitted mainly due to toxicity studies that only study the effect of the exposure to these toxic substances in animal models for short time periods. An enzyme test for the ACSM2B enzyme and the ACSM2B protein itself is therefore needed to enable future research to characterize the ACSM2B protein. The first aim of this study was therefore to optimize an enzyme test for the ACSM2B enzyme. The ACSM2B enzyme is produced in the human liver however, it is not expressed in human liver cancerous cell lines. It is therefore impractical to isolate the enzyme from natural sources. The second aim of this study was to recombinantly produce the ACSM2B protein. The ACSM2B gene was synthesized and cloned into various expression plasmids. The ACSM2B gene is 98.8% identical to the ACSM2A gene. These two genes formed due to a chromosomal doubling event of the ACMS2 gene. The biological role of the ACSM2A enzyme is unknown. The high similarity of these two enzymes begs the question if these two enzymes take part in the same enzymatic pathway and if these enzymes’ substrate affinity overlaps. The characterization of the ACSM2A enzyme is therefore also important, because it can have an effect in glycine conjugation. The ACSM2A gene was cloned in the same manner as the ACSM2B gene and both enzymes were recombinantly expressed. An enzyme test for the ACSM2 enzymes were identified, downscaled and optimized to be performed on a plate reader. Various bacterial expression methods were tested for the ability to produce soluble ACSM2A an ACSM2B proteins and a method that produced soluble ACSM2 enzymes was identified and partially optimized