title: Functional expression of novel human and murine AKR1B genes.
abstract: The Aldo Keto Reductases (AKRs) are a superfamily of enzymes that catalyze the reduction of biogenic and xenobiotic aldehydes and ketones. AKR1B family has 2 known members in humans and 3 in rodents. Two novel gene loci, hereafter referred to as AKR1B15 in human and Akr1b16 in mouse have been predicted to exist within the AKR1B clusters. AKR1B15 displays 91% and 67% sequence identity with human genes AKR1B10 and AKR1B1, respectively while Akr1b16 shares 82-84% identity with murine Akr1b8 and Akr1b7. We tested the hypothesis that AKR1B15 and Akr1b16 genes are expressed as functional proteins in human and murine tissues, respectively. Using whole tissue mRNA, we were able to clone the full-length open reading frames for AKR1B15 from human eye and testes, and Akr1b16 from murine spleen, demonstrating that these genes are transcriptionally active. The corresponding cDNAs were cloned into pET28a and pIRES-hrGFP-1α vectors for bacterial and mammalian expression, respectively. Both genes were expressed as 36kDa proteins found in the insoluble fraction of bacterial cell lysate. These proteins, expressed in bacteria showed no enzymatic activity. However, lysates from COS-7 cells transfected with AKR1B15 showed a 4.8-fold (with p-nitrobenzaldehyde) and 3.3-fold (with dl-glyceraldehyde) increase in enzyme activity compared with untransfected COS-7 cells. The Akr1b16 transcript was shown to be ubiquitously expressed in murine tissues. Highest levels of transcript were found in heart, spleen, and lung. From these observations we conclude that the predicted AKR1B15 and 1b16 genes are expressed in several murine and human tissues. Further studies are required to elucidate their physiological roles.
authors.affiliation: Diabetes and Obesity Center, School of Medicine, University of Louisville, Louisville, KY 40202, United States.
authors.forename: Joshua K
authors.initials: JK
authors.lastname: Salabei
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Li
authors.forename: J Mark
authors.initials: JM
authors.lastname: Petrash
authors.forename: Aruni
authors.initials: A
authors.lastname: Bhatnagar
authors.forename: Oleg A
authors.initials: OA
authors.lastname: Barski
mesh_terms: D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000166:cytology; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D056426:Genetic Loci / Q000235:genetics; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D050837:Oxidoreductases Acting on Aldehyde or Oxo Group Donors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; C560504:AKR1B15 protein, human; C560505:Akr1b10 protein, mouse; D000074408:Aldo-Keto Reductases; D050837:Oxidoreductases Acting on Aldehyde or Oxo Group Donors