title: A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay.
abstract: Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated using E coli- or insect-based expression systems is either insoluble or enzymatically unstable, and the LH2 enzymatic activity assays that are currently available measure radioactive CO2released from14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems.
authors.affiliation: Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States.
authors.forename: Hou-Fu
authors.initials: HF
authors.lastname: Guo
authors.affiliation: Division of Medicinal Chemistry, Targeted Therapeutic Drug Discovery and Development Program, College of Pharmacy, The University of Texas at Austin, Austin, TX, United States.
authors.forename: Eun Jeong
authors.initials: EJ
authors.lastname: Cho
authors.affiliation: Division of Medicinal Chemistry, Targeted Therapeutic Drug Discovery and Development Program, College of Pharmacy, The University of Texas at Austin, Austin, TX, United States.
authors.forename: Ashwini K
authors.initials: AK
authors.lastname: Devkota
authors.affiliation: Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States.
authors.forename: Yulong
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Departments of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, United States.
authors.forename: William
authors.initials: W
authors.lastname: Russell
authors.affiliation: Department of Biosciences and Chemistry, Rice University, Houston, TX, United States.
authors.forename: George N
authors.initials: GN
authors.lastname: Phillips
authors.affiliation: Oral and Craniofacial Health Sciences, School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Yamauchi
authors.affiliation: Division of Medicinal Chemistry, Targeted Therapeutic Drug Discovery and Development Program, College of Pharmacy, The University of Texas at Austin, Austin, TX, United States; Division of Chemical Biology & Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX, United States. Electronic address: dalby@austin.utexas.edu.
authors.forename: Kevin N
authors.initials: KN
authors.lastname: Dalby
authors.affiliation: Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States. Electronic address: jkurie@mdanderson.org.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Kurie
mesh_terms: D000818:Animals; D016466:CHO Cells; D002244:Carbon / Q000737:chemistry; D002245:Carbon Dioxide / Q000737:chemistry; D002853:Chromatography, Liquid; D003094:Collagen / Q000737:chemistry; D003412:Cricetulus; D017077:Culture Media, Conditioned / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007656:Ketoglutaric Acids / Q000737:chemistry*; D008156:Luciferases / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis; D019802:Succinic Acid / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D017077:Culture Media, Conditioned; D007656:Ketoglutaric Acids; D011994:Recombinant Proteins; D002245:Carbon Dioxide; D002244:Carbon; D003094:Collagen; D019802:Succinic Acid; D008156:Luciferases; C479138:PLOD2 protein, human; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; D008239:Lysine
keywords: Chinese hamster ovary cell; Collagen; High-throughput assay; Lysyl hydroxylase 2; Oxygenase; Succinate detection