Human liver glycolate oxidase Gene identification and protein studies Emma Louise Wiiliams UCL A thesis submitted for the degree of Doctor of Philosophy University College London Abstract Glycolate oxidase (GO) is a peroxisomal flavoenzyme which catalyses the oxidation of short chain a-hydroxy acids, notably glycolate. The reaction product, glyoxylate, is an oxalate precursor and GO is thus of potential interest for its role in the pathogenesis of the primary hyperoxalurias. The project aims were to identify human GO, characterise the kinetics and substrate specificity of the enzyme and establish methods for the analysis of relevant metabolic pathways in vitro. The gene for human GO was cloned from liver and expressed in bacterial cells. The cDNA is 1128 bp in length and has a 1113 bp open reading frame encoding a 372 amino acid protein. The genomic sequence comprises eight exons and spans -57 kb of chromosome 20pl2. Recombinant human GO protein shares 53% and 89% sequence similarity to GO from spinach and rat respectively, shows a-hydroxy acid oxidase activity in vitro and has been purified to homogeneity. Polyclonal anti-GO antibody detects a band of 43 kDa in human liver and, consistent with northern blot analysis, expression is not detected in other tissues including kidney and leucocytes. Kinetic analysis with a range of a-hydroxy acids indicates GO has highest affinity for glycolate as substrate (K^ = 0.54 mM) and 10 fold less affinity for glyoxylate (K^,= 5.1 mM). Site directed mutagenesis of active site residues demonstrates the importance of chain length for substrate affinity. Thus mutation of a Trp residue, conserved between spinach and human GO to a less bulky amino acid, permits the catalysis of longer chain length a-hydroxy acids. HPLC methods were developed for the separation and quantitation of glyoxylate, hydroxypyruvate and pyruvate, enabling analysis of metabolites produced by GO and neighboring enzymes in the metabolic pathway. These assays will be invaluable for future studies in which the pathways of glyoxylate metabolism are constructed in vitro. Abbreviations Enzymes and EC numbers: AGT alanine:glyoxylate aminotransferase (EC 2.6.1.44) ALDH aldehyde dehydrogenase (EC 1.2.1.21) CL 2-oxoglutarate:glyoxylate carboligase (EC 4.1.3.15) DAO D-amino acid oxidase (EC 1.4.3.3) D-GDH D-glycerate dehydrogenase (EC 1.1.1.29) DGK D-glycerate kinase (EC 2.7.1.31) FCBz flavocytochrome hi (EC 1.1.2.3) GDH glycolate dehydrogenase (EC 1.1.99.14) GGT glutamate:glyoxylate aminotransferase (EC 2.6.1.4) GO glycolate oxidase (EC 1.1.3.15) GR glyoxylate reductase (EC 1.1.1.79) HAO A hydroxy acid oxidase type A (EC 1.1.3.15) HAO B hydroxy acid oxidase type B (EC 1.1.3.15) HPD hydroxypyruvate dehydrogenase (EC 4.1.1.40) HPR hydroxypyruvate reductase (EC 1.1.1.79) LDH lactate dehydrogenase (EC 1.1.1.27) LMO lactate mono-oxygenase (EC 1.13.12.4) MDH mandelate dehydrogenase (EC 1.1.2.3) PGDH phosphoglycerate dehydrogenase (EC 1.1.1.95) PGI phosphoglycerate isomerase (EC 5.4.2.1 ) PSA phosphoserine transaminase (EC 2.6.1.52) PSP phosphoserine phosphatase (EC 3.1.3.3) RUBISCO ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) SPT serineipyruvate aminotransferase (EC 2.6.1.51) XAO xanthine oxidase (EC 1.1.3.22) General abbreviations: cDNA complementary DNA DNA deoxyribonucleic acid dNTP deoxynucleoside triphosphate EDTA ethylenediaminetetra-acetic acid FMN flavin mononucleotide HPLC high performance liquid chromatography mRNA messenger RNA NAD+ p-Nicotinamide adenine dinucleotide NADH p-Nicotinamide adenine dinucleotide, reduced form NADP+ p-Nicotinamide adenine dinucleotide phosphate NADPH p-Nicotinamide adenine dinucleotide phosphate, reduced form OPD o-phenylenediamine PHI primary hyperoxaluria type 1 PH2 primary hyperoxaluria type 2 PHZ phenylhydrazine hydrochloride PTS peroxisomal targeting signal PAGE polyacrylamide gel electrophoresis RNA ribonucleic acid RT-PCR Reverse transcriptase polymerase chain reaction SDS sodium dodecyl sulphate Table of Contents Chapter Index Tit l e ....................................................................................................................................................... 1 A bstract ...............................................................................................................................................2 A bbreviations ....................................................................................................................................4 Table of C o n te n t s ........................................................................................................................... 7 CHAPTER INDEX.................................................................................................................................. 7 LIST OF FIGURES.................................................................................................................................10 LIST OF TABLES..................................................................................................................................13 A cknowledgements .......................................................................................................................14 1.0 C h a pter O ne - I ntroduction ...........................................................................................15 1.1 Ox a l a t e .................................................................................................................................... 16 1.1.1 Dietary intake and absorption o f oxalate .................................................................... 17 1.1.2 Renal handling o f oxalate ................................................................................................18 1.2 Endogenous Oxalate P roduction .................................................................................20 1.2.1 Glyoxylate synthesis .......................................................................................................... 21 1.2.2 Glyoxylate utilisation ........................................................................................................27 1.3 H yperoxaluria ...................................................................................................................... 34 1.4 Secondary H yperoxaluria .............................................................................................. 35 1.5 P rimary H yperoxaluria .................................................................................................... 37 1.5.1 Primary hyperoxaluria type 1 ........................................................................................ 37 1.5.2 Primary hyperoxaluria type 2 ........................................................................................ 43 1.5.2 The treatment o f primary hyperoxaluria ...................................................................... 48 1.5.4 Atypical hyperoxaluria .....................................................................................................51 1.6 G lycolate Oxid ase ..............................................................................................................53 1.6.1 GO in plants ........................................................................................................................56 1.6.2 GO in anim als .................................................................................................................... 57 2.0 C h a pter T w o - G enera l M e t h o d s ................................................................................59 2.1 Introduction ..........................................................................................................................60 2.2 M a ter ia ls ................................................................................................................................60 2.2.1 Chemicals .............................................................................................................................60 2.2.2 Buffers ...................................................................................................................................60 2.2.3 Bacterial media and antibiotics .....................................................................................63 2.3 M eth ods ....................................................................................................................................65 2.3.1 Isolation of human liver RNA ..........................................................................................65 2.3.2 Reverse transcriptase-polymerase chain reaction (RT-PCR) ................................ 65 2.3.3 TA cloning of PCR products ...........................................................................................66 2.3.4 Sequencing .......................................................................................................................... 67 2.3.5 Northern blot analysis ......................................................................................................68 2.3.6 Transfection intoBL21 competent cells ....................................................................... 68 2.3.7 Expression o f recombinant protein in BL21 cells .....................................................69 2.3.8 Protein SDS-PAGE ............................................................................................................69 2.3.9 Protein staining with Coomassie brilliant blue ..........................................................70 2.3.10