Cregeen, David Peter; (2001) Molecular and kinetic analysis of human glyoxylate/hydroxypyruvate reductase. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes the elucidation of the genetic basis of primary hyperoxaluria type 2 (PH2), an autosomal recessive disease characterised by recurrent calcium oxalate kidney stone formation leading to renal failure and, in some cases to an early death. The enzyme deficient in this condition, glyoxylate / hydroxypyruvate reductase (GRHPR), has been partially purified and characterised from human liver and is distinct from lactate dehydrogenase which also has hydroxypyruvate reductase activity. GRHPR utilises hydroxypyruvate and glyoxylate as substrates with a preference for hydroxypyruvate with NADPH as preferred cofactor. Chromatofocused partially purified human liver proteins show no OR activity associated with LDH, using NADPH as cofactor. Although sharing low protein sequence similarity to the GR/HPR enzymes of plants and bacteria, a human cDNA encoding GRHPR has been identified from an EST database. Purified recombinant GRHPR has similar kinetic properties to the partially purified liver protein, with a preference for hydroxypyruvate as substrate and NADPH as cofactor at pH7.0. Antibodies raised against this recombinant enzyme identify a single protein in human liver of approximately 38kDa which is absent in patients with PH2. The protein demonstrates charge heterogeneity on chromatofocusing and isoelectric focusing, although at a different pi range unless subunits are chemically crosslinked as a dimer (the preferred enzyme conformation). Northern blot analysis of GRHPR mRNA shows its primary location to be the liver but with RNA present in many other tissues. This ubiquitous expression is not reflected by distribution of immunoreactive protein which was essentially restricted to the liver. DNA from patients with PH2 has been screened for mutations in the GRHPR gene by direct sequencing of cDNA where available, or SSCP analysis and sequencing of genomic DNA. 6 mutations and a single polymorphism have been identified including a 1 bp deletion in codon 35, found in 33% of all PH2 alleles.

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