Functional Synergism between the Most Common Polymorphism in Human Alanine:Glyoxylate Aminotransferase and Four of the Most Common Disease-causing Mutations

Abstract

The autosomal recessive disorder primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the liver-specific pyridoxal-phosphate-dependent enzyme alanine:glyoxylate aminotransferase (AGT). Numerous mutations and polymorphisms in the gene encoding AGT have been identified, but in only a few cases has the causal relationship between genotype and phenotype actually been demonstrated. In this study, we have determined the effects of the most common naturally occurring amino acid substitutions (both normal polymorphisms and disease-causing mutations) on the properties, especially specific catalytic activity, of purified recombinant AGT. The results presented in this paper show the following: 1) normal human His-tagged AGT can be expressed at high levels in Escherichia coli and purified in a correctly folded, dimerized and catalytically active state; 2) presence of the common P11L polymorphism decreases the specific activity of purified recombinant AGT by a factor of three; 3) AGTs containing four of the most common PH1-specific mutations (G41R, F152I, G170R, and I244T) are all soluble and catalytically active in the absence of the P11L polymorphism, but in its presence all lead to protein destabilization and aggregation into inclusion bodies; 4) naturally occurring and artificial amino acid substitutions that lead to peroxisome-to-mitochondrion AGT mistargeting in mammalian cells also lead to destabilization and aggregation in E. coli; and 5) the PH1-specific G82E mutation abolishes AGT catalytic activity by interfering with cofactor binding, as does the artificial K209R mutation at the putative site of cofactor Shiff base formation. These results are discussed in the light of the high allelic frequency (ϳ20%) of the P11L polymorphism and its importance in determining the phenotypic manifestations of mutations in PH1.

Cloning and mutation analysis of ZFP276 as a candidate tumor suppressor in breast cancer

“…It has been documented that single nucleotide polymorphisms (SNPs) can potentially affect the way genes function (Lumb and Danpure, 2000). Nonsynonymous changes (amino acid changes) may affect protein folding and thus the function of the proteins.…”

Molecular defects of the glycine 41 variants of alanine glyoxylate aminotransferase associated with primary hyperoxaluria type I

“…Photo-induced cross-linking with TBPR was performed as previously described (7). SEC experiments were done on an Akta FPLC system (GE Healthcare) using a custom packed Sephacryl S-300 10∕600 column.…”

“…To date, well over 100 pathogenic mutations associated to AGT-Ma and/or AGT-Mi are known (6). Three categories of enzymatic phenotypes causing PH1 can be identified: deficiency of AGT catalytic activity but not AGT immunoreactivity, catalytic activity and immunoreactivity deficiency, and mistargeting to mitochondria (4,7). Notably, clinical data for single PH1 AGT mutations are generally limited to a small number of individuals, which may interfere with identifying clear correlations between disease characteristics and properties of mutant proteins.…”

Peroxisomal Import of Human Alanine:glyoxylate Aminotransferase Requires Ancillary Targeting Information Remote from Its C Terminus

“…Val 326 3 Ile segregates with a variant called the minor (African) AGXT allele (43). Although the functional effects of the two PTS1A polymorphisms have not been well characterized, it is known that Ile 340 3 Met can partially counteract the untoward effects of the Pro 11 3 Leu polymorphism, which include partial mistargeting, slowing down dimerization, and decreasing specific catalytic activity (3,44). In some of the mammals discussed in this report (i.e.…”