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

Abstract

G41 is an interfacial residue located within the α-helix 34-42 of alanine:glyoxylate aminotransferase (AGT). Its mutations on the major (AGT-Ma) or the minor (AGT-Mi) allele give rise to the variants G41R-Ma, G41R-Mi, and G41V-Ma causing hyperoxaluria type 1. Impairment of dimerization in these variants has been suggested to be responsible for immunoreactivity deficiency, intraperoxisomal aggregation, and sensitivity to proteasomal degradation. However, no experimental evidence supports this view. Here we report that G41 mutations, besides increasing the dimer-monomer equilibrium dissociation constant, affect the protein conformation and stability, and perturb its active site. As compared to AGT-Ma or AGT-Mi, G41 variants display different near-UV CD and intrinsic emission fluorescence spectra, larger exposure of hydrophobic surfaces, sensitivity to Met53-Tyr54 peptide bond cleavage by proteinase K, decreased thermostability, reduced coenzyme binding affinity, and catalytic efficiency. Additionally, unlike AGT-Ma and AGT-Mi, G41 variants under physiological conditions form insoluble inactive high-order aggregates (∼5; 000 nm) through intermolecular electrostatic interactions. A comparative molecular dynamics study of the putative structures of AGT-Mi and G41R-Mi predicts that G41 → R mutation causes a partial unwinding of the 34-42 α-helix and a displacement of the first 44 N-terminal residues including the active site loop 24-32. These simulations help us to envisage the possible structural basis of AGT dysfunction associated with G41 mutations. The detailed insight into how G41 mutations act on the structure-function of AGT may contribute to achieve the ultimate goal of correcting the effects of these mutations.dimer interface | pathogenic variant | protein aggregation | pyridoxal 5'-phosphate

Four of the Most Common Mutations in Primary Hyperoxaluria Type 1 Unmask the Cryptic Mitochondrial Targeting Sequence of Alanine:glyoxylate Aminotransferase Encoded by the Polymorphic Minor Allele

“…in the absence of the P11L replacement). Previous studies on purified recombinant AGT expressed in E. coli suggest that, except for G41R, they do not (18,21,34). Therefore it is likely that peroxisome-to-mitochondrion mistargeting caused by these mutations, all result from a similar synergistic interaction to that previously identified for G170R.…”

Phenotype-Genotype Correlations and Estimated Carrier Frequencies of Primary Hyperoxaluria

“…These results emphasize the incompletely penetrant nature/ variable expressivity of p.G170R and suggest a different pathomechanism for other MiR alleles, such as monomer aggregation or active site disruption, as indicated for p.G41R. 29,44 This is important in considering pyridoxine therapy for patients with other MiR alleles because this cofactor is thought to stabilize the monomer/dimer. Nevertheless, anecdotal evidence suggests pyridoxine response in a variety of AGXT mutation patients (including p.F152I or two truncating alleles), indicating that alternative therapeutic benefits of pyridoxine may exist (e.g., influence on gene expression and/or enzyme activity).…”

Rapid Profiling of Disease Alleles Using a Tunable Reporter of Protein Misfolding

“…G41E and G41R, located at the dimerization interface of AGT, were included in this group. G41R has been characterized in depth in vitro, where it has been shown to affect dimerization, reduce affinity of AGT for PLP (Cellini et al 2010b), and significantly increase aggregation under physiological conditions. Molecular dynamics simulations of this mutant revealed unwinding of the 34-42 a-helix and displacement of the first 44 amino acid residues, including an active site loop at residues 24-32 (Cellini et al 2010b).…”

“…G41R has been characterized in depth in vitro, where it has been shown to affect dimerization, reduce affinity of AGT for PLP (Cellini et al 2010b), and significantly increase aggregation under physiological conditions. Molecular dynamics simulations of this mutant revealed unwinding of the 34-42 a-helix and displacement of the first 44 amino acid residues, including an active site loop at residues 24-32 (Cellini et al 2010b). While not involved in coordinating PLP, Gly47 is also near the active site of the enzyme, and the replacement of the smaller glycine in the G47R variant may locally perturb the structure.…”