Correlation between the molecular effects of mutations at the dimer interface of alanine–glyoxylate aminotransferase leading to primary hyperoxaluria type I and the cellular response to vitamin B₆

Abstract

Primary hyperoxaluria type I (PH1) is a rare disease caused by the deficit of liver alanine-glyoxylate aminotransferase (AGT). AGT prevents oxalate formation by converting peroxisomal glyoxylate to glycine. When the enzyme is deficient, progressive calcium oxalate stones deposit first in the urinary tract and then at the systemic level. Pyridoxal 5'-phosphate (PLP), the AGT coenzyme, exerts a chaperone role by promoting dimerization, as demonstrated by studies at protein and cellular level. Thus, variants showing a destabilized dimeric structure should, in principle, be responsive to vitamin B, a precursor of PLP. However, models to predict the extent of responsiveness of each variant are missing. We examined the effects of pathogenic interfacial mutations by combining bioinformatic predictions with molecular and cellular studies on selected variants (R36H, G42E, I56N, G63R, and G216R), in both their holo- (i.e., with bound PLP) and apo- (i.e., without bound PLP) form. We found that all variants displayed structural alterations mainly related to the apoform and consisting of an altered tertiary and quaternary structure. G216R also shows a strongly reduced catalytic efficiency. Moreover, all but G216R respond to vitamin B, as shown by their increased specific activity and expression level in a cellular disease model. A global analysis of data unraveled a possible inverse correlation between the degree of destabilization/misfolding induced by a mutation and the extent of B6 responsiveness. These results provide a first explanation of factors influencing B6 response in PH1, a model possibly valuable for other rare diseases caused by protein deficits.

Modulation of Guanylate Cyclase Activating Protein 1 (GCAP1) Dimeric Assembly by Ca2+ or Mg2+: Hints to Understand Protein Activity

“… * ΔΔG 0 was calculated by using an empirical correlation between ZD-s and ΔG 0 as explained in [ 27 ] and in [ 40 ]. …”

Novel mutations in response to vitamin B6 in primary hyperoxaluria type 1 after only kidney transplantation: a case report

“…Actually, as a precursor of the PLP coenzyme of AGT, B6 has been confirmed to contribute to the dimerization and stability of AGT. Except for mutations which involve residues directly interacting with PLP, such as S81L, G82E, H83R, W108R, S158L, and D183N, most mutations should be sensitive to B6 treatment in theory (11). The mutations A186V, R197Q, and I340M in our report were all out of the PLP-binding domain.…”

“…A prospective clinical trial confirmed the efficacy of B6 application in 50% of the patients with particular mutations in the AGXT gene, mainly those with G170R and F152I (13). Another in vitro study also verified that B6 treatment exerted effects on R36H, G42E, I56N, and G63R (11). However, evidence of the response to B6 treatment in these genotypes is still lacking.…”

“…Most of these mutations influence mainly protein folding, dimerization, and intracellular localization, thereby leading to AGT deficiency (9,10). Dimerization is considered a crucial event that influences protein stability and intracellular fate (11). As an essential cofactor of AGT, pyridoxal phosphate (PLP) has been confirmed to be necessary for dimerization and protein stability at the protein and cellular levels.…”

Primary hyperoxaluria diagnosed after kidney transplantation: a case report and literature review

“…Three patients with PH reported a reversal of the continuous increase in creatinine by oral vitamin B6 therapy [ 5 , 10 , 18 ]. This may be due to the fact that some patients with specific mutations in AGXT are sensitive to B6 treatment [ 18 , 27 ]. To the best of our knowledge, this is the first case to report the plasma oxalate concentration of pre-continuous and post-continuous renal replacement therapy (72 h) in a patient with PH diagnosed after kidney transplantation.…”