SHEDDING LIGHT ON THE MOLECULAR DEFECT OF TWOALANINE:GLYOXYLATE AMINOTRANSFERASE PATHOGENIC VARIANTS:A BIOCHEMICAL APPROACH.

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder characterized by the deposition of insoluble calcium oxalate crystals at first in the kidneys and urinary tract and then, in the absence of appropriate treatments, in the whole body. PH1 is caused by the deficiency of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that converts glyoxylate to glycine, thus preventing glyoxylate oxidation to oxalate and therefore the formation of calcium oxalate. Normal human AGT is encoded by the AGXT gene that exists in human populations in two polymorphic forms: the major allele (AGT-Ma) and the minor allele (AGT-Mi), which is characterized by two point mutations, leading to the Pro11Leu and Ile340Met substitutions, and a 74 bp-duplication in intron 1. Although the presence of the minor allele polymorphism is not pathogenic “per se”, it makes AGT more susceptible to the effect of some PH1-causing mutation that are expected to be not pathogenic when associated with the major allele. Thus, there is a great interest in defining the properties of AGT-Mi, as the base to unravel the molecular mechanism underlying the synergism between AGT-Mi and the pathogenic mutations that cosegregate with it. In this work, by an “in vitro” approach on purified proteins, we studied the effects on the biochemical features of AGT of the two combined polymorphic mutations typical of the minor allele as well as of two PH1-causing mutations associated with the minor allele, Phe152Ile and Gly170Arg. The data obtained have shown that: 1) AGT-Mi displays spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma. However, its dimeric structure is characterized by a low resistance to both chemical and thermal stress. This appears to be due to the P11L mutation since the P11L variant exhibits a denaturation pattern comparable to that of AGT-Mi; 2) The PH1-causing F152I mutation leads to a ~200 fold decrease in the affinity of AGT for pyridoxamine 5’-phosphate and, when associated with the minor allele polymorphism, to a time-dependent inactivation and aggregation at physiological temperature; 3) The pathogenic mutation G170R does not affect neither the spectroscopic nor the kinetic properties of AGT-Mi under native conditions. However, it makes the dimeric structure of apoG170R-Mi more susceptible to dissociation than the corresponding apoAGT-Mi. Overall, the obtained data: (i) reveal the biochemical differences between AGT-Ma and AGT-Mi; (ii) allow to shed light on the molecular defect associated with the F152-Mi and the G170R-Mi variants; (iii) permit to speculate on the responsiveness to pyridoxine therapy of the patients bearing these mutations.