S81L and G170R mutations causing Primary Hyperoxaluria type I in homozygosis and heterozygosis: an example of positive interallelic complementation

Abstract

Primary Hyperoxaluria type I (PH1) is a rare disease due to the deficit of peroxisomal alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal-5′-phosphate (PLP) enzyme present in humans as major (Ma) and minor (Mi) allele. PH1-causing mutations are mostly missense identified in both homozygous and compound heterozygous patients. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, whereas the molecular aspects of the genotype-enzymatic-clinical phenotype relationship in compound heterozygous patients are completely unknown. Here, for the first time, we elucidate the enzymatic phenotype linked to the S81L mutation on AGT-Ma, relative to a PLP-binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functionality, is present in the second allele. By using a bicistronic eukaryotic expression vector, we demonstrate that (i) S81L-Ma is mainly in its apo-form and has a significant peroxisomal localization and (ii) S81L and G170R monomers interact giving rise to the G170R-Mi/S81L-Ma holo-form, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the heterodimer and the homodimeric counterparts in their purified recombinant form, (i) highlight the molecular basis of the pathogenicity of S81L-Ma and (ii) provide evidence for a positive interallelic complementation between the S81L and G170R monomers. Our study represents a valid approach to investigate the molecular pathogenesis of PH1 in compound heterozygous patients.

Liver peroxisomal alanine:glyoxylate aminotransferase and the effects of mutations associated with Primary Hyperoxaluria Type I: An overview

“…This raises the question whether the different effects are all relevant for the in vivo pathogenesis and which effect plays the major role. Moreover, it is becoming increasingly evident that many mutations affect mainly or exclusively the apo-form of AGT (as found for the G170R, F152I, G161R, G161C, G161S, I244T and A295T variants) [12,17,45,49,51,52,57], thus highlighting the fact that the pathogenic mechanisms leading to AGT deficiency range probably wider than previously thought and that the study of each variant in both the holo-and apo-forms is a necessary step for the identification of its molecular defect.…”

“…However, each approach explores a different facet of a mutation's effect. Expression in mammalian cells is considered as the closest model of in vivo conditions and gives information on the expression level, intracellular stability and targeting of the AGT variants [17,28,35,40,[46][47][48][49][50]. On the other hand, the purification and characterization of the variants expressed in E. coli are suitable to dissect and quantify the effect of a mutation on the functional properties of AGT as well as on its secondary, tertiary and quaternary structures [11][12][13]17,18,35,36,[51][52][53][54][55][56][57][58].…”

“…A reduced catalytic efficiency for the transamination reaction (due to a reduced k cat and/or to an increased K m for the substrate as shown in Supplementary Table 2) or an altered PLP/PMP binding (in terms of affinity and/or binding mode) are the main functional effects. They are typically caused by mutations of residues that directly interact with PLP or are located in the proximity of the active site, such as Ser81, Gly82, His83, Trp108, Phe152, Ser158, Asp183 and Ser218 [11,16,49,52,57]. However, a remote conformational change of the active site can be also generated by mutations of residues located far from the active site such as Ser187, Ile244 and Gly350, as demonstrated by crystallographic and biochemical studies [17,56,57].…”

“…However, the median age at ESRD of patients bearing the G170R mutation (47 years in homozygous and 35 years in compound heterozygous) is higher than that (21 years) of patients bearing other mutations [65]. A first effort toward the understanding of the correlation between the enzymatic and clinical phenotypes of PH1 compound heterozygous patients is represented by a study collecting clinical, biochemical and cell biology data on the effects of the S81L mutation on the major allele (S81L-Ma) and the G170R mutation on the minor allele (G170R-Mi) under homozygous and heterozygous conditions [49]. Studies performed by individually expressing each mutant, i.e.…”

SNP Variants in RET and PAX2 and Their Possible Contribution to the Primary Hyperoxaluria Type 1 Phenotype

“…Heterozygosity of missense alleles adds another layer of potential heterogeneity as interallelic complementation may generate heterodimers with improved function compared to the individual homodimers (Montioli et al 2014).…”