Primary hyperoxaluria type 1 in the Canary Islands: A conformational disease due to I244T mutation in the P11L-containing alanine:glyoxylate aminotransferase

Abstract

Primary hyperoxaluria type 1 (PH1) is an inborn error of metabolism resulting from a deficiency of alanine:glyoxylate aminotransferase (AGXT; EC 2.6.1.44). Most of the PH1 alleles detected in the Canary Islands carry the Ile-244 3 Thr (I244T) mutation in the AGXT gene, with 14 of 16 patients homozygous for this mutation. Four polymorphisms within AGXT and regional microsatellites also were shared in their haplotypes (AGXT*LTM), consistent with a founder effect. The consequences of these amino acid changes were investigated. Although I244T alone did not affect AGXT activity or subcellular localization, when present in the same protein molecule as Leu-11 3 Pro (L11P), it resulted in loss of enzymatic activity in soluble cell extracts. Like its normal counterpart, the AGXT*LTM protein was present in the peroxisomes but it was insoluble in detergent-free buffers. The polymorphism L11P behaved as an intragenic modifier of the I244T mutation, with the resulting protein undergoing stable interaction with molecular chaperones and aggregation. This aggregation was temperature-sensitive. AGXT*LTM expressed in Escherichia coli, as a GST-fusion protein, and in insect cells could be purified and retained enzymatic activity. Among various chemical chaperones tested in cell culture, betaine substantially improved the solubility of the mutant protein and the enzymatic activity in cell lysates. In summary, I244T, the second most common mutation responsible for PH1, is a protein conformational disease that may benefit from new therapies with pharmacological chaperones or small molecules to minimize protein aggregation.

In Vivo and in Vitro Examination of Stability of Primary Hyperoxaluria-associated Human Alanine:Glyoxylate Aminotransferase

“…In these studies, we also examined protection by PLP. Although previous studies, including our yeast experiments, were unable to demonstrate protection of AGT by PLP in vivo, indirect effects of PLP on stability have been observed in vitro by several groups (6,7,13). Purified proteins were preincubated for 1 h at the indicated temperatures in the presence or absence of 50 M PLP.…”

“…Interacting mutations include I244T, found in ϳ9% of PH1 patients. This mutation is not disease-causing itself but leads to protein aggregation and disease when combined with the minor allele polymorphism (6). The mutation G170R, found in ϳ30% of PH1 patients, is also not by itself deleterious but when combined with the minor allele is thought to reduce stability, delay dimerization, and result in protein mislocalization (5).…”

“…Previous studies have characterized wild-type AGT and PH1-associated variants biochemically and in cell culture (5)(6)(7)(8)(9)(10). The minor allele itself appears to have a particularly complex phenotype; studies suggest that it has reduced catalytic activity (5,8) and reduced stability (5,7) and that the P11L mutation generates a cryptic mitochondrial targeting signal that affects protein trafficking (4,10,11).…”

“…Although the trafficking differences between the major and minor allele proteins are well established (2), there is only indirect evidence supporting that the minor allele is destabilized, and some of these experiments are difficult to interpret because they assessed the minor allele in combination with other mutations or characterized proteins containing only one of the two minor allele mutations. Evidence for minor allele destabilization includes in vitro transcription/translation studies demonstrating that AGTmi is more aggregation-prone than AGTma (7) and protease protection and overexpression results showing that variants containing the P11L mutation are more susceptible to proteolytic digestion (6) and prone to aggregation (5).…”

“…Pyridoxine is broken down in the cell to form pyridoxal 5Ј-phosphate (PLP), a cofactor for AGT. Several in vitro studies suggest that PLP stabilizes AGT; PLP was found to increase the solubility of a variant (AGTmi-I244T) expressed in COS cells (6), to improve the yield and increase dimerization of in vitro transcribed/translated protein (7), to protect AGT mutants from proteasomal degradation (7), and to protect wild-type AGT from heat inactivation (13). None of these studies, however, incorporated direct stability measurements, and the issue is further complicated because attempts to observe an effect of PLP in vivo (similar to the effect seen with betaine, for example) have failed (9).…”