Bacterial expression of mutant argininosuccinate lyase reveals imperfect correlation of in‐vitro enzyme activity with clinical phenotype in argininosuccinic aciduria

Abstract

The bacterially expressed enzymes proved that the mutations found in patients and studied here indeed are detrimental. However, as in the case of red cell ASL activity assays, some mutations found in genetically homozygous patients with mild presentations resulted in virtual loss of enzyme activity in the bacterial system, suggesting a more protective environment for the mutant enzyme in the liver than in the heterologous expression system and/or in the highly dilute assays utilized here.

Understanding the Role of Argininosuccinate Lyase Transcript Variants in the Clinical and Biochemical Variability of the Urea Cycle Disorder Argininosuccinic Aciduria

“…The ASL enzyme activity was determined spectrophotometrically in cell extracts after transient transfection or cotransfection of P-WT, P-ex2del, and/or P-ex7del, using a coupled assay with arginase and measuring urea production as described before (33). Briefly, 100 l of 34 mM argininosuccinate (argininosuccinic acid disodium salt hydrate) in water and 100 l of arginase (50 units) (both from Sigma-Aldrich) in 66.7 mM phosphate buffer (11.1 mM potassium dihydrogenphosphate and 55.6 mM disodium hydrogenphosphate, pH 7.5) were incubated at 37°C for 5 min.…”

“…The clinical and biochemical phenotype of ASA is highly variable ranging from asymptomatic cases with only a biochemical phenotype (23)(24)(25), some of them diagnosed through newborn screening, to severe neonatal-onset hyperammonemic encephalopathy (26,27). The molecular basis for the diversity of ASA is not fully understood, and several explanations have been suggested, including tissue-specific ASL expression (27,28), genetic heterogeneity at the ASL locus (29), intragenic comple-mentation (7, 30 -32), different levels of residual ASL activity (33,34), the developmental control of the ASL gene by DNA methylation (35), and alternative splicing events at the ASL locus leading to frequent exon deletions (5,36,37).…”

Unstable argininosuccinate lyase in variant forms of the urea cycle disorder argininosuccinic aciduria

“…In addition to measurements in patients' samples, there are some in vitro assays investigating naturally occurring ASL mutations in bacterial (E. coli) (Engel et al 2012;Sampaleanu et al 2001;Yu et al 2001), yeast (Barbosa et al 1991;Doimo et al 2012;Trevisson et al 2009) and eukaryotic (COS1-cells) (Walker et al 1990(Walker et al , 1997 expression systems. While identification of severely affected ASL proteins was feasible in all of these, there was overall no satisfying sensitivity for residual ASL activities and hence the predictive value was limited.…”

Overview of Recent Progress in Protein-Expression Technologies for Small-Molecule Screening

“…Lack of efficacy in these downstream assays can occur for many reasons, including limited understanding of disease biology, 3 but any potentially significant differences between the endogenous target and the protein used for screening must also be considered, especially because the properties of the same protein expressed in different systems may not be similar. 4,5 With the rapid proliferation of targets resulting from an explosion of genomic data combined with recent advances in affinity screening technologies that access significantly larger regions of chemical space than traditional screening decks, 6,7 it is critical at the outset of any new screening campaign to consider expression technologies designed to produce proteins that structurally and functionally resemble endogenous targets. Such systems should also be more suitable for production of what have unfortunately been labeled as "challenging" targets, such as those that require complex posttranslational modifications or the presence of cell-type-specific protein partners to fold into their native conformation and multispanning membrane targets.…”