Co-expression of different subunits of human phenylalanine hydroxylase: Evidence of negative interallelic complementation

Abstract

To study the interaction between two different subunits of the heteromeric human phenylalanine hydroxylase (hPAH), present in hyperphenylalaninemic (HPA) compound heterozygous patients, heteroallelic hPAH enzymes were produced. A dual vector expression system was used (PRO Bacterial Expression System) in which each mutant subunit was expressed from a separate compatible vector, with different epitope tags, in a single bacterial host. Experimental conditions were selected in order that each plasmid produced equivalent levels of mutant subunits. In this study, we demonstrated that both subunits were expressed and that the purified heteroallelic enzymes, were catalytically active. As expected, the produced proteins displayed enzymatic activities levels lower than the predicted catalytic activity, calculated by averaging in vitro PAH activities from both alleles, and were strongly dependent on the proteins subunit composition. The obtained data suggest that interactions between the studied hPAH subunits, namely the I65T, R261Q, R270K and V388M, and the wild-type protein occurred. As postulated, this phenomenon could be a source of phenotypic variation in genetic diseases involving multimeric proteins.

Assessment of tetrahydrobiopterin (BH4)-responsiveness and spontaneous phenylalanine reduction in a phenylalanine hydroxylase deficiency population

“…Interestingly, a patient with IVS4 +5/p.E178G genotype (the latter mutation has a PRA = 39%) and an MHP phenotype, was not shown to be BH 4 responsive, despite the relatively low Phe levels achieved with a protein rich diet (just over 400 μmol/L). A negative inter-allelic complementation could have a role, preventing better response [51]. In addition, IVS8 − 7 mutation was shown not to be BH 4 responsive in a functionally hemizygous patient, which is in accordance with a single previous report [48].…”

Variations in genotype-phenotype correlations in phenylketonuria patients

“…The R270K mutation gives low residual enzyme activity (2.1%; Leandro et al, 2006). Patients that were homozygotic for either of these two mutations had classic PKU in our study.…”

Tetrahydrobiopterin, its Mode of Action on Phenylalanine Hydroxylase, and Importance of Genotypes for Pharmacological Therapy of Phenylketonuria

“…However, most PKU patients are compound heterozygotes carrying different mutations and two‐hybrid analysis may allow the semi‐quantitative assessment of protein–protein interactions in living cells. Coexpression systems were reported with bacteria [Leandro et al., ; ], yeast and mammalian cells as host [Perez et al., ; Waters et al., ]. These systems allow the study of disease‐causing mutations under normal assembly of the tetrameric PAH.…”