Functional consequences of PRODH missense mutations

Abstract

PRODH maps to 22q11 in the region deleted in the velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS) and encodes proline oxidase (POX), a mitochondrial inner-membrane enzyme that catalyzes the first step in the proline degradation pathway. At least 16 PRODH missense mutations have been identified in studies of type I hyperprolinemia (HPI) and schizophrenia, 10 of which are present at polymorphic frequencies. The functional consequences of these missense mutations have been inferred by evolutionary conservation, but none have been tested directly. Here, we report the effects of these mutations on POX activity. We find that four alleles (R185Q, L289M, A455S, and A472T) result in mild (<30%), six (Q19P, A167V, R185W, D426N, V427M, and R431H) in moderate (30%-70%), and five (P406L, L441P, R453C, T466M, and Q521E) in severe (>70%) reduction in POX activity, whereas one (Q521R) increases POX activity. The POX encoded by one severe allele (T466M) shows in vitro responsiveness to high cofactor (flavin adenine dinucleotide) concentrations. Although there is limited information on plasma proline levels in individuals of known PRODH genotype, extant data suggest that severe hyperprolinemia (>800 microM) occurs in individuals with large deletions and/or PRODH missense mutations with the most-severe effect on function (L441P and R453C), whereas modest hyperprolinemia (300-500 microM) is associated with PRODH alleles with a moderate reduction in activity. Interestingly, three of the four alleles associated with or found in schizophrenia (V427M, L441P, and R453C) resulted in severe reduction of POX activity and hyperprolinemia. These observations plus the high degree of polymorphism at the PRODH locus are consistent with the hypothesis that reduction in POX function is a risk factor for schizophrenia.

Figure 1

Diagram of PRODH (upper panel) and ΨPRODH (lower panel). The exons, represented by the gray rectangles, are numbered and shown roughly to scale. The intronic sequence is represented by the black heavy line and is not to scale. The exonic location of the missense mutations, by exon, is shown above the rectangles. The translation start site in exon 2 is indicated by the right-angle arrow; the heavy dashed line below the rectangles in the upper panel indicates the ∼10-kb PRODH-specific PCR product. The deletion in ΨPRODH is indicated by the dashed lines in the lower panel.

Figure 2

Schematic representation of proline metabolic pathways. The rectangle represents a mitochondrion (see text for additional details).

Figure 3

Alignment of the predicted PRODH amino acid sequences from various eukaryotic species. Residues conserved across three or four of the sequences are indicated by a gray background, those conserved in all five eukaryotic examples by a black background. Also shown are the 40 residues of E. coli PutA669 that are within 5 Å of substrate and/or cofactor (Lee et al. 2003). These are spaced as they are in PutA669, but, for the sake of clarity, we have not shown the intervening residues. The shading scheme for the E. coli residues follows the convention for the eukaryotic sequences. The locations of the human missense mutations studied in this article are indicated at the top of the figure. The arrowhead indicates the predicted cleavage site for the mitochondrial targeting sequence. The overline indicates a possible leucine zipper motif. Hs=Homo sapiens, Mm=Mus musculus, Dm=Drosophila melanogaster, Ce=Caenorhabditis elegans, Sc=Saccharomyces cerevisiae, and Ec=E. coli.

Figure 4

Immunoblot analysis of POX and GFP in sonicates of cells transfected with the indicated recombinant pTracer construct. For each panel, POX is shown above, GFP below. Each lane contains 20 μg of crude cell sonicate. POX migrates as an ∼66-kDa protein.

Figure 5

Proline oxidase activity of PRODH alleles. For each transfected allele, the activity was normalized to transfection efficiency and was expressed as a percentage of the wild-type allele (see the “Material and Methods” section). The thin vertical lines indicate the range of mean activity measured in two-to-five independent transient transfection experiments. Bars without these vertical lines indicate the mean of a triplicate assay performed in one transfection experiment. For some alleles (L441P, R453C/T466M, and A472T), the value is from multiple experiments, but the variance is so narrow that it cannot be seen on this scale.

Figure 6

Structural models of the active site of human POX, as predicted from the solved structure of E. coli PutA669 (Lee et al. 2003). The location of residues altered in certain of the missense mutations is shown: A, T466M; B, Q521E; C, L441P. The white arrows indicate potential disruptive forces.