Abstract
Among the human diseases that result from chromosomal aberrations, a de novo deletion in chromosome 11p13 is clinically associated with a syndrome characterized by Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR). Not all genes in the deleted region have been characterized biochemically or functionally. We have recently identified the first Class III cyclic nucleotide phosphodiesterase, Rv0805, from Mycobacterium tuberculosis, which biochemically and structurally belongs to the superfamily of metallophosphoesterases. We performed a large scale bioinformatic analysis to identify orthologs of the Rv0805 protein and identified many eukaryotic genes that included the human 239FB gene present in the region deleted in the WAGR syndrome. We report here the first detailed biochemical characterization of the rat 239FB protein and show that it possesses metallophosphodiesterase activity. Extensive mutational analysis identified residues that are involved in metal interaction at the binuclear metal center. Generation of a rat 239FB protein with a mutation corresponding to a single nucleotide polymorphism seen in human 239FB led to complete inactivation of the protein. A close ortholog of 239FB is found in adult tissues, and biochemical characterization of the 239AB protein demonstrated significant hydrolytic activity against 2',3'-cAMP, thus representing the first evidence for a Class III cyclic nucleotide phosphodiesterase in mammals. Highly conserved orthologs of the 239FB protein are found in Caenorhabditis elegans and Drosophila and, coupled with available evidence suggesting that 239FB is a tumor suppressor, indicate the important role this protein must play in diverse cellular events.
Highlights
Remain uncharacterized at the biochemical and functional level, and current annotation can provide a general prediction of the function of some proteins, understanding finer aspects of their regulation require individual analysis at a single gene level
Association of WT1 and PAX6 genes with Wilms’ tumor and aniridia (9 –12), respectively, suggested that the other genes in this region could be involved in the development or function of the central nervous system, and deletion of these genes could be responsible for the mental retardation seen in WAGR patients
The 239FB gene is present between the FSHB and PAX6 genes, and earlier studies have shown that the chromosomal locus of 239FB gene is present within a deletion interval that had been associated with the mental retardation phenotype of WAGR syndrome [19]. 239FB mRNA is predominantly expressed in the fetal brain [20], and the high degree of similarity between the predicted protein sequence of 239FB and two Caenorhabditis elegans cDNA clones showed an extensive evolutionary conservation of this protein [21]
Summary
Remain uncharacterized at the biochemical and functional level, and current annotation can provide a general prediction of the function of some proteins, understanding finer aspects of their regulation require individual analysis at a single gene level. Chromosome 11p13-14 has been extensively studied because of its association with various tumors, among them lung and bladder cancers [1, 2] as well as several developmental disorders such as the WAGR syndrome Extensive studies on this contiguous gene syndrome and association of 11p13 interstitial deletions with the WAGR syndrome suggest that this region contained a cluster of developmentally significant genes [6,7,8,9]. It is not readily possible to classify the MPEs as either monoesterases or diesterases by inspection of the sequences of the proteins
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