LOC56985 discussion

Evolutionary analysis of LOC56985 suggest that this protein is inherited through vertical transmission, with only one one exception seen in Trypanosoma. The eukaryotic Trypanosoma was grouped in with the bacteria and still retained a relatively high bootstrap value of 75%. Further analysis of the Trypanosoma protein sequence showed that it's protien is has a considerably larger sequenece than all others. This finding may lead one to hypothesise about a possible insertion of the protein within another gene in Trypanosoma through means of lateral transmission.

Clustal alignment also showed various amino acid residues that are conserved throughout all species, suggesting an important role for these residues in the structure or function of the protein. (thats my bit on evolution for now, might add to it later.)

this is just a part of the first draft, i'll be putting more up soon. just post what you got here, i'll get rid of the rendundat bits and combine the whole thing

Our results provide a structural, phylogenetic, and biochemical basis for the functional annotation of the human gene C17orf48. These findings can also be extrapolated to the multitude of hypothetical metallophosphoesterase orthologs recently identified in mammalian and plant genomes. High-throughput gene expression data demonstrate highly controlled and tissue specific expression of LOC56985 orthologs, suggesting potential significance to disease states. However, there is minimal experimental evidence concerning hypothetical protein LOC56985 in the literature to ascertain cellular roles and substrate and metal ion specificities by bioinformatical analysis alone.

SCOP and Pfam analysis of the loc sequence identified a lone metallphosphoesterase domain and hence classified it as a member of the metallophosphoesterase superfamily. This is a relatively well characterized superfamily which counts paps, DNA double-strand break repair nucleases and protein serine/threonine phosphatases among its constituent families. The superfamily server failed to assign loc to any established SCOP family (no E-value <0.01 was obtained, closest match was PAPs, E-value = 0.016).

Comparison of 2nxf structure to all protein structures currently available via the PBD showed that it is most similar to paps. As mentioned above, both paps and loc orthlogs are metallophophoesterases and hence are likely exhibit significant similarities in topology, fold organization active-site organization. The biochemistry behind metal binding and phosphoesterase activity is identical among all members of the superfamily. Each family and subsequently each family member is unique in its metal ion and substrate specificities.

Whist Zn ions were used as ligands during crystallization of 2nxf for structural studies, the in vivo metal ions which ligand with 2nxf was unknown. It is likely that loc orthologs are specific for Mn (II), as shown in canoles et al., where the rat ortholog failed to activiate in the absence of Mn(II). The putative active centre of loc is within the cavity where PO4 lies bound to a pair of metal ions as shown in the crystal structure of 2nxf. Protein cavity prediction using CastP identified the PO4-bound cavity as the largest in loc. cavity residues which stabilize the metal ions and bind PO4 are also likely to be involved in the catalytic step. For example, His267 (part of the catalytically relevant GNHE region in all metallophosphoesterases) interacts with the oxygen of the phosphoanhydride linkage which is duly hydrolysed.

Substrate specificity should be accounted for by loc residues which interact with the non-phosphate regions of the substrate. It must be noted that the PO4 complexed to 2nxf is not a complete representation of the enzyme-substrate complex. It is rather a potential representation of PO4 bound to the enzyme subsequent to the catalytic step.

Homology searches using blastp and sequence comparison of homologs using clustalx2 show that while all PAPs and loc orthologs contain the GNH[D/E] pattern which identify them as metallophosphoesterases, there is relatively minimal conservation across the remainder of the sequence. In contrast, additional conserved patterns are present in all loc orthologs in both eukaryotes and prokaryotes. Likely prokaryotic precursors of loc orthologs were identified in alphaproteobacteria, green sulfur bacteria and bacteriodetes. Even though these prokaryotic homologs do not exhibit all the conserved patterns seen among vertebrates, plants and green algae, they still exhibit greater homology to loc than any paps. Loc ortholog expression is limited to vertebrates, plants and green algae, whereas, paps are a widespread metallophosphoesterase family with well characterized members widely distributed among eukaryotic phyla and a larger variety of prokaryotes than loc orthologs. The structural similarities seen beteween locs and paps indicate that both protein types likely originated from a gene duplication event of a common ancestor. however the relatively poor sequence similarity observed between locs and paps, differing metal ion specificities, and poor HMM matches indicate that loc orthologs cannot be defined as typical paps.

Until very recently, there was no experimental documentation of the celllular role, and substrate and metal ion specificities of loc orthologs. the only information available to date is from a recently submitted, unreviewed paper which characterises the product of the orthologous rat gene A9Y0H8. This protein has been identified as a Mn(II)-dependant ADP-ribose/ CDP-alcohol pyrophosphatase (ADPRibase-Mn). ADP-ribose is a potent intracellular regulator of ion channel activity. Until now, it was assumed that Mg2+ dependant hydrolases in Nudix superfamily, particularly ADPRibase-I and ADPRibase-II, regulate ADP-Ribose concentration in all mammalian cells. Loc orthologs show no sequence or structural similarities to Nudix hydrolases. ADPRibase-Mn was isolated from rat liver supernatants after seperation from Nudix hydrolases devoid of CDP-alcohol pyrophosphatase activity. In fact, ADPRibase-Mn is the only known hydrolase which can catalyse hydrolysis of ADP-Ribose as well as CDP-alcohol. Interestingly, the crystal structure 2nxf exhibits a smaller cavity, distinct from the PO4 binding site, where two residues are involved in binding an ethanol molecule. However, sequence alignments fail show to show any conservation of these two residues in other organisms.

A microarray experiment (listed on the Gene Expression Omnibus (GEO) database, GEO dataset GDS2068), aimed at identifying mouse immune genes from 8734 genomic features showed that LOC56 ortholog Rik is among 360 which show preferential expression in the thymus, spleen, peripheral blood mononuclear cells, lymph nodes and in vitro activated T cells when compared to non-immune tissues. Unigene’s EST profile viewer showed that Rik is predominantly expressed in the thymus. However, expression levels in spleen samples were not as prominent as was observed in the GEO listed microarray. Additional evidence which suggest an immune specific role came from fractionation of tissue supernatants from rats to quantitate and compare expression and activity of Nudix hydrolases (ADPRibase-I & ADPRibase-II) and ADPRibase-Mn using northern blots and acitivity assays. ADPRibase-Mn expression was significantly higher in thymus and spleen than in non-immune tissues. ADPRibase acitivity was found to be 2.5-5 fold higher in thymus and spleen than liver and muscle, and 4-8 fold higher in splenocytes than in non-immune tissues. Nudix hydrolases did not exhibit an immune specific expression profile.

Furthermore, CDP-alcohol pyrophosphatase activity may have a role in the biosynthesis pathways of CDP-choline or CDP-ethanolamine. CDP-choline, an intermediate in the generation of phosphatidylcholine from choline, is currently used in the treatment of Alzheimer’s disease as pyschostimulant/ nootropic.

The structural homology results obtained from the Dali server showed strong homology between the protein studied here and purple acid phosphatases. Purple acid phosphatases have been characterised as enzymes that catalyse phosphate ester hydrolysis under acidic conditions, are ~35kDa in size and contain two Fe ions at their catalytic centres (Wilcox, 1996). Similar acid phosphatases found in kidney bean, sweet potato and other plants contain Zn and Fe, two Fe or Mn at their cores.

Although the physiological role of purple acid phosphatases in the cell in not known, some of the proposed biological functions include iron transport, generation of reactive oxygen species and bone resorption (Oddie, et al., 2000); plant functions may include phosphate metabolism and generation of reactive oxygen species (Leung, Teixeira, Guddat, Mitić, & Schenk, 2007). Their localizations in the lysosomes of osteoclasts and macrophages support an intracellular monophosphate function in animals (Doi, Antanaitis, & Aisen, 1988). What is interesting from the results obtained here is that the ortholog from Zebrafish does not contain the characteristic iron core. The structure shows two Zinc ions at the catalytic site with the phosphate ion ligand. This may be the first case where a purple acid phosphatase does not contain this iron core.

Abstract | Introduction | Results | Discussion | Conclusion | Method | References

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