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Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases.

Leslie NR, McLennan AG, Safrany ST - BMC Biochem. (2002)

Bottom Line: Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10.We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2.These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap5A and Ap6A act as intracellular messengers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cell Signalling, School of Life Sciences, The University of Dundee, Dundee, DD1 5EH, UK. n.r.leslie@dundee.ac.uk

ABSTRACT

Background: The human genome contains at least 18 genes for Nudix hydrolase enzymes. Many have similar functions to one another. In order to understand their roles in cell physiology, these proteins must be characterised.

Results: We have characterised two novel human gene products, hAps1, encoded by the NUDT11 gene, and hAps2, encoded by the NUDT10 gene. These cytoplasmic proteins are members of the DIPP subfamily of Nudix hydrolases, and differ from each other by a single amino acid. Both metabolise diadenosine-polyphosphates and, weakly, diphosphoinositol polyphosphates. An apparent polymorphism of hAps1 has also been identified, which leads to the point mutation S39N. This has also been characterised. The favoured nucleotides were diadenosine 5',5"'-pentaphosphate (kcat/Km = 11, 8 and 16 x 10(3) M(-1) x s(-1) respectively for hAps1, hAps1-39N and hAps2) and diadenosine 5',5"'-hexaphosphate (kcat/Km = 13, 14 and 11 x 10(3) M(-1) x s(-1) respectively for hAps1, hAps1-39N and hAps2). Both hAps1 and hAps2 had pH optima of 8.5 and an absolute requirement for divalent cations, with manganese (II) being favoured. Magnesium was not able to activate the enzymes. Therefore, these enzymes could be acutely regulated by manganese fluxes within the cell.

Conclusions: Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10. We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2. These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap5A and Ap6A act as intracellular messengers.

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Comparison of hAps1 with translated EST sequences from mouse and cow. No full-length bovine EST for the hAps1 homologue is available, hence the composite of two separate sequences is shown. A predicted rat protein from genomic sequencing is also shown.
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Figure 3: Comparison of hAps1 with translated EST sequences from mouse and cow. No full-length bovine EST for the hAps1 homologue is available, hence the composite of two separate sequences is shown. A predicted rat protein from genomic sequencing is also shown.

Mentions: An initial BLAST search of the GenBank expressed sequence tag (EST) database with the hDIPP-1 sequence identified two closely related but previously uncharacterised predicted proteins, which we have called hAps1 and hAps2 (human ApsixA hydrolases 1 and 2). Alignment of the cDNA sequences with the human genome indicated that the genomic sequences encoding these proteins, FLJ10628 and LOC139770, lie about 150 kb apart on the X chromosome at Xp11.23 and are transcribed in opposite directions (Fig. 1). In accordance with the guidelines for the Nudix protein family, the genes for hAps1 and hAps2 have been designated NUDT11 and NUDT10 respectively by the HUGO Gene Nomenclature Committee (see ). These proteins are each 164 amino acids long and are most similar to hDIPP-2β, hAps1 showing 76, 90 and 91% identity at the amino acid level to hDIPP-1, 2α and 2β, respectively. In particular, they both possess the additional Gln residue (Q86 in DIPP-2β) that distinguishes hDIPP-2β from hDIPP-2α and hDIPP-1 and which is responsible for the reduced activity of hDIPP-2β towards diphosphoinositol polyphosphates compared to the other DIPPs. hAps1 and hAps2 are also identical to the hDIPP-2 enzymes throughout the Nudix motif (Fig. 2). These observations led us to anticipate that hAps1 and hAps2 would be potent dinucleoside polyphosphate hydrolases with reduced activity towards diphosphoinositol polyphosphates. The existence of two such similar expressed gene products was surprising. Comparison of the open reading frames (ORFs) of hAps1 and hAps2 revealed 6 differences at the nucleotide level, leading to five silent changes (T8, E28, E69, D88 and stop), and an Arg-Pro substitution at position 89. A comparison of the cDNAs for the two gene products showed further divergence in the 3' untranslated region. It appears that the evolution of these closely related genes occurred recently, as hAps1 and hAps2 show greater similarity with each other than with the Aps-like sequences from mouse (of which two genes, XM_135784 and XM_135786 appear to yield the same protein product) or cow (Fig 3). Further examination of all available hAps1 ESTs revealed a sequence divergence, leading to a point mutation S39N, in the coding region. This is present in two ESTs (Table 1) and was detected in 10% (3 from 30) of polymerase chain reaction (PCR) products generated from a range of normal human cDNA samples from diverse sources. In all other respects investigated, including the 3' untranslated region, it appeared to be identical to hAps1. This would suggest that this minor variant, hAps1-39N, represents a polymorphism present in the human population. Its properties were also examined in this study.


Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases.

Leslie NR, McLennan AG, Safrany ST - BMC Biochem. (2002)

Comparison of hAps1 with translated EST sequences from mouse and cow. No full-length bovine EST for the hAps1 homologue is available, hence the composite of two separate sequences is shown. A predicted rat protein from genomic sequencing is also shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117780&req=5

Figure 3: Comparison of hAps1 with translated EST sequences from mouse and cow. No full-length bovine EST for the hAps1 homologue is available, hence the composite of two separate sequences is shown. A predicted rat protein from genomic sequencing is also shown.
Mentions: An initial BLAST search of the GenBank expressed sequence tag (EST) database with the hDIPP-1 sequence identified two closely related but previously uncharacterised predicted proteins, which we have called hAps1 and hAps2 (human ApsixA hydrolases 1 and 2). Alignment of the cDNA sequences with the human genome indicated that the genomic sequences encoding these proteins, FLJ10628 and LOC139770, lie about 150 kb apart on the X chromosome at Xp11.23 and are transcribed in opposite directions (Fig. 1). In accordance with the guidelines for the Nudix protein family, the genes for hAps1 and hAps2 have been designated NUDT11 and NUDT10 respectively by the HUGO Gene Nomenclature Committee (see ). These proteins are each 164 amino acids long and are most similar to hDIPP-2β, hAps1 showing 76, 90 and 91% identity at the amino acid level to hDIPP-1, 2α and 2β, respectively. In particular, they both possess the additional Gln residue (Q86 in DIPP-2β) that distinguishes hDIPP-2β from hDIPP-2α and hDIPP-1 and which is responsible for the reduced activity of hDIPP-2β towards diphosphoinositol polyphosphates compared to the other DIPPs. hAps1 and hAps2 are also identical to the hDIPP-2 enzymes throughout the Nudix motif (Fig. 2). These observations led us to anticipate that hAps1 and hAps2 would be potent dinucleoside polyphosphate hydrolases with reduced activity towards diphosphoinositol polyphosphates. The existence of two such similar expressed gene products was surprising. Comparison of the open reading frames (ORFs) of hAps1 and hAps2 revealed 6 differences at the nucleotide level, leading to five silent changes (T8, E28, E69, D88 and stop), and an Arg-Pro substitution at position 89. A comparison of the cDNAs for the two gene products showed further divergence in the 3' untranslated region. It appears that the evolution of these closely related genes occurred recently, as hAps1 and hAps2 show greater similarity with each other than with the Aps-like sequences from mouse (of which two genes, XM_135784 and XM_135786 appear to yield the same protein product) or cow (Fig 3). Further examination of all available hAps1 ESTs revealed a sequence divergence, leading to a point mutation S39N, in the coding region. This is present in two ESTs (Table 1) and was detected in 10% (3 from 30) of polymerase chain reaction (PCR) products generated from a range of normal human cDNA samples from diverse sources. In all other respects investigated, including the 3' untranslated region, it appeared to be identical to hAps1. This would suggest that this minor variant, hAps1-39N, represents a polymorphism present in the human population. Its properties were also examined in this study.

Bottom Line: Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10.We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2.These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap5A and Ap6A act as intracellular messengers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cell Signalling, School of Life Sciences, The University of Dundee, Dundee, DD1 5EH, UK. n.r.leslie@dundee.ac.uk

ABSTRACT

Background: The human genome contains at least 18 genes for Nudix hydrolase enzymes. Many have similar functions to one another. In order to understand their roles in cell physiology, these proteins must be characterised.

Results: We have characterised two novel human gene products, hAps1, encoded by the NUDT11 gene, and hAps2, encoded by the NUDT10 gene. These cytoplasmic proteins are members of the DIPP subfamily of Nudix hydrolases, and differ from each other by a single amino acid. Both metabolise diadenosine-polyphosphates and, weakly, diphosphoinositol polyphosphates. An apparent polymorphism of hAps1 has also been identified, which leads to the point mutation S39N. This has also been characterised. The favoured nucleotides were diadenosine 5',5"'-pentaphosphate (kcat/Km = 11, 8 and 16 x 10(3) M(-1) x s(-1) respectively for hAps1, hAps1-39N and hAps2) and diadenosine 5',5"'-hexaphosphate (kcat/Km = 13, 14 and 11 x 10(3) M(-1) x s(-1) respectively for hAps1, hAps1-39N and hAps2). Both hAps1 and hAps2 had pH optima of 8.5 and an absolute requirement for divalent cations, with manganese (II) being favoured. Magnesium was not able to activate the enzymes. Therefore, these enzymes could be acutely regulated by manganese fluxes within the cell.

Conclusions: Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10. We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2. These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap5A and Ap6A act as intracellular messengers.

Show MeSH