Substrate-specifying determinants of the nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2

Abstract

The nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2/autotaxin are structurally related eukaryotic ecto-enzymes, but display a very different substrate specificity. NPP1 releases nucleoside 5'-monophosphates from various nucleotides, whereas NPP2 mainly functions as a lysophospholipase D. We have used a domain-swapping approach to map substrate-specifying determinants of NPP1 and NPP2. The catalytic domain of NPP1 fused to the N- and C-terminal domains of NPP2 was hyperactive as a nucleotide phosphodiesterase, but did not show any lysophospholipase D activity. In contrast, chimaeras of the catalytic domain of NPP2 and the N- and/or C-terminal domains of NPP1 were completely inactive. These data indicate that the catalytic domain as well as both extremities of NPP2 contain lysophospholipid-specifying sequences. Within the catalytic domain of NPP1 and NPP2, we have mapped residues close to the catalytic site that determine the activities towards nucleotides and lysophospholipids. We also show that the conserved Gly/Phe-Xaa-Gly-Xaa-Xaa-Gly (G/FXGXXG) motif near the catalytic site is required for metal binding, but is not involved in substrate-specification. Our data suggest that the distinct activities of NPP1 and NPP2 stem from multiple differences throughout the polypeptide chain.

Figure 1. Expression of chimaeras of NPP1 and NPP2

(A) Domain structure of NPP1 and NPP2. The vertical black lines indicate the position where silent sites were introduced for domain-swapping. Also indicated is the position of the catalytic-site threonine (arrowhead). (B) Western blot analysis with anti-myc antibodies of immunoprecipitates of NPP (chimaeras). HA- and myc-tagged NPP1, NPP2 and the indicated chimaeras were expressed in COS-1 cells. NPP1-1-1 (wt NPP1), NPP1-1-2, NPP1-2-2 and NPP1-2-1 were immunoprecipitated with anti-HA antibodies from the cell lysates, whereas NPP2-2-2 (wt NPP2), NPP2-1-1, NPP2-1-2 and NPP2-1-1 were immunoprecipitated from the culture medium with anti-myc antibodies. The immunoprecipitated proteins were used at the same molar concentrations for the enzymic assays shown in Figure 2.

Figure 2. Effect of domain swapping on enzymic activities of NPP1 and NPP2

Immunoprecipitated NPP1, NPP2 and the NPP chimaeras were assayed at equal molar concentrations, as detected by immunoblotting (Figure 1B). The enzymes were assayed for nucleotide phosphodiesterase activities with pNP-TMP as a substrate (A) and for lysophospholipase D activities with lysophosphatidylcholine as a substrate (B). Data are means±S.E.M. (n=3–7).

Figure 3. Conservation of residues near the catalytic site of NPP1 and NPP2

(A) Alignments of the catalytic site of NPP1 and NPP2 for the indicated species. The residues that are conserved in both NPP1 and NPP2 are in bold. The catalytic-site threonine is underlined and the conserved isoform-specific residues are boxed. (B) Alignments of the G/FXGXXG motif, which was predicted to be close to the catalytic site [2]. The residues that are conserved in both NPP1 and NPP2 are in bold and the first residue of G/FXGXXG motif is boxed. Dr, Danio rerio; Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Xl, Xenopus laevis. GenBank® accession numbers are P22413 (HsNPP1), P06802 (MmNPP1), BX293547.6 (DrNPP1), Q13822 (HsNPP2), Q64610 (RnNPP2) and AAH44675 (XlNPP2).

Figure 4. Mutagenesis of residues in the catalytic site of NPP1

The indicated mutants of NPP1 were expressed in COS-1 cells and immunoprecipitated from the cell lysates with anti-HA antibodies. Immunoprecipitated NPP1 variants were assayed at the same protein concentration, as detected by immunoblot analysis (A) and were assayed for nucleotide phosphodiesterase activities with pNP-TMP as a substrate (B). Data are means±S.E.M. (n=3).

Figure 5. Mutagenesis of residues in the catalytic site of NPP2

The indicated mutants of NPP2 were expressed in COS-1 cells and immunoprecipitated with anti-myc antibodies from the culture medium. The immunoprecipitated NPP2 variants were diluted to a similar molar concentration, as detected by immunoblot analysis (A) and assayed for nucleotide phosphodiesterase activities with pNP-TMP as a substrate (B, upper panel), and for lysophospholipase D activities with lysophosphatidylcholine as a substrate (B, lower panel). Data are means±S.E.M. (n=3).

Figure 6. GXGXXG motif is essential for catalysis by NPP1

The indicated mutants of NPP1 were expressed in COS-1 cells and immunoprecipitated from the cell lysates with anti-HA antibodies. The immunoprecipitated NPP1 variants were diluted to the same molar concentration, as detected by immunoblot analysis (A) and assayed for nucleotide phosphodiesterase activities with pNP-TMP as a substrate (B). Data are means±S.E.M. (n=3).

Figure 7. The FXGXXG motif is essential for catalysis by NPP2

The indicated mutants of NPP2 were expressed in COS-1 cells and immunoprecipitated with anti-myc antibodies from the culture medium. The immunoprecipitated NPP2 variants were diluted to a similar molar concentration, as detected by immunoblot analysis (A) and assayed for nucleotide phosphodiesterase activities with pNP-TMP as a substrate (B, upper panel) and for lysophospholipase D activities with lysophosphatidylcholine as a substrate (B, lower panel). Data are means±S.E.M. (n=3).

Figure 8. Partial recovery of the activity of a glycine motif mutant of NPP1 by ZnCl₂

(A) The nucleotide pyrophosphatase activities of immunoprecipitated NPP1-wt and NPP1-G513A/G515A/G518A were measured with [γ-³²P]ATP as substrate, in the absence or presence of ZnCl₂, as indicated. The control refers to an incubation without added enzyme. The Figure shows an autoradiogram after separation of ATP and PP_i by TLC. (B) The nucleotide phosphodiesterase activity of immunoprecipitated NPP1-wt (○) and NPP1-G513A/G515A/G518A (•) was measured with pNP-TMP as substrate in the presence of the indicated concentrations of ZnCl₂. The activities were expressed as a percentage of the activities at 1 mM ZnCl₂ (logarithmic scale). The activity of NPP1-G513A/G515A/G518A at 1 mM ZnCl₂ amounted to 27% of the activity of NPP1-wt under the same conditions.