Functional characterization of ecto-5′-nucleotidases and apyrases in Drosophila melanogaster

Ecto-5′-nucleotidases are glycosyl phosphatidylinositol (GPI)-linked membrane-bound glycoproteins that convert extracellular AMP to adenosine. They play important roles in the inflammatory response where they modulate levels of pro-inflammatory extracellular ATP and anti-inflammatory extracellular adenosine. They are found in the saliva of blood feeding insects and also have a role in male reproduction. Drosophila possesses five genes with eight alternative transcripts encoding proteins with sequence homology to mammalian ecto-5′-nucleotidases. Here we show that two of them – NT5E-1 (CG4827) and NT5E-2 (CG30104) – are GPI-linked proteins with ecto-5′-nucleotidase activity but that they can also be released from the GPI anchor and exhibit secreted 5′-nucleotidase activity in growth media. The third locus in the cluster, CG30103, most likely also encodes a GPI-anchored membrane-bound protein but without 5′-nucleotidase activity, possibly due to the numerous substitutions in the amino acid sequence. Together with NT5E-2, CG30103 is also expressed in the testis offering an interesting model to investigate ecto-5′-nucleotidase enzymatic and extra-enzymatic function in male reproduction. CG42249 locus encoding two alternative transcripts is sequentially similar to family of apyrases related to 5′-nucleotidases and we show here that together with CG5276 belonging to another family of calcium-activated nucleotidases function as apyrases converting extracellular ATP to ADP and AMP. The last locus, CG11883, encodes most likely a cytoplasmic/mitochondrial protein.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (47 K)Download as PowerPoint slideHighlights► Ecto-5′-nucleotidases are GPI-linked membrane-bound glycoproteins converting extracellular AMP to adenosine. ► Drosophila possesses five genes encoding proteins with sequence homology to mammalian ecto-5′-nucleotidases. ► Two genes – NT5E-1 and NT5E-2 – encode GPI-linked active ecto-5′-nucleotidases. ► CG30103 might play non-enzymatic role in male reproduction. ► CG42249 encodes an apyrase belonging to ecto-5′-nucleotidase family.