First apyrase splice variants have different enzymatic properties

LALP70 is a novel lysosomal membrane protein belonging to the apyrase prote in family. The apyrase protein family comprises enzymes capable of cleaving nucleotidetri- and diphosphates in a calcium- or magnesium-dependent manne r, not being altered by P-type, F-type, or V-type NTPase inhibitors. In thi s study we have cloned and sequenced the human LALP70 gene to deter mine th e genomic structure. The gene is organized in 11 introns and 12 exons cover ing a genomic region of approximately 16 kilobase pairs. By fluorescence in , situ hybridization analysis, the hLALP70 gene was mapped to the human chr omosome 8p21.1-p21.3. We further show that there is at least one alternativ ely spliced variant, hLALP70v, which can be generated via an alternative sp lice side at the 3'-end of exon 7, leading to a protein variant differing i n 8 amino acids (VSFASSQQ). This is the first splice variant that has been described in the apyrase protein family. Reverse transcriptase polymerase c hain reaction analysis showed an ubiquitous expression of both variants, wi th different relative mRNA expression levels in different tissues. Comparis on of the enzymatic properties of the splice variants revealed a broader su bstrate specificity for hLALP70v with CTP, UDP, CDP, GTP, and GDP as prefer red substrates, while hLALP70 utilized UTP and TTP preferentially. Furtherm ore, enzyme activity of hLALP70v was equally dependent on Ca2+ and Mg2+, be ing saturated already at 1 mM concentration. In contrast, hLALP70 enzymatic activity were unsaturated up to 10 mM Ca2+, while Mg2+ showed a saturation at already 1 mhz concentration with 2-3-fold lower enzymatic activity as o bserved with Ca2+. Our data suggest that the presence or absence of the 8-a mino acid motif VSFASSQQ provoke differences in substrate specificity and d ivalent cation dependence of hLALP70/hLALP70v.