Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that release nucleoside 5-monophosphate from a variety of nucleotides and nucleotide derivatives. The mammalian NPP family comprises seven members, but only three of these (NPP1-3) have been studied in some detail. Previously we showed that lysophospholipase D, which hydrolyzes lysophosphatidylcholine (LPC) to produce lysophosphatidic acid, is identical to NPP2. More recently an uncharacterized novel NPP member (NPP7) was shown to have alkaline sphingomyelinase activity. These findings raised the possibility that other members of the NPP family act on phospholipids. Here we show that the sixth member of the NPP family, NPP6, is a choline-specific glycerophosphodiester phosphodiesterase. The sequence of NPP6 encodes a transmembrane protein containing an NPP domain with significant homology to NPP4, NPP5, and NPP7/ alkaline sphingomyelinase. When expressed in HeLa cells, NPP6 was detected in both the cells and the cell culture medium as judged by Western blotting and by enzymatic activity. Recombinant NPP6 efficiently hydrolyzed the classical substrate for phospholipase C, p-nitrophenyl phosphorylcholine, but not the classical nucleotide phosphodiesterase substrate, p-nitrophenyl thymidine 5-monophosphate. In addition, NPP6 hydrolyzed LPC to form monoacylglycerol and phosphorylcholine but not lysophosphatidic acid, showing it has a lysophospholipase C activity. NPP6 showed a preference for LPC with short (12:0 and 14:0) or polyunsaturated (18:2 and 20:4) fatty acids. It also hydrolyzed glycerophosphorylcholine and sphingosylphosphorylcholine efficiently. In mice, NPP6 mRNA was predominantly detected in kidney with a lesser expression in brain and heart, and in human it was detected in kidney and brain. The present results suggest that NPP6 has a specific role through the hydrolysis of polyunsaturated LPC, glycerophosphorylcholine, or sphingosylphosphorylcholine in these organs.Nucleotide pyrophosphatases/phosphodiesterases (NPPs) 1 are ubiquitous membrane-associated or secreted ectoenzymes that have a role in regulating extracellular nucleotide metabolism (1, 2). They act by hydrolyzing a variety of nucleotides and nucleotide derivatives such as ATP and ADP. The mammalian NPP family has had five members (NPP1-5) (1, 2) that fall within two subgroups. NPP1-3 are type II transmembrane glycoproteins (ϳ900 amino acids) that have similar modular structures composed of a short amino-terminal intracellular domain, a single transmembrane domain, two somatomedin B-like motifs, a conserved catalytic site, a nuclease-like sequence, and a putative carboxyl-terminal "EF-hand" motif (Fig. 1D). In contrast, NPP4 (1, 2) and NPP5 (1-3) have a shorter structure (ϳ450 amino acids), a predicted type I transmembrane orientation, a short intracellular carboxyl-terminal domain, and a conserved catalytic site. The extracellular domains of NPP4 and NPP5 contain only a phosphodiesterase motif (Fig. 1D). Recently two additional ...

Infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7, which causes diarrhea and hemorrhagic colitis in humans, often results in fatal systemic complications, such as neurological damage and hemolytic-uremic syndrome. Because Stx circulating in the blood is a major causative factor of these complications, the development of a Stx neutralizer that functions in the circulation holds promise as a viable therapy. Here we developed a series of carbosilane dendrimers, in which trisaccharides of globotriaosyl ceramide, a receptor for Stx, were variously oriented at their termini (referred to as SUPER TWIG), and identified a SUPER TWIG with six trisaccharides as a Stx neutralizer functioning in the circulation. This SUPER TWIG specifically bound to Stx with high affinity (Kd ‫؍‬ 1.1 ؋ 10 ؊6 M) and inhibited the incorporation of the toxin into target cells. Intravenous administration of the SUPER TWIG along with Stx to mice substantially reduced the fatal brain damage and completely suppressed the lethal effect of Stx. Moreover, the SUPER TWIG protected mice from challenge with a fatal dose of E. coli O157:H7, even when administered after the establishment of the infection. The SUPER TWIG neutralized Stx in vivo by a mechanism in which the accumulation and immediate degradation of Stx by phagocytic macrophages present in the reticuloendothelial system were induced. Taken together, our findings indicate that this SUPER TWIG is therapeutic agent against infections by Stx-producing E. coli.

Adipocyte-derived leucine aminopeptidase (A-LAP) is a recently identified novel member of the M1 family of zinc-metallopeptidases. Transfection of the A-LAP cDNA into COS-7 cells resulted in the secretion of the enzyme. In this study, recombinant A-LAP was expressed in Chinese hamster ovary cells, purified to homogeneity and its enzymatic properties were characterized. The purified enzyme was active towards a synthetic substrate, L-leucyl-p-nitroanilide, yielding a V(max) of 3.55 micromol/min/mg and a K(m) of 1.28 mM, and was shown to be a monomeric protein with molecular mass of 120 kDa in solution. By monitoring the sequential N-terminal amino acid liberation, it was found that the enzyme hydrolyzes a variety of bioactive peptides, including angiotensin II and kallidin. Immunohistochemical analysis indicated that the enzyme is expressed in the cortex of the human kidney, where tissue kallikrein is localized. Taken together, these results indicate that A-LAP possesses a broad substrate specificity towards naturally occurring peptide hormones and suggest that it plays a role in the regulation of blood pressure through the inactivation of angiotensin II and/or the generation of bradykinin in the kidney.

Shiga toxin (Stx) is a major virulence factor of Stx-producing Escherichia coli. Recently, we developed a therapeutic Stx neutralizer with 6 trisaccharides of globotriaosyl ceramide, a receptor for Stx, in its dendrimer structure (referred to as "SUPER TWIG [1]6") to function in the circulation. Here, we determined the optimal structure of SUPER TWIG for it to function in the circulation and identified a SUPER TWIG with 18 trisaccharides, SUPER TWIG (2)18, as another potent Stx neutralizer. SUPER TWIGs (1)6 and (2)18 shared a structural similarity, a dumbbell shape in which 2 clusters of trisaccharides were connected via a linkage with a hydrophobic chain. The dumbbell shape was found to be required for formation of a complex with Stx that enables efficient uptake and degradation of Stx by macrophages and, consequently, for potent Stx-neutralizing activity in the circulation. We also determined the binding site of the SUPER TWIGs on Stx.