Synaptotagmins (Syts) are transmembrane proteins with two Ca2+-binding C2 domains in their cytosolic region. Syt I, the most widely studied isoform, has been proposed to function as a Ca2+ sensor in synaptic vesicle exocytosis. Several of the twelve known Syts are expressed primarily in brain, while a few are ubiquitous (Sudhof, T.C., and J. Rizo. 1996. Neuron. 17: 379–388; Butz, S., R. Fernandez-Chacon, F. Schmitz, R. Jahn, and T.C. Sudhof. 1999. J. Biol. Chem. 274:18290–18296). The ubiquitously expressed Syt VII binds syntaxin at free Ca2+ concentrations ([Ca2+]) below 10 μM, whereas other isoforms require 200–500 μM [Ca2+] or show no Ca2+-dependent syntaxin binding (Li, C., B. Ullrich, Z. Zhang, R.G.W. Anderson, N. Brose, and T.C. Sudhof. 1995. Nature. 375:594–599). We investigated the involvement of Syt VII in the exocytosis of lysosomes, which is triggered in several cell types at 1–5 μM [Ca2+] (Rodríguez, A., P. Webster, J. Ortego, and N.W. Andrews. 1997. J. Cell Biol. 137:93–104). Here, we show that Syt VII is localized on dense lysosomes in normal rat kidney (NRK) fibroblasts, and that GFP-tagged Syt VII is targeted to lysosomes after transfection. Recombinant fragments containing the C2A domain of Syt VII inhibit Ca2+-triggered secretion of β-hexosaminidase and surface translocation of Lgp120, whereas the C2A domain of the neuronal- specific isoform, Syt I, has no effect. Antibodies against the Syt VII C2A domain are also inhibitory in both assays, indicating that Syt VII plays a key role in the regulation of Ca2+-dependent lysosome exocytosis.

Trypanosoma brucei contains a soluble serine oligopeptidase (OP-Tb) that is released into the host bloodstream during infection, where it has been postulated to participate in the pathogenesis of African trypanosomiasis. Here, we report the identification of a single copy gene encoding the T. brucei oligopeptidase and a homologue from the related trypanosomatid pathogen Leishmania major. The enzymes encoded by these genes belong to an emerging subgroup of the prolyl oligopeptidase family of serine hydrolases, referred to as oligopeptidase B. The trypanosomatid oligopeptidases share 70% amino acid sequence identity with oligopeptidase B from the intracellular pathogen Trypanosoma cruzi, which has a demonstrated role in mammalian host cell signaling and invasion. OP-Tb exhibited no activity toward the prolyl oligopeptidase substrate H-Gly-Pro-7-amido-4-methylcoumarin. Instead, it had activity toward substrates of trypsin-like enzymes, particularly those that have basic amino acids in both P 1 and P 2 (e.g. benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin k cat /K m ‫؍‬ 529 s ؊1 M ؊1). The activity of OP-Tb was enhanced by reducing agents and by polyamines, suggesting that these agents may act as in vivo regulators of OP-Tb activity. This study provides the basis of the characterization of a novel subgroup of serine oligopeptidases from kinetoplastid protozoa with potential roles in pathogenesis.

Aminopeptidases are emerging as exciting novel drug targets and vaccine candidates in parasitic infections. In this study, we describe for the first time an aminopeptidase from three highly pathogenic Leishmania species. Intronless genes encoding a leucyl aminopeptidase (lap) were cloned from Leishmania amazonensis, Leishmania donovani, and Leishmania major, which encoded 60-kDa proteins that displayed homology to leucyl aminopeptidases from Gram-negative bacteria, plants, and mammals. The lap genes were present as a single copy in each genome, and lap mRNA was detected by reverse transcription-PCR in all life-cycle stages of L. amazonensis. Lap assembled into catalytically competent 360-kDa hexamers and demonstrated potent amidolytic activity against synthetic aminopeptidase substrates containing leucine, methionine, and cysteine residues, representing the most restricted substrate specificity of any leucyl aminopeptidase described to date. Optimal activity was observed against L-leucyl-7-amido-4-methylcoumarin (k cat /K m Ϸ 63 s ؊1 ⅐mM ؊1 ) with a pH optimum of 8.5. Leishmania Lap activity was inhibited by metal ion chelators and enhanced by divalent manganese, cobalt, and nickel cations, although only zinc was detected in the purified Lap by inductively coupled plasma atomic emission spectroscopy, indicating that zinc is the natural Lap cofactor. Activity was potently inhibited by bestatin and apstatin in a slow binding competitive fashion, with K i * values of 3 and 44 nM, respectively. Actinonin was a tight binding competitive inhibitor (K i Ϸ 1 nM), whereas arphamenine A (K i Ϸ 70 M) and Lleucinol (K i Ϸ 100 M) were non-tight binding competitive inhibitors. Lap was not secreted by Leishmania in vitro and was localized to the parasite cytosol.Protozoan parasites of the genus Leishmania cause visceral, cutaneous, and mucosal diseases in humans, collectively referred to as leishmaniasis. Leishmaniasis is prevalent in 88 countries, with 12 million people currently infected, a further 350 million at risk, and 2 million new cases reported per year.No vaccine exists, and therapies are inadequate (1). There is a pressing need for the identification of novel leishmanial virulence factors, drug targets, and vaccines to improve our understanding, prevention, and treatment of leishmaniasis.The peptidases of parasitic protozoans (for review, see Ref.2) are becoming increasingly important as virulence factors, drug targets, and vaccine candidates in parasitic infections. However, only three peptidases from Leishmania, lysosomal cysteine peptidases (3), a cell-surface metallopeptidase (4), and a cytosolic serine oligopeptidase (5) have received attention.Aminopeptidases, which catalyze the removal of N-terminal amino acid residues from peptides and proteins (6), are emerging as novel and exciting anti-parasite targets. Vaccination of sheep with Fasciola leucyl aminopeptidase (Lap) 1 elicited high anti-Lap titers that conferred protection against fascioliasis and fascioliasis-related liver damage (7). Synthetic broad-spec...

Lesions caused by Leishmania amazonensis normally heal, but relapses occur due to parasite persistence in host tissues. It has been proposed that infection of fibroblasts plays an important role in this process by providing the parasites with a safe haven in which to replicate. However, most previous studies have focused on the entry of Leishmania into macrophages, a process mediated by serum opsonins. To gain insight into a possible role of nonopsonic entry in the intracellular persistence of amastigotes, we examined the invasion of Chinese hamster ovary (CHO) cells. Amastigotes entered CHO cells by a cytochalasin D, genistein, wortmannin, and 2,3-butanedione monoxime-sensitive pathway and replicated within phagolysosomes. However, unlike most phagocytic processes described to date, amastigote internalization in CHO cells involved activation of the GTPases Rho and Cdc42 but not Rac-1. When uptake was mediated by fibronectin or when amastigotes were opsonized with immunoglobulin G and internalized by Fc receptor-expressing CHO cells, Rac-1 activation was restored and found to be required for parasite internalization. Given the essential role of Rac in assembly of the respiratory burst oxidase, invasion through this nonopsonic, Rac-1-independent pathway may play a central role in the intracellular survival of Leishmania in immune hosts.