Crystal structures of HER3 extracellular domain 4 in complex with the designed ankyrin-repeat protein D5.

The members of the human epidermal growth factor receptor (HER) family are among the most intensely studied oncological targets. HER3 (ErbB3), which had long been neglected, has emerged as a key oncogene, regulating the activity of other receptors and being involved in progression and tumor escape in multiple types of cancer. Designed ankyrin-repeat proteins (DARPins) serve as antibody mimetics that have proven to be useful in the clinic, in diagnostics and in research. DARPins have previously been selected against EGFR (HER1), HER2 and HER4. In particular, their combination into bivalent binders that separate or lock receptors in their inactive conformation has proved to be a promising strategy for the design of potent anticancer therapeutics. Here, the selection of DARPins targeting extracellular domain 4 of HER3 (HER3d4) is described. One of the selected DARPins, D5, in complex with HER3d4 crystallized in two closely related crystal forms that diffracted to 2.3 and 2.0 Šresolution, respectively. The DARPin D5 epitope comprises HER3d4 residues 568-577. These residues also contribute to interactions within the tethered (inactive) and extended (active) conformations of the extracellular domain of HER3.

Induction of Extracellular Aminopeptidase Production by Peptides in Some Marine Bacterial Species.

Bacterial extracellular aminopeptidases are key enzymes in protein processing in oligotrophic seawater. To the best of our knowledge, the regulation of aminopeptidase production in microbes inhabiting seawater has not yet been reported. The present study attempted to experimentally clarify which organic materials affect bacterial extracellular aminopeptidase production by nutrient-rich and starved cells growing in artificial seawater using Photobacterium, Alteromonas, Ruegeria, and Sulfitobacter. In all four species, we found that peptides induced bacterial extracellular aminopeptidase production. Amino acids led to cell growth with markedly lower aminopeptidase production by Photobacterium and Sulfitobacter, but not by Alteromonas and Ruegeria. These results suggest that the extracellular aminopeptidases of marine bacteria are primarily produced on demand in response to the presence of relevant substrates (peptides) in seawater. Peptidyl substances may be regulatory nutrients for marine bacterial growth in aquatic environments.

Exosomal and extracellular HMGB1 have opposite effects on SASH1 expression in rat astrocytes and glioma C6 cells.

Exosomes are a type of extracellular vesicles derived from cells and mediators of intercellular communication. Different cell types have their own unique exosomes for exchanging information. We previously found that SASH1, a tumor suppressor, was lowly expressed or absent in glioma tissues and glioma C6 cells, but the structure and function of the corresponding exosomes had been unclear. Hence, we aimed to investigate whether exosomes generated from normal glial cells and glioma cells form different protein patterns and whether those derived from normal glial cells affect SASH1 expression in glioma cells. We collected exosomes from astrocytes and C6 cells and identified their exosomal proteins through mass spectrometry. We also performed gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses, whose results showed that both the total and unique exosomal proteins from each cell type were similar. Moreover, the KEGG analysis revealed different clusters of unique exosomal proteins in glial cells and glioma cells. In the normal glial cells, the top clusters were mainly involved in processes with RNA transcripts and proteins, whereas in glioma cells the clusters were attributed to PI3K-Akt signaling, cell adhesion, and cancer-related pathways. Western blot analysis showed that HMGB1 exists in exosomes derived from cultured astrocytes, although its expression was higher in glioma C6 cells. Furthermore, we found that exosomes extracted from astrocytes could increase SASH1 expression in C6 cells (P = 0.040), whereas those derived from HMGB1-depleted astrocytes could not (P = 0.6133). The expression levels of SASH1 decreased after the addition of extracellular recombinant HMGB1 protein, whereas that of TLR4 increased. Our study is the first to demonstrate that HMGB1 plays different roles depending on its form: as an extracellular protein, HMGB1 decreases SASH1 expression, but as an exosomal protein, HMGB1 increases SASH1 expression. Nevertheless, the mechanism, which partly depends on the TLR4 pathway, behind these opposing effects requires further study. Our novel findings on the structure-dependent roles of the cytokine HMGB1 in promoting or inhibiting cancer provide a fresh insight into the interactions of cancer cells with the microenvironment.

Germ cell-specific apoptosis by extracellular clusterin in cryptorchid dog testes.

Mammalian testes are maintained at a relatively lesser temperature than the abdominal region so that normal spermatogenesis can occur. Germ cell apoptosis has resulted in heat-damaged testes that occurs as a result of cryptorchidism, but the mechanism is not yet fully understood. To elucidate the cause of germ-cell death by cryptorchidism, cryptorchidism was surgically induced in dog testes and histological and molecular analyses were performed. Histological data indicated that the seminiferous tubules of cryptorchid testes and epididymis contained fewer germ cells. Total RNA sequencing was performed to screen for overexpressed genes in cryptorchid dog testes. Clusterin RNA was in greater abundance (approximately 12.8-fold) in cryptorchid testes than in normal testes. In addition, cleaved caspase-3 and -8 were detected in greater abundance in cryptorchid dog testes. Real time RT-PCR and western blotting analysis indicated there was a greater abundance of clusterin in cryptorchid dog testes. Furthermore, clusterin was detected in extracellular regions of cryptorchid dog testes during the 4 weeks after surgery. Thus, germ-cell specific apoptosis and expression of clusterin genes occur with a resulting presence of this protein in extracellular regions of cryptorchid dog testes. This result will facilitate further study of spermatogenesis and the specific mechanisms by which cryptorchidism results in male infertility.

Extracellular genomic biomarkers of osteoarthritis.

INTRODUCTION: Osteoarthritis (OA), a chronic, debilitating and degenerative disease of the joints, is the most common form of arthritis. The seriousness of this prevalent and chronic disease is often overlooked. Disease modifying OA drug development is hindered by the lack of soluble biomarkers to detect OA early. The objective of OA biomarker research is to identify early OA prior to the appearance of radiographic signs and the development of pain. Areas covered: This review has focused on extracellular genomic material that could serve as biomarkers of OA. Recent studies have examined the expression of extracellular genomic material such as miRNA, lncRNA, snoRNA, mRNA and cell-free DNA, which are aberrantly expressed in the body fluids of OA patients. Changes in genomic content of peripheral blood mononuclear cells in OA could also function as biomarkers of OA. Expert commentary: There is an unmet need for soluble biomarkers for detecting and then monitoring OA disease progression. Extracellular genomic material research may also reveal more about the underlying pathophysiology of OA. Minimally-invasive liquid biopsies such as synovial fluid and blood sampling of genomic material may be more sensitive over radiography in the detection, diagnosis and monitoring of OA in the future.

Splice variants of the extracellular region of RON receptor tyrosine kinase in lung cancer cell lines identified by PCR and sequencing.

BACKGROUND: Altered expression of receptor tyrosine kinases (RTKs) is a major driver of growth and metastasis of cancers. Recepteur d'origine nantais (RON) receptor is a single-pass transmembrane RTK aberrantly expressed in a number of cancers. Efforts to block deregulated RON signaling in tumors using small molecule kinase inhibitors or antibodies are complicated by the presence of unknown number/types of isoforms of RON, which, despite having similar sequences, are localized differently and mediate varied functions. The objective of this study was to identify splice variants of RON transcripts between exons 1 and 10 that code for the extracellular region. METHODS: Direct cDNA sequencing was performed for the transcript between exons 1-10 of RON by Sanger sequencing in various lung cancer cell lines. RESULTS: PCR amplification and bi-directional sequencing of cDNA for section between exons 1 and 10 from lung cancer cell lines revealed the presence of several splice variants of RON transcripts; the variants were formed by skipping of exons 2, 2-3, 5-6, 6 and 8-9. Each of these transcript variants were found in one or more cell lines. While the variants formed by skipping of exons 2, 2-3 and 5-6 resulted in loss of 63, 106 and 109 amino acids, respectively, and didn't cause reading-frameshift, the transcripts formed by skipping of exons 6 and 8-9 caused reading-frameshift. Splice variant lacking exons 8-9 was found in 13 out of 23 cell lines tested. CONCLUSION: Lung cancer cell lines contain several splice variants of RON which involve skipping of exons coding for extracellular region. Some of the splicing changes result in reading-frameshift and the N-terminally truncated isoforms are expected to be secreted out. The ubiquitous nature of alternative splicing events in RON suggests the need for isoform specific approaches to functional analysis and therapeutic targeting of RON.

Purification, characterization, and cloning of an extracellular laccase with potent dye decolorizing ability from white rot fungus Cerrena unicolor GSM-01.

A novel laccase was purified from fermentation broth of the white rot fungus Cerrena unicolor strain GSM-01 following three ion-exchange chromatography steps and one gel-filtration step. The purified enzyme was determined to be a monomeric protein of 63.2kDa and demonstrated high oxidation activity of 2.05×104U/mg towards ABTS. Its cDNA, gene, and amino acid sequences were obtained. It possessed high sequence similarity with that of other laccases but different enzymatic properties. It manifested optimal pH and temperature of 2.6 and 45°C, respectively. Fe3+ and Fe2+ were the most efficient inhibitors towards Cerrena unicolor laccase (CUL), while Mn2+ can slightly enhance the laccase activity of 3.8-10.5%. The Km and Vmax of CUL were estimated to 302.7µM and 13.6µMm-1, respectively. CUL was effective in the decolorization of bromothymol blue, evans blue, methyl orange, and malachite green with decolorization efficiencies of 50%-85%. It possesses potential application in textile and environmental industries.

Liquid Profiling in Lung Cancer - Quantification of Extracellular miRNAs in Bronchial Lavage.

Extracellular miRNAs cannot only be isolated from different body fluids like plasma and serum, but also from bronchial lavage samples (BL) obtained by bronchoscopy. Alterations in the expression of microRNAs might be useful for a discrimination of lung cancer patients from patients with a benign lung disease. We profiled extracellular microRNAs from three BL pools of lung cancer patients and three BL pools from a control group (patients with a benign lung disease) with TaqMan MicroRNA Array cards. For the confirmation of these results, we analyzed a panel of eight miRNAs in a qRT-PCR of the BL of 30 different lung cancer and non-cancerous patients. For the data normalization, we used exogenously added cel-miR-39 RNA. Using microRNA arrays, we found a panel of eight microRNAs (hsa-miR 19b-1, 1285, 1289, 1303, 217, 29a-5p, 548-3p, 650) that were differentially expressed between the lung cancer and the non-cancerous group. Further investigation by qPCR revealed five microRNAs (U6 snRNA, hsa-miR 1285, 1303, 29a-5p, 650) that were significantly up-regulated in patients with lung cancer. In bronchial lavage samples, the five microRNAs identified in this study may have a diagnostic potential to be used as biomarkers in lung cancer.

Academia Meets Industry.

Researchers working in industrial laboratories as well as in academic laboratories discussed topics related to the use of extracellular nucleic acids in different fields. These included areas like non-invasive prenatal diagnosis, the application of different methods for the analysis and characterization of patients with benign and malignant diseases and technical aspects associated with extracellular nucleic acids. In addition, the possibilities and chances for a cooperation of researchers working in different worlds, i.e. academia and industry, were discussed.

Extracellular DNA: A Bridge to Cancer.

DNase I is a secreted enzyme whose function has been presumed to control "waste management" in the human system, by degrading DNA that leaks from dead and dying cells. Emerging studies have instead yielded evidence that DNase I plays a central role in newly defined dynamics of immune and autoimmune diseases, as well as cancer and vascular disorders, including thrombosis. Cancer cells have been reported to be associated with distinctive extracellular structures that facilitate aggregation and implantation. The fact that DNA is a component of such structures and that it plays a role in cancer development is illustrated by direct evidence: DNase I added to tumor cells eliminates the structures and inhibits tumorigenicity of some cancer cell lines. DNase I injected into experimental animals, moreover, results in significant inhibition of metastasis. Despite independent observations of such phenomena in diverse cancers for over 50 years, the potential for using DNase I as a clinical tool to prevent or treat cancer remains unexplored. The discovery of neutrophil extracellular traps has yielded a conceptual framework for interpreting how extracellular DNA may function in cancer development and why it may prove to be an important clinical target in stopping cancer outside the cell.

Extracellular microRNAs in bronchoalveolar lavage samples from patients with lung diseases as predictors for lung cancer.

OBJECTIVE: The detection of tumor-associated microRNA expression in bronchial lavage (BL) samples of lung cancer patients could improve the non-invasive tumor diagnostic. METHODS: The profile of extracellular microRNAs in bronchial lavage was evaluated using three pools for lung cancer group (malignant) and three pools for non-cancerous group (benign) of 10 patients each. To confirm the results for the selected microRNAs in a qRT-PCR the BL of 30 different lung cancer and non-cancerous patients was used. We examined total-RNA from cell-free supernatant of BL samples. For normalization we added exogenous cel-miR 39. RESULTS: Using microRNA arrays we found a panel of eight microRNAs (hsa-miR 19b-1, 1285, 1289, 1303, 217, 29a-5p, 548-3p, 650) that were differentially expressed between the lung cancer and the non-cancerous group. Further investigation by qPCR revealed five microRNAs (U6 snRNA, hsa-miR 1285, 1303, 29a-5p, 650) that were significantly up-regulated in patients with lung cancer. CONCLUSIONS: In bronchial lavage samples the five microRNAs identified in this study have a diagnostic potential for use as noninvasive biomarkers in lung cancer.

Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines.

Intercellular communication can be mediated by extracellular small regulatory RNAs (sRNAs). Circulating sRNAs are being intensively studied for their promising use as minimally invasive disease biomarkers. To date, most attention is centered on exosomes and microRNAs as the vectors and the secreted species, respectively. However, this field would benefit from an increased understanding of the plethora of sRNAs secreted by different cell types in different extracellular fractions. It is still not clear if specific sRNAs are selected for secretion, or if sRNA secretion is mostly passive. We sequenced the intracellular sRNA content (19-60 nt) of breast epithelial cell lines (MCF-7 and MCF-10A) and compared it with extracellular fractions enriched in microvesicles, exosomes and ribonucleoprotein complexes. Our results are consistent with a non-selective secretion model for most microRNAs, although a few showed secretion patterns consistent with preferential secretion. On the contrary, 5' tRNA halves and 5' RNA Y4-derived fragments of 31-33 were greatly and significantly enriched in the extracellular space (even in non-mammary cell lines), where tRNA halves were detected as part of ∼45 kDa ribonucleoprotein complexes. Overall, we show that different sRNA families have characteristic secretion patterns and open the question of the role of these sRNAs in the extracellular space.

Emergence of Multiple EGFR Extracellular Mutations during Cetuximab Treatment in Colorectal Cancer.

PURPOSE: Patients with colorectal cancer who respond to the anti-EGFR antibody cetuximab often develop resistance within several months of initiating therapy. To design new lines of treatment, the molecular landscape of resistant tumors must be ascertained. We investigated the role of mutations in the EGFR signaling axis on the acquisition of resistance to cetuximab in patients and cellular models. EXPERIMENTAL DESIGN: Tissue samples were obtained from 37 patients with colorectal cancer who became refractory to cetuximab. Colorectal cancer cells sensitive to cetuximab were treated until resistant derivatives emerged. Mutational profiling of biopsies and cell lines was performed. Structural modeling and functional analyses were performed to causally associate the alleles to resistance. RESULTS: The genetic profile of tumor specimens obtained after cetuximab treatment revealed the emergence of a complex pattern of mutations in EGFR, KRAS, NRAS, BRAF, and PIK3CA genes, including two novel EGFR ectodomain mutations (R451C and K467T). Mutational profiling of cetuximab-resistant cells recapitulated the molecular landscape observed in clinical samples and revealed three additional EGFR alleles: S464L, G465R, and I491M. Structurally, these mutations are located in the cetuximab-binding region, except for the R451C mutant. Functionally, EGFR ectodomain mutations prevent binding to cetuximab but a subset is permissive for interaction with panitumumab. CONCLUSIONS: Colorectal tumors evade EGFR blockade by constitutive activation of downstream signaling effectors and through mutations affecting receptor-antibody binding. Both mechanisms of resistance may occur concomitantly. Our data have implications for designing additional lines of therapy for patients with colorectal cancer who relapse upon treatment with anti-EGFR antibodies.

Hyaluronan deficiency due to Has3 knock-out causes altered neuronal activity and seizures via reduction in brain extracellular space.

Hyaluronan (HA), a large anionic polysaccharide (glycosaminoglycan), is a major constituent of the extracellular matrix of the adult brain. To address its function, we examined the neurophysiology of knock-out mice deficient in hyaluronan synthase (Has) genes. Here we report that these Has mutant mice are prone to epileptic seizures, and that in Has3(-/-) mice, this phenotype is likely derived from a reduction in the size of the brain extracellular space (ECS). Among the three Has knock-out models, namely Has3(-/-), Has1(-/-), and Has2(CKO), the seizures were most prevalent in Has3(-/-) mice, which also showed the greatest HA reduction in the hippocampus. Electrophysiology in Has3(-/-) brain slices demonstrated spontaneous epileptiform activity in CA1 pyramidal neurons, while histological analysis revealed an increase in cell packing in the CA1 stratum pyramidale. Imaging of the diffusion of a fluorescent marker revealed that the transit of molecules through the ECS of this layer was reduced. Quantitative analysis of ECS by the real-time iontophoretic method demonstrated that ECS volume was selectively reduced in the stratum pyramidale by ∼ 40% in Has3(-/-) mice. Finally, osmotic manipulation experiments in brain slices from Has3(-/-) and wild-type mice provided evidence for a causal link between ECS volume and epileptiform activity. Our results provide the first direct evidence for the physiological role of HA in the regulation of ECS volume, and suggest that HA-based preservation of ECS volume may offer a novel avenue for development of antiepileptogenic treatments.

Phosphorylation of α3 glycine receptors induces a conformational change in the glycine-binding site.

Inflammatory pain sensitization is initiated by prostaglandin-induced phosphorylation of α3 glycine receptors (GlyRs) that are specifically located in inhibitory synapses on spinal pain sensory neurons. Phosphorylation reduces the magnitude of glycinergic synaptic currents, thereby disinhibiting nociceptive neurons. Although α1 and α3 subunits are both expressed on spinal nociceptive neurons, α3 is a more promising therapeutic target as its sparse expression elsewhere implies a reduced risk of side-effects. Here we compared glycine-mediated conformational changes in α1 and α3 GlyRs to identify structural differences that might be exploited in designing α3-specific analgesics. Using voltage-clamp fluorometry, we show that glycine-mediated conformational changes in the extracellular M2-M3 domain were significantly different between the two GlyR isoforms. Using a chimeric approach, we found that structural variations in the intracellular M3-M4 domain were responsible for this difference. This prompted us to test the hypothesis that phosphorylation of S346 in α3 GlyR might also induce extracellular conformation changes. We show using both voltage-clamp fluorometry and pharmacology that Ser346 phosphorylation elicits structural changes in the α3 glycine-binding site. These results provide the first direct evidence for phosphorylation-mediated extracellular conformational changes in pentameric ligand-gated ion channels, and thus suggest new loci for investigating how phosphorylation modulates structure and function in this receptor family. More importantly, by demonstrating that phosphorylation alters α3 GlyR glycine-binding site structure, they raise the possibility of developing analgesics that selectively target inflammation-modulated GlyRs.

Structural stability of Burkholderia cenocepacia biofilms is reliant on eDNA structure and presence of a bacterial nucleic acid binding protein.

Cystic fibrosis (CF) is the most common lethal inherited genetic disorder affection Caucasians. Even with medical advances, CF is life-shortening with patients typically surviving only to age 38. Infection of the CF lung by Burkholderia cenocepacia presents exceptional challenges to medical management of these patients as clinically this microbe is resistant to virtually all antibiotics, is highly transmissible and infection of CF patients with this microbe renders them ineligible for lung transplant, often the last lifesaving option. Here we have targeted two abundant components of the B. cenocepacia biofilm for immune intervention: extracellular DNA and DNABII proteins, the latter of which are bacterial nucleic acid binding proteins. Treatment of B. cenocepacia biofilms with antiserum directed at one of these DNABII proteins (integration host factor or IHF) resulted in significant disruption of the biofilm. Moreover, when anti-IHF mediated destabilization of a B. cenocepacia biofilm was combined with exposure to traditional antibiotics, B. cenocepacia resident within the biofilm and thereby typically highly resistant to the action of antibiotics, were now rendered susceptible to killing. Pre-incubation of B. cenocepacia with anti-IHF serum prior to exposure to murine CF macrophages, which are normally unable to effectively degrade ingested B. cenocepacia, resulted in a statistically significant increase in killing of phagocytized B. cenocepacia. Collectively, these findings support further development of strategies that target DNABII proteins as a novel approach for treatment of CF patients, particularly those whose lungs are infected with B. cenocepacia.

Secretion of truncated recombinant rabies virus glycoprotein with preserved antigenic properties using a co-expression system in Hansenula polymorpha.

Rabies virus infection remains a serious public health threat in the developing world, where cost-concerns make wide-scale public health interventions impractical. The development of novel and inexpensive ELISA diagnostic antigens is critical in early detection and prevention of complications. The transmembrane glycoprotein (G) of rabies virus (RV) contains an external domain capable of inducing the synthesis of anti-rabies, virus-neutralizing antibodies, in infected or immunized hosts. In our study, the external G domain was synthesized and fused in-frame with a polyhistidine-tag coding sequence present in the expression plasmid. Soluble truncated recombinant G was secreted in Hansenula polymorpha (H. polymorpha) using H. polymorpha-derived calnexin (HpCNE1) overproduction and found to be correctly N-glycosylated. The truncated recombinant G was purified from cell culture supernatant by Ni-agarose affinity chromatography and when compared with the full-length glycoprotein, found to be similarly immunogenic in vaccinated rabbits. These results subsequently led us to explore the potential of truncated recombinant G as a diagnostic antigen in ELISA. Our results show that the truncated recombinant G can detect antibodies directed to both whole virion and native glycoprotein. More sophisticated applications of truncated recombinant G would profit from the correctly N-glycosylated and soluble monomer.

Expression pattern of recombinant organophosphorus hydrolase from Flavobacterium sp. ATCC 27551 in Escherichia coli.

Concerned with the influence of tagging system on the expression of heterogeneous protein in Escherichia coli, we attempted to express the organophosphorus hydrolase (OPH) of Flavobacterium sp. ATCC 27551 in E. coli. Recombinant OPH was overproduced successfully in E. coli when modified without the use of a tobacco etch virus (TEV) protease cleavage sequence. In addition, though there has never been a report on the extracellular secretion of recombinant OPH harboring native Tat signal peptides in E. coli, the produced protein was observed to be secreted extracellularly. Through the use of reverse transcriptional quantitative real-time PCR and comparison of the predicted folding rate, it was determined that OPH expression may be affected by the existence of a TEV protease cleavage sequence at the C-terminus during the process of translated protein folding, leading to the suppressed OPH activity. With the potential compatibility between native Tat signal peptides of OPH and E. coli Tat pathway secretion system, we report a successful expression of recombinant OPH harboring native Tat signal peptides in E. coli, for the first time.

Modification in media composition to obtain secretory production of STxB-based vaccines using Escherichia coli.

Shiga toxin B-subunit (STxB) from Shigella dysenteriae targets in vivo antigen to cancer cells, dendritic cells (DC) and B cells, which preferentially express the globotriaosylceramide (Gb3) receptor. This pivotal role has encouraged scientists to investigate fusing STxB with other clinical antigens. Due to the challenges of obtaining a functional soluble form of the recombinant STxB, such as formation of inclusion bodies during protein expression, scientists tend to combine STxB with vaccine candidates rather than using their genetically fused forms. In this work, we fused HPV16 E7 as a vaccine candidate to the recombinantly-produced STxB. To minimize the formation of inclusion bodies, we investigated a number of conditions during the expression procedure. Then various strategies were used in order to obtain high yield of soluble recombinant protein from E. coli which included the use of different host strains, reduction of cultivation temperature, as well as using different concentrations of IPTG and different additives (Glycin, Triton X-100, ZnCl(2)). Our study demonstrated the importance of optimizing incubation parameters for recombinant protein expression in E. coli; also showed that the secretion production can be achieved over the course of a few hours when using additives such as glycine and Triton X-100. Interestingly, it was shown that when the culture mediums were supplemented by additives, there was an inverse ratio between time of induction (TOI) and the level of secreted protein at lower temperatures. This study determines the optimal conditions for high yield soluble E7-STxB expression and subsequently facilitates reaching a functionally soluble form of STxB-based vaccines, which can be considered as a potent vaccine candidate for cervical cancer.

Different secreted phosphatase activities in Leishmania amazonensis.

Leishmania has strong acid phosphatase activity on the external surface of the plasma membrane and secreted into the extracellular milieu. Secreted acid phosphatase (sAcP), which is the most abundant secreted protein of Leishmania, is also a virulence factor that plays a role in vertebrate infection and survival in sand flies. In this study, we characterized the secreted phosphatase activities in Leishmania amazonensis. Both acidic and alkaline secreted phosphatase activities were observed with ß-glycerophosphate and p-nitrophenyl phosphate (p-NPP) hydrolysis and were inhibited with sodium tartrate and sodium orthovanadate. Cytochemical labeling revealed a significant difference in the localization of the electron-dense precipitates depending on the substrate. ß-Glycerophosphate electron-dense precipitates were concentrated on both the cell surface and flagellar pocket, whereas p-NPP labeling occurred primarily within intracellular organelles. Orthovanadate-treated metacyclic promastigotes were less infective and were confined to a tight parasitophorous vacuole (PV), which is not characteristic of this Leishmania species. Based on the results, we characterized the presence of different secreted phosphatase activities in L. amazonensis, the influence of the substrate in cytochemical labeling, and the potential involvement of secreted phosphatase activity in both PV maturation and amastigote survival.