ß-1,3-Glucan recognition by Acanthamoeba castellanii as a putative mechanism of amoeba-fungal interactions.

In this study, we conducted an in-depth analysis to characterize potential Acanthamoeba castellanii (Ac) proteins capable of recognizing fungal ß-1,3-glucans. Ac specifically anchors curdlan or laminarin, indicating the presence of surface ß-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to Ac was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, Histoplasma capsulatum (Hc) G217B, possessing a ß-1,3-glucan outer layer, showed significant adhesion to Ac compared to a Hc G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble ß-1,3-glucan substantially inhibited this adhesion, indicating the involvement of ß-1,3-glucan recognition. Biotinylated ß-1,3-glucan-binding proteins from Ac exhibited higher binding to Hc G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the ß-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identified several Ac proteins capable of binding ß-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identified proteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinity to ß-1,3-glucans. These findings underscore the complexity of binding via ß-1,3-glucan and suggest the existence of alternative fungal recognition pathways in Ac.IMPORTANCEAcanthamoeba castellanii (Ac) and macrophages both exhibit the remarkable ability to phagocytose various extracellular microorganisms in their respective environments. While substantial knowledge exists on this phenomenon for macrophages, the understanding of Ac's phagocytic mechanisms remains elusive. Recently, our group identified mannose-binding receptors on the surface of Ac that exhibit the capacity to bind/recognize fungi. However, the process was not entirely inhibited by soluble mannose, suggesting the possibility of other interactions. Herein, we describe the mechanism of ß-1,3-glucan binding by A. castellanii and its role in fungal phagocytosis and survival within trophozoites, also using macrophages as a model for comparison, as they possess a well-established mechanism involving the Dectin-1 receptor for ß-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.

Identification and characterization of an ectophosphatase activity involved in Acanthamoeba castellanii adhesion to host cells.

Acanthamoeba castellanii is a free-living amoeba and an opportunistic pathogen for humans that can cause encephalitis and, more commonly, Acanthamoeba keratitis. During its life cycle, A. castellanii may present as proliferative and infective trophozoites or resistant cysts. The adhesion of trophozoites to host cells is a key first step in the pathogenesis of infection. A major virulence protein of Acanthamoeba is a mannose-binding protein (MBP) that mediates the adhesion of amoebae to cell surfaces. Ectophosphatases are ecto-enzymes that can dephosphorylate extracellular substrates and have already been described in several microorganisms. Regarding their physiological roles, there is consistent evidence that ectophosphatase activities play an important role in parasite-host interactions. In the present work, we identified and biochemically characterized the ectophosphatase activity of A. castellanii. The ectophosphatase activity is acidic, stimulated by magnesium, cobalt and nickel, and presents the following apparent kinetic parameters: Km = 2.12 ± 0.54 mM p-NPP and Vmax = 26.12 ± 2.53 nmol p-NP × h-1 × 10-6 cells. We observed that sodium orthovanadate, ammonium molybdate, sodium fluoride, and inorganic phosphate are able to inhibit ectophosphatase activity. Comparing the two stages of the A. castellanii lifecycle, ectophosphatase activity is significantly higher in trophozoites than in cysts. The ectophosphatase activity is stimulated by mannose residues and is significantly increased when trophozoites interact with LLC-MK2 cells. The inhibition of ectophosphatase by pretreatment with sodium orthovanadate also inhibits the adhesion of trophozoites to epithelial cells. These results allow us to conclude that the ectophosphatase activity of A. castellanii is somehow important for the adhesion of trophozoites to their host cells. According to our data, we believe that the activation of MBP by mannose residues triggers the stimulation of ectophosphatase activity to facilitate the adhesion process.

Mechanism of high-mannose N-glycan breakdown and metabolism by Bifidobacterium longum.

Bifidobacteria are early colonizers of the human gut and play central roles in human health and metabolism. To thrive in this competitive niche, these bacteria evolved the capacity to use complex carbohydrates, including mammalian N-glycans. Herein, we elucidated pivotal biochemical steps involved in high-mannose N-glycan utilization by Bifidobacterium longum. After N-glycan release by an endo-ß-N-acetylglucosaminidase, the mannosyl arms are trimmed by the cooperative action of three functionally distinct glycoside hydrolase 38 (GH38) α-mannosidases and a specific GH125 α-1,6-mannosidase. High-resolution cryo-electron microscopy structures revealed that bifidobacterial GH38 α-mannosidases form homotetramers, with the N-terminal jelly roll domain contributing to substrate selectivity. Additionally, an α-glucosidase enables the processing of monoglucosylated N-glycans. Notably, the main degradation product, mannose, is isomerized into fructose before phosphorylation, an unconventional metabolic route connecting it to the bifid shunt pathway. These findings shed light on key molecular mechanisms used by bifidobacteria to use high-mannose N-glycans, a perennial carbon and energy source in the intestinal lumen.

Structural Characterization of L-Galactose Dehydrogenase: An Essential Enzyme for Vitamin C Biosynthesis.

In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learned concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase [Spinacia oleracea GDH (SoGDH) from spinach], from the D-mannose/L-galactose (Smirnoff-Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homolog from camu camu, a super-accumulator of vitamin C found in the Peruvian Amazon. Both enzymes are monomers in solution and have a pH optimum of 7, and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (ß/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favors the oxidation reaction that ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes toward a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.

Garvicins AG1 and AG2: Two Novel Class IId Bacteriocins of Lactococcus garvieae Lg-Granada.

Lactococcus garvieae causes infectious diseases in animals and is considered an emerging zoonotic pathogen involved in human clinical conditions. In silico analysis of plasmid pLG50 of L. garvieae Lg-Granada, an isolate from a patient with endocarditis, revealed the presence of two gene clusters (orf46-47 and orf48-49), each one encoding a novel putative bacteriocin, i.e., garvicin AG1 (GarAG1; orf46) and garvicin AG2 (GarAG2; orf48), and their corresponding immunity proteins (orf47 and orf49). The chemically synthesised bacteriocins GarAG1 and GarAG2 presented inhibitory activity against pathogenic L. garvieae strains, with AG2 also being active against Listeria monocytogenes, Listeria ivanovii and Enterococcus faecalis. Genetic organisation, amino acid sequences and antimicrobial activities of GarAG1 and GarAG2 indicate that they belong to linear non-pediocin-like one-peptide class IId bacteriocins. Gram-positive bacteria that were sensitive to GarAG2 were also able to ferment mannose, suggesting that this bacteriocin could use the mannose phosphotransferase transport system (Man-PTS) involved in mannose uptake as a receptor in sensitive strains. Intriguingly, GarAG1 and GarAG2 were highly active against their own host, L. garvieae Lg-Granada, which could be envisaged as a new strategy to combat pathogens via their own weapons.

A novel fission yeast platform to model N-glycosylation and the bases of congenital disorders of glycosylation type I.

Congenital disorders of glycosylation type I (CDG-I) are inherited human diseases caused by deficiencies in lipid-linked oligosaccharide (LLO) synthesis or the glycan transfer to proteins during N-glycosylation. We constructed a platform of 16 Schizosaccharomyces pombe strains that synthesize all possible theoretical combinations of LLOs containing three to zero glucose (Glc) residues and nine to five mannose (Man) residues. The occurrence of unexpected LLOs suggested the requirement of specific Man residues for glucosyltransferase activities. We then quantified protein hypoglycosylation in each strain and found that in S. pombe the presence of Glc in the LLO is more relevant to the transfer efficiency than the number of Man residues. Surprisingly, a decrease in the number of Man residues in glycans somehow improved the glycan transfer. The most severe hypoglycosylation was produced in cells that synthesized LLOs completely lacking Glc and having a high number of Man residues. This deficiency could be reverted by expressing a single-subunit oligosaccharyltransferase with a broad range of substrate specificity. Our work shows the usefulness of this new S. pombe set of mutants as a platform to model the molecular bases of human CDG-I diseases. This article has an associated First Person interview with the first authors of the paper.

Purification and characterization of a highly thermostable GlcNAc-binding lectin from Collaea speciosa seeds.

Lectins from plants of the Diocleinae subtribe often exhibit specificity towards mannose/glucose and derived sugars, with some plants also displaying a second lectin specific to lactose/GalNAc. Here, we present a novel lectin from Collaea speciosa, named CsL, that displays specificity for GlcNAc/glucose. The lectin was extracted from Collaea speciosa seeds and purified by a single chromatographic step on a Sephadex G-50 matrix. In solution, the lectin appears as a dimeric protein composed of 25 kDa monomers. The protein is stable at pH 7-8 and dependent on divalent cations. CsL maintained its agglutination activity after heating to 90 °C for 1 h. Glycan array studies revealed that CsL binds to N-glycans with terminal GlcNAc residues, chitobiose and chitotriose moieties. The partial amino acid sequence of the lectin is similar to that of some lactose-specific lectins from the same subtribe. In contrast to other ConA-like lectins, CsL is not toxic to Artemia. Because of its remarkably different properties and specificity, this lectin could be the first member of a new group inside the Diocleinae lectins.

ArtinM: Purification and Evaluation of Biological Activities.

The immunomodulatory activity of plant lectins has been evaluated because of their high selectivity for glycans linked to receptors on innate and adaptative immune cells. ArtinM is a mannosyl-binding lectin, obtained from the seeds of Artocarpus heterophyllus, that induces the differentiation of CD4+ T cells and macrophages by interacting with CD3 and TLR2/CD14, respectively. This ArtinM property ultimately favors the combat of intracellular pathogens, opening new perspectives on the lectins application as immunomodulatory agents. The current section describes protocols for purification and evaluation of ArtinM biological activity. The purification is based on the ArtinM-D-mannose affinity. The effect of inducing IL-12 production by murine macrophages cell line is adopted to evaluate the ArtinM biological activity.

SRL pathogenicity island contributes to the metabolism of D-aspartate via an aspartate racemase in Shigella flexneri YSH6000.

In recent years, multidrug resistance of Shigella strains associated with genetic elements like pathogenicity islands, have become a public health problem. The Shigella resistance locus pathogenicity island (SRL PAI) of S. flexneri 2a harbors a 16Kbp region that contributes to the multidrug resistance phenotype. However, there is not much information about other functions such as metabolic, physiologic or ecological ones. For that, wild type S. flexneri YSH6000 strain, and its spontaneous SRL PAI mutant, 1363, were used to study the contribution of the island in different growth conditions. Interestingly, when both strains were compared by the Phenotype Microarrays, the ability to metabolize D-aspartic acid as a carbon source was detected in the wild type strain but not in the mutant. When D-aspartate was added to minimal medium with other carbon sources such as mannose or mannitol, the SRL PAI-positive strain was able to metabolize it, while the SRL PAI-negative strain did not. In order to identify the genetic elements responsible for this phenotype, a bioinformatic analysis was performed and two genes belonging to SRL PAI were found: orf8, coding for a putative aspartate racemase, and orf9, coding for a transporter. Thus, it was possible to measure, by an indirect analysis of racemization activity in minimal medium supplemented only with D-aspartate, that YSH6000 strain was able to transform the D-form into L-, while the mutant was impaired to do it. When the orf8-orf9 region from SRL island was transformed into S. flexneri and S. sonnei SRL PAI-negative strains, the phenotype was restored. Although, when single genes were cloned into plasmids, no complementation was observed. Our results strongly suggest that the aspartate racemase and the transporter encoded in the SRL pathogenicity island are important for bacterial survival in environments rich in D-aspartate.

Evaluating glucose and mannose profiles in Candida species using quantum dots conjugated with Cramoll lectin as fluorescent nanoprobes.

Glycoconjugates found on cell walls of Candida species are fundamental for their pathogenicity. Laborious techniques have been employed to investigate the sugar composition of these microorganisms. Herein, we prepared a nanotool, based on the fluorescence of quantum dots (QDs) combined with the specificity of Cramoll lectin, to evaluate glucose/mannose profiles on three Candida species. The QDs-Cramoll conjugates presented specificity and bright fluorescence emission. The lectin preserved its biological activity after the conjugation process mediated by adsorption interactions. The labeling of Candida species was analyzed by fluorescence microscopy and quantified by flow cytometry. Morphological analyses of yeasts labeled with QDs-Cramoll conjugates indicated that C. glabrata (2.7 µm) was smaller when compared to C. albicans (4.0 µm) and C. parapsilosis sensu stricto (3.8 µm). Also, C. parapsilosis population was heterogeneous, presenting rod-shaped blastoconidia. More than 90% of cells of the three species were labeled by conjugates. Inhibition and saturation assays indicated that C. parapsilosis had a higher content of exposed glucose/mannose than the other two species. Therefore, QDs-Cramoll conjugates demonstrated to be effective fluorescent nanoprobes for evaluation of glucose/mannose constitution on the cell walls of fungal species frequently involved in candidiasis.

Novel transglycosylation activity of ß-N-acetylglucosaminidase of Lecanicillium lecanii produced by submerged culture.

N-acetylglucosaminidase produced from Lecanicillium lecanii on submerged culture displayed hydrolytic and transglycosylation activities. The highest specific activity for the enzyme was 1.87 U/mg after 120 h of culture. The chromatographic purification for a single protein fraction showed a molecular weight of 50.4 kDa and hydrolytic N-acetylglucosaminidase activity of 17.59 U/mg at 37 °C and pH 6. This enzyme was able to transglycosylate and to synthesize oligosaccharides from 2 to 6 units with a degree of acetylation between 100 and 26% employing glucose, mannose, N-acetyl-D-glucosamine and N-acetyl-D-lactosamine as donor substrates. Optimal conditions of temperature and pH were determined for both types of enzymatic activities.

Insights into anti-pathogenic activities of mannose lectins.

Carbohydrate-binding proteins, also known as lectins, are valuable tools for biotechnology, including pharmacological uses. Mannose lectins obtained from plant and animal sources are applied to protection and characterization of autoimmune diseases as well as defense proteins against pathogens. The presence of mannose-binding lectins in plants that also recognize glucose could be entitled Man/Glc lectins; such specificity has allowed employing these vegetal lectins for several applications. Animal mannose-binding lectins are synthesized in the liver and secreted into the blood stream where both concentration and activity are greatly affected due to gene polymorphisms; these serum proteins play important roles in the immune system by recognizing mannose-like carbohydrate ligands found exclusively on pathogenic microorganisms. Mannose lectins already showed strong binding to relevant bacteria, viruses, protozoa and helminth species, initiating potent host defense mechanisms by inducing growth inhibition or death of such organisms; the ability to prevent the formation or destruction of microbial biofilms has also been reported. Mannose-binding lectins have attracted considerable attention against carcinogenesis and atherogenesis. The aim of this review article is to approach biotechnology characteristics of these lectins from different sources and microorganism/cell surface interactions with mannose; in addition, aspects of mechanisms associated to lectin antipathogenic activities are described.

Assays with recombinant soluble isoforms of DC-SIGN, a dengue virus ligand, show variation in their ability to bind to mannose residues.

The DC-SIGN glycoprotein is responsible for the initial adhesion of dengue virus (DENV) to immune cells by the carbohydrate recognition domain (CRD). There are thirteen soluble and membrane-bound DC-SIGN isoforms, but the role of soluble isoforms in the DENV internalization process is not known. Five isoforms with an altered or absent CRD were identified, and three different soluble isoforms were used to confirm the interactions with mannose residues. The results show the loss of binding ability of one soluble isoform and binding ability of two of them. All of them will be used to verify their role in the DENV internalization process.

Unravelling the interactions of the environmental host Acanthamoeba castellanii with fungi through the recognition by mannose-binding proteins.

Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.

Glycosylation Profile of the Transferrin Receptor in Gestational Iron Deficiency and Early-Onset Severe Preeclampsia.

OBJECTIVE: To examine the expression of hypoxia-inducible factor-1α (HIF-1α), TfR1, and TfR1-attached terminal monosaccharides in placentas of women with IDAP and severe preeclampsia. METHODS: TfR1 and HIF-1α were detected by western blot. Immunoadsorption of TfR1 was performed to characterize the terminal monosaccharides by specific lectin binding. RESULTS: There was no difference in the expression of TfR1 and HIF-1α between groups. Lectin blot analysis pointed out an overexpression of galactose ß1-4 N-acetylglucosamine (Gal-GlcNAc) and mannose in severe preeclampsia. CONCLUSION: The increase in Gal-GlcNAc may be due to the increased presence of antennary structures and the mannose glycans of TfR1 may indicate the presence of misfolded or incomplete proteins. These findings may be associated with the low expression of placental TfR1 in women with preeclampsia.

Labeling membrane receptors with lectins and evaluation of the midgut histochemistry of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) populations with different levels of susceptibility to formulated Bt.

BACKGROUND: Studies show that insects can adapt to the toxins of Bacillus thuringiensis under field and laboratory conditions through the development of resistance to the bacterium and its formulations. This has been demonstrated in the failure to control Tuta absoluta populations in Brazil. This study evaluated membrane receptors using peroxidase-labeled lectins and the midgut histochemistry of T. absoluta populations to assess susceptibility to the insecticides Bt fomulations. The histochemistry analysis used Periodic Acid-Schiff for glycogen and Ponceau Xylidine for total proteins. The presence of glucose/mannose and N-acetylgalactosamine (GalNAc) was analyzed using specific lectins. One susceptible and one tolerant population were used in the study; insects were exposed to the insecticide concentrations recommended by the manufacturers. The midgut was collected after an interval of 20 min and analyzed using optical microscopy. RESULTS: Bt fomulation interferes with the glycogen content, whereas XenTari® interferes with the protein content, irrespective of the level of susceptibility. High expression of GalNAc residues was observed using soybean lectin labeling, indicating a direct relationship between the glycosylation pattern and susceptibility to Bt fomulation in the Pelotas population. CONCLUSION: The use of Bt fomulation caused greater alterations in the larval intestinal histophysiology compared to the use of XenTari® . © 2018 Society of Chemical Industry.

Lectin from Canavalia villosa seeds: A glucose/mannose-specific protein and a new tool for inflammation studies.

With important carbohydrate binding properties, lectins are proteins able to decipher the glycocode, and as such, they can be used in bioassays involving cell-cell communication, protein targeting, inflammation, and hypernociception, among others. In this study, a new glucose/mannose-specific lectin from Canavalia villosa seeds (Cvill) was isolated by a single affinity chromatography step in a Sephadex® G-50 column, with a purification yield of 19.35mg of lectin per gram of powdered seed. Analysis of intact protein by mass spectrometry showed the lectin is composed of three polypeptide chains, including a 25.6kDa α chain, 12.9KDa ß, and 12.6 KDa γ fragments, similar to the profile of ConA-like glucose/mannose-specific lectins. Partial sequence of the protein was obtained by MS-MALDI TOF/TOF covering 41.7% of its primary structure. Cvill presented sugar specificity to d-glucose, α-methyl-d-mannoside, d-mannose, and glycoproteins fetuin and ovoalbumin. The lectin characterization showed that Cvill presents high stability within a broad range of pH and temperature, also showing average toxicity against Artemia nauplii. The proinflammatory effect of Cvill was observed by induction of paw edema and hypernociception in mice, with the participation of the carbohydrate binding site, showing its potential to be used as tool in inflammation studies.

Recombinant ArtinM activates mast cells.

BACKGROUND: Mast cells are hematopoietically derived cells that play a role in inflammatory processes such as allergy, as well as in the immune response against pathogens by the selective and rapid release of preformed and lipid mediators, and the delayed release of cytokines. The native homotetrameric lectin ArtinM, a D-mannose binding lectin purified from Artocarpus heterophyllus seeds, is one of several lectins that are able to activate mast cells. Besides activating mast cells, ArtinM has been shown to affect several biological responses, including immunomodulation and acceleration of wound healing. Because of the potential pharmacological application of ArtinM, a recombinant ArtinM (rArtinM) was produced in Escherichia coli. The current study evaluated the ability of rArtinM to induce mast cell degranulation and activation. RESULTS: The glycan binding specificity of rArtinM was similar to that of jArtinM. rArtinM, via its CRD, was able to degranulate, releasing ß-hexosaminidase and TNF-α, and to promote morphological changes on the mast cell surface. Moreover, rArtinM induced the release of the newly-synthesized mediator, IL-4. rArtinM does not have a co-stimulatory effect on the FcεRI degranulation via. The IgE-dependent mast cell activation triggered by rArtinM seems to be dependent on NFkB activation. CONCLUSIONS: The lectin rArtinM has the ability to activate and degranulate mast cells via their CRDs. The present study indicates that rArtinM is a suitable substitute for the native form, jArtinM, and that rArtinM may serve as an important and reliable pharmacological agent.

Comparative analysis of carbohydrate residues in the midgut of phlebotomines (Diptera: Psychodidae) from colony and field populations from Amazon, Brazil.

Leishmaniasis are worldwide diseases that occur in 98 countries including Brazil, transmitted by the bite of female phlebotomines during blood feeding. In Brazil it is known that some species of sand flies as Lutzomyia longipalpis sensun latum (vector of Leishmania infantum chagasi), Lutzomyia flaviscutellata (vector of Leishmania (Leishmania) amazonensis) and Lutzomyia antunesi [suspected vector of Leishmania (Viannia) lindenbergi] are incriminated of transmitting the parasite Leishmania for the vertebrate host. The phlebotomine-parasite is mediated by the attachment of the promastigote lipophosphoglycan (LPG) to the midgut epithelium. However, another mechanism that is LPG-independent and mediated by N-acetyl-galactosamine (GalNAc) seems to occur in some species of phlebotomines that are classified as permissive. The aim of this study was to characterize the carbohydrate residues that, probably, play a role in parasite attachment to the midgut of phlebotomine from colony and field populations from the Brazilian Amazonian region. We observed the presence of GalNAc, mannose, galactose and GlcNAc in all phlebotomine species. A binding assay between L. (L.) amazonensis and L. i.chagasi to the midguts of different species of phlebotomines was performed. The attachment of both Leishmania and vector species suggests the presence of GalNAc on the midgut surfaces. Thus, these results suggested that GalNAc is a possible binding sites of Leishmania in sand flies from the Brazilian Amazonian region.

High-level expression and characterization of a glycosylated human cementum protein 1 with lectin activity.

This work aims to contribute to the knowledge of human cementum protein 1 (CEMP1), its conformational characteristics and influence during the biomineralization process. The results revealed that hrCEMP1 expressed in Pichia pastoris is a 2.4% glycosylated, thermostable protein which possesses a molecular mass of 28,770 Da. The circular dichroism spectrum indicated a secondary structure content of 28.6% of alpha-helix, 9.9% of beta-sheet and 61.5% of random-coil forms. Biological activity assays demonstrated that hrCEMP1 nucleates and regulates hydroxyapatite crystal growth. Hereby, it is demonstrated for the first time that CEMP1 has a (C-type) lectin-like activity and specifically recognizes mannopyranoside. The information produced by this biochemical and structural characterization may contribute to understand more fully the biological functions of CEMP1.