Fungal Glycosidases in Sporothrix Species and Candida albicans.

Glycoside hydrolases (GHs) are enzymes that participate in many biological processes of fungi and other organisms by hydrolyzing glycosidic linkages in glycosides. They play fundamental roles in the degradation of carbohydrates and the assembly of glycoproteins and are important subjects of studies in molecular biology and biochemistry. Based on amino acid sequence similarities and 3-dimensional structures in the carbohydrate-active enzyme (CAZy), they have been classified in 171 families. Members of some of these families also exhibit the activity of trans-glycosydase or glycosyl transferase (GT), i.e., they create a new glycosidic bond in a substrate instead of breaking it. Fungal glycosidases are important for virulence by aiding tissue adhesion and colonization, nutrition, immune evasion, biofilm formation, toxin release, and antibiotic resistance. Here, we review fungal glycosidases with a particular emphasis on Sporothrix species and C. albicans, two well-recognized human pathogens. Covered issues include a brief account of Sporothrix, sporotrichosis, the different types of glycosidases, their substrates, and mechanism of action, recent advances in their identification and characterization, their potential biotechnological applications, and the limitations and challenges of their study given the rather poor available information.

Long-term exposure to particulate matter from air pollution alters airway ß-defensin-3 and -4 and cathelicidin host defense peptides production in a murine model.

Epidemiological studies have associated long-term exposure to environmental air pollution particulate matter (PM) with the development of diverse health problems. They include infectious respiratory diseases related to the deregulation of some innate immune response mechanisms, such as the host defense peptides' expression. Herein, we evaluated in BALB/c mice the effect of long-standing exposure (60 days) to urban-PM from the south of Mexico City, with aerodynamic diameters below 2.5 µm (PM2.5) and 10 µm (PM10) on the lung's gene expression and production of three host defense peptides (HDPs); murine beta-defensin-3, -4 (mBD-3, mBD-4) and cathelin-related antimicrobial peptide (CRAMP). We also evaluated mRNA levels of Il1b and Il10, two cytokines related to the expression of host defense peptides. Exposure to PM2.5 and PM10 differentially induced lung inflammation, being PM2.5, which caused higher inflammation levels, probably associated with a differential deposition on the airways, that facilitate the interaction with alveolar macrophages. Inflammation levels were associated with an early upregulation of the three HDPs assessed and an increment in Il1b mRNA levels. Interestingly, after 28 days of exposure, Il10 mRNA upregulation was observed and was associated with the downregulation of HDPs and Il1b mRNA levels. The upregulation of Il10 mRNA and suppression of HDPs might facilitate microbial colonization and the development of diseases associated with long-term exposure to PM.

Analysis of biofilm formation by Sporothrix schenckii.

The development of mature biofilms is an aid in numerous aspects of the life cycle of fungi. It is well known that Sporothrix schenckii complex causes a benign subcutaneous mycosis, but recent studies have suggestedthat biofilm formation may be one of the important factors involved in its virulence. Here we report the study of the biomass organization and a model of the stages of S. schenckii biofilm development: adsorption, active adhesion, microcolony formation, maturation, and dispersal of biofilm fragments. During the development, the biofilm is surrounded by extracellular matrix, which contains glycoprotein (mannose rich), carbohydrates, lipids, and nucleic acid. In addition, the extracellular DNA increases in extracellular matrix as a key component to structural integrity and antifungal resistance. The study of S. schenckii biofilm contributes to a better understanding of growth biofilm and physiology, adding new insights into the mechanisms of virulence and persistence of pathogenic microorganisms.

Tal6 From Trichoderma atroviride Is a LysM Effector Involved in Mycoparasitism and Plant Association.

LysM effectors play a relevant role during the plant colonization by successful phytopathogenic fungi, since they enable them to avoid either the triggering of plant defense mechanisms or their attack effects. Tal6, a LysM protein from Trichoderma atroviride, is capable of binding to complex chitin. However, until now its biological function is not completely known, particularly its participation in plant-Trichoderma interactions. We obtained T. atroviride Tal6 null mutant and Tal6 overexpressing strains and determined the role played by this protein during Trichoderma-plant interaction and mycoparasitism. LysM effector Tal6 from T. atroviride protects the hyphae from chitinases by binding to chitin of the fungal cell wall, increases the fungus mycoparasitic capacity, and modulates the activation of the plant defense system. These results show that beneficial fungi also employ LysM effectors to improve their association with plants.

Role of Arf-like proteins (Arl1 and Arl2) of Mucor circinelloides in virulence and antifungal susceptibility.

Mucor circinelloides is an etiologic agent of mucormycosis, a fungal infection produced by Mucorales often associated with mortality due to unavailability of antifungal drugs. Arl proteins belong to the Arf family and are involved in vesicle trafficking and tubulin assembly. This study identified two Arl (Arf-like)-encoding genes, arl1 and arl2, in M. circinelloides and explored their function in morphogenesis, virulence, and antifungal susceptibility. Although Arl1 and Arl2 proteins shared 55% amino acid sequence identity, arl1 and arl2 genes showed distinct transcriptional expression patterns. arl1 was expressed at higher levels than arl2 and induced in mycelia, suggesting a role in morphological transitions. Disruption of the arl1 and arl2 genes led to heterokaryon (Δarl1(+)(-)) and homokaryon (Δarl2) genotypes, respectively. The incapacity to generate homokaryon mutants for arl1 suggested that it is essential for growth of M. circinelloides. Deletion of each gene reduced the expression of the other, suggesting the existence of a positive cross-regulation between them. Thus, deletion of arl2 resulted in a ~60% reduction of arl1 expression, whereas the Δarl1(+)(-) showed ∼90% reduction of arl1 expression. Mutation of arl2 showed no phenotype or a mild phenotype between Δarl1(+)(-) and wild-type (WT), suggesting that all observed phenotypes in both mutant strains corresponded to arl1 low expression. The Δarl1(+)(-) produced a small amount of spores that showed increased sensitivity to dodecyl-sulfate and azoles, suggesting a defect in the cell wall that was further supported by decrease in saccharide content. These defects in the cell wall were possibly originated by abnormal vesicle trafficking since FM4-64 staining of both mutants Δarl1(+)(-) and Δarl2 revealed less well-localized endosomes compared to the WT. Moreover, aberrant vesicle trafficking may be responsible for the secretion of specific virulence-related proteins since cell-free medium from Δarl1(+)(-) were found to increase killing of Caenorhabditis elegans compared to WT.

Analysis of some immunogenic properties of the recombinant Sporothrix schenckii Gp70 expressed in Escherichia coli.

AIM: Sporothrix schenckii is the causative agent of sporotrichosis. A 70-kDa glycoprotein, Gp70, is a candidate for the development of prophylactic alternatives to control the disease, and its gene (GP70) is predicted to encode for a protein of 43 kDa, contrasting with the molecular weight of the native protein. MATERIALS & METHODS: The GP70 was expressed in bacteria, the recombinant protein purified, used in immunoassays and injected to Galleria mellonella. RESULTS & CONCLUSION: The recombinant protein was detected by anti-Gp70 antibodies, confirming that the Gp70 backbone is a 43-kDa peptide. This protein showed enzyme activity of cyclase and was recognized by sera of patients with sporotrichosis. Although it was not useful for serodiagnosis of sporotrichosis, it conferred protection to animals against experimental sporotrichosis.

Proteases of Sporothrix schenckii: cytopathological effects on a host-cell model / Proteasas de Sporothrix schenckii: efectos citopatológicos en un modelo de células huésped

Background. Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. The adhesion of the fungus to the host tissue has been considered the key step in the colonization and invasion, but little is known about the early events in the host-parasite interaction. Aims. To evaluate the proteolytic activity of S. schenckii on epithelial cells. Methods. The proteolytic system (at pH 5 and 7) was evaluated using azocoll and zymograms. The host-parasite interaction and epithelial cell response were also analyzed by examining the microfilament cytoskeleton using phalloidin-FITC and transmission electron microscopy. Finally, the metabolic activity was determined using an XTT assay. Results. The zymograms showed that S. schenckii yeast cells possess high intracellular and extracellular proteolytic activities (Mr≥200, 116, 97, and 70kDa) that are pH dependent and are inhibited by PMSF and E64, which act on serine and cysteine-type proteases. During the epithelial cell-protease interaction, the cells showed alterations in the microfilament distribution, as well as in the plasma membrane structure. Moreover, the metabolic activity of the epithelial cells decreased 60% without a protease inhibitor. Conclusions. Our data demonstrate the complexity of the cellular responses during the infection process. This process is somehow counteracted by the action of proteases inhibitors. Furthermore, the results provide critical information for understanding the nature of host-fungus interactions and for searching a new effective antifungal therapy, which includes protease inhibitors (AU)

Antecedentes. La esporotricosis es una infección fúngica causada por el complejo Sporothrix schenckii. La adhesión del hongo al tejido hospedero se ha considerado un paso clave en la colonización e invasión, sin embargo poco se conoce de los eventos tempranos en la interacción hospedero-parasito. Objetivos. Evaluar la actividad proteolítica de S. schenckii en células epiteliales. Métodos. El sistema proteolítico (bajo los valores pH 5 y 7) fue evaluado mediante azocoll y zimogramas. Además, la interacción hospedero-parasito y la respuesta celular fueron analizadas con el examen de los microfilamentos del citoesqueleto mediante faloidina-FITC y microscopia electrónica de transmisión. Finalmente, la actividad metabólica (viabilidad celular) fue determinada por un ensayo de XTT. Resultados. Los zimogramas de S. schenckii muestran que posee una alta actividad proteolítica intracelular y extracelular (Mr≥200, 116, 97 y 70kD) dependientes de pH e inhibidas por PMSF y E64, que actúan sobre serin- y cistein proteasas. Durante la interacción de las células epiteliales-proteasas, las células mostraron alteraciones en la distribución de los microfilamentos y la estructura de la membrana plasmática. Además, la actividad metabólica (viabilidad celular) de las células epiteliales disminuyó un 60% sin inhibidores de proteasas. Conclusiones. Nuestros datos demuestran la complejidad de la respuesta celular durante el proceso de infección, proceso que puede ser en parte contrarrestado por la acción de los inhibidores de proteasas. Además, los resultados proporcionan información crítica para el entendimiento de la naturaleza en la interacción hospedero-hongo y para una nueva terapia antifúngica eficaz que incluya inhibidores de proteasas (AU)

Proteases of Sporothrix schenckii: Cytopathological effects on a host-cell model.

BACKGROUND: Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. The adhesion of the fungus to the host tissue has been considered the key step in the colonization and invasion, but little is known about the early events in the host-parasite interaction. AIMS: To evaluate the proteolytic activity of S. schenckii on epithelial cells. METHODS: The proteolytic system (at pH 5 and 7) was evaluated using azocoll and zymograms. The host-parasite interaction and epithelial cell response were also analyzed by examining the microfilament cytoskeleton using phalloidin-FITC and transmission electron microscopy. Finally, the metabolic activity was determined using an XTT assay. RESULTS: The zymograms showed that S. schenckii yeast cells possess high intracellular and extracellular proteolytic activities (Mr≥200, 116, 97, and 70kDa) that are pH dependent and are inhibited by PMSF and E64, which act on serine and cysteine-type proteases. During the epithelial cell-protease interaction, the cells showed alterations in the microfilament distribution, as well as in the plasma membrane structure. Moreover, the metabolic activity of the epithelial cells decreased 60% without a protease inhibitor. CONCLUSIONS: Our data demonstrate the complexity of the cellular responses during the infection process. This process is somehow counteracted by the action of proteases inhibitors. Furthermore, the results provide critical information for understanding the nature of host-fungus interactions and for searching a new effective antifungal therapy, which includes protease inhibitors.

Purification and characterization of an extracellular ß-glucosidase from Sporothrix schenckii.

An extracellular ß-glucosidase (E.C. 3.2.1.21), induced by cellulose in the mycelial form of human pathogen fungus Sporothrix schenckii, was purified to homogeneity using hydroxyapatite (HAp) adsorption chromatography in batch and Sephacryl S200-HR size exclusion chromatography. The molecular mass of the purified enzyme was estimated to be 197 kDa by size exclusion chromatography with a subunit of 96.8 kDa determined by SDS/PAGE. The ß-glucosidase exhibited optimum catalytic activity at pH 5.5/45 °C and was relatively stable for up to 24 h at 45 °C. Isoelectric focusing displayed an enzyme with a pI value of 4.0. Its activity was inhibited by Fe2+ but not by any other ions or chelating agents. Km and Vmax values of the purified enzyme were 0.012 mm and 2.56 nmol·min-1·mg-1, respectively, using 4-methylumbelliferyl ß-D-glucopyranoside (4-MUG) as the substrate and 44.14 mm and 22.49 nmol·min-1·mg-1 when p-nitrophenyl ß-D-glucopyranoside (p-NPG) was used. The purified ß-glucosidase was active against cellobioside, laminarin, 4-MUG, and p-NPG and slightly active against 4-methylumbelliferyl ß-D-cellobioside and p-nitrophenyl ß-D-cellobioside but did not hydrolyze 4-methylumbelliferyl ß-D-xyloside, 4-methylumbelliferyl ß-D-galactopyranoside nor 4-methylumbelliferyl α-D-glucopyranoside. In addition, the enzyme showed transglycosylation activity when it was incubated along with different oligosaccharides. Whether the transglycosylation and cellulase activities function in vivo as a mechanism involved in the degradation of cellulolytic biomass in the saprophytic stage of S. schenckii remains to be determined.

Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae.

Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.

Candida species: new insights into biofilm formation.

Biofilms of Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis are associated with high indices of hospital morbidity and mortality. Major factors involved in the formation and growth of Candida biofilms are the chemical composition of the medical implant and the cell wall adhesins responsible for mediating Candida-Candida, Candida-human host cell and Candida-medical device adhesion. Strategies for elucidating the mechanisms that regulate the formation of Candida biofilms combine tools from biology, chemistry, nanoscience, material science and physics. This review proposes the use of new technologies, such as synchrotron radiation, to study the mechanisms of biofilm formation. In the future, this information is expected to facilitate the design of new materials and antifungal compounds that can eradicate nosocomial Candida infections due to biofilm formation on medical implants. This will reduce dissemination of candidiasis and hopefully improve the quality of life of patients.

Purification and partial biochemical characterization of a membrane-bound type II-like α-glucosidase from the yeast morphotype of Sporothrix schenckii.

The early steps of glycoprotein biosynthesis involve processing of the N-glycan core by endoplasmic reticulum α-glucosidases I and II which sequentially trim the outermost α1,2-linked and the two more internal α1,3-linked glucose units, respectively. We have demonstrated the presence of some components of the enzymic machinery required for glycoprotein synthesis in Sporothrix schenckii, the etiological agent of human and animal sporotrichosis. However, information on this process is still very limited. Here, a distribution analysis of α-glucosidase revealed that 38 and 50% of total enzyme activity were present in a soluble and in a mixed membrane fraction, respectively. From the latter, the enzyme was solubilized, purified to apparent homogeneity and biochemically characterized. Analysis of the enzyme by denaturing electrophoresis and size exclusion chromatography revealed molecular masses of 75.4 and 152.7 kDa, respectively, suggesting a homodimeric structure. Purified α-glucosidase cleaved the fluorogenic substrate 4-methylumbelliferyl-α-D: -glucopyranoside with high affinity as judged from K(m) and V(max) values of 0.3 µM and 250 nmol of MU/min/mg protein, respectively. Analysis of linkage specificity using a number of glucose α-disaccharides as substrates demonstrated a clear preference of the enzyme for nigerose, an α1,3-linked disaccharide, over other substrates such as kojibiose (α1,2), trehalose (α1,1) and isomaltose (α1,6). Use of selective inhibitors of processing α-glucosidases such as 1-deoxynojirimycin, castanospermine and australine provided further evidence of the possible type of α-glucosidase. Accordingly, 1-deoxynojirimycin, a more specific inhibitor of α-glucosidase II than I, was a stronger inhibitor of hydrolysis of 4-methylumbelliferyl-α-D: -glucopyranoside and nigerose than castanospermine, a preferential inhibitor of α-glucosidase I. Inhibition of hydrolysis of kojibiose and maltose by 1-deoxynojirimycin and castanoespermine was significantly lower than that of nigerose. Taken together, these properties are consistent with a type II-like α-glucosidase probably involved in N-glycan processing. To our knowledge, this is the first report of such an activity in a truly dimorphic fungus.

Sporothrix schenckii complex and sporotrichosis, an emerging health problem.

Sporothrix schenckii, now named the S. schenckii species complex, has largely been known as the etiological agent of sporotrichosis, which is an acute or chronic subcutaneous mycosis of humans and other mammals. Gene sequencing has revealed the following species in the S. schenckii complex: Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana and S. schenckii. The increasing number of reports of Sporothrix infection in immunocompromised patients, mainly the HIV-infected population, suggests sporotrichosis as an emerging global health problem concomitant with the AIDS pandemic. Molecular studies have demonstrated a high level of intraspecific variability. Components of the S. schenckii cell wall that act as adhesins and immunogenic inducers, such as a 70-kDa glycoprotein, are apparently specific to this fungus. The main glycan peptidorhamnomannan cell wall component is the only O-linked glycan structure known in S. schenckii. It contains an α-mannobiose core followed by one α-glucuronic acid unit, which may be mono- or di-rhamnosylated. The oligomeric structure of glucosamine-6-P synthase has led to a significant advance in the development of antifungals targeted to the enzyme's catalytic domain in S. schenckii.

Entamoeba histolytica: identification and partial characterization of alpha-mannosidase activity.

Despite their well recognized importance in pathogenesis of Entamoeba histolytica there are few studies dealing with the assembly and secretion of glycoproteins that participate in the adhesion to target cells and in the dissemination of the parasite in infected tissues. Some of these studies refer to the identification and, in some cases, the characterization of glycosyl transferases and glycosidases involved in the biosynthesis of these macromolecules as well as to compartments involved in the amoeba dolichol-linked glycosylation pathway. While an N-glycan trimming alpha-mannosidase has been demonstrated in E. histolytica, little is known on its cellular distribution and properties. Here we describe the presence and partial biochemical characterization of soluble and MMF-associated forms of alpha-mannosidase and the separation of at least three internal membrane structures enriched with this glycosidase. Results are discussed in terms of the possible identity of alpha-mannosidase activity and the potential precursor-product relationship between the two enzyme forms.

Sporothrix schenckii: purification and partial biochemical characterization of glucosamine-6-phosphate synthase, a potential antifungal target.

The first committed step of the biosynthetic pathway leading to uridine-5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is catalyzed by glucosamine-6-phosphate synthase (GlcN-6-P synthase), an enzyme proposed as a potential antifungal chemotherapy target. Here, we describe the purification and biochemical characterization of the native enzyme from the dimorphic pathogenic fungus Sporothrix schenckii. The availability of the pure protein facilitated its biochemical characterization. The enzyme exhibited subunit and native molecular masses of 79 and 350+/-5 kDa, respectively, suggesting a homotetrameric structure. Isoelectric point was 6.26 and K(m) values for fructose-6-phosphate and L-glutamine were 1.12+/-0.3 and 2.2+/-0.7 mM, respectively. Inhibition of activity by UDP-GlcNAc was enhanced by Glc-6-P and phosphorylation stimulated GlcN-6-P synthase activity without affecting the enzyme sensitivity to the aminosugar. A glutamine analogue, FMDP [N(3)-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid] was a more potent inhibitor of activity than ADMP (2-Amino-2-deoxy-D-mannitol-6-phosphate) but the latter was a stronger inhibitor of growth in two culture media. To our knowledge, this is the first report on the purification and biochemical characterization of a non-recombinant GlcN-6-P synthase from a true dimorphic fungus. Inhibition of enzyme activity and fungal growth by specific inhibitors of GlcN-6-P synthase strongly reinforces the role of this enzyme as a potential target for antifungal chemotherapy.

Protein glycosylation in Candida.

Candidiasis is a significant cause of invasive human mycosis with associated mortality rates that are equivalent to, or worse than, those cited for most cases of bacterial septicemia. As a result, considerable efforts are being made to understand how the fungus invades host cells and to identify new targets for fungal chemotherapy. This has led to an increasing interest in Candida glycobiology, with an emphasis on the identification of enzymes essential for glycoprotein and adhesion metabolism, and the role of N- and O-linked glycans in host recognition and virulence. Here, we refer to studies dealing with the identification and characterization of enzymes such as dolichol phosphate mannose synthase, dolichol phosphate glucose synthase and processing glycosidases and synthesis, structure and recognition of mannans and discuss recent findings in the context of Candida albicans pathogenesis.

Isolation and some properties of a glycoprotein of 70 kDa (Gp70) from the cell wall of Sporothrix schenckii involved in fungal adherence to dermal extracellular matrix.

Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis and an emerging disease in immunocompromised patients. Adherence to target cells is a prerequisite for fungal dissemination and systemic complications. However, information on the cell surface components involved in this interaction is rather scarce. In this investigation, the extraction of isolated cell walls from the yeast phase of S. schenckii with SDS and separation of proteins by SDS-PAGE led to the identification of a periodic acid-Schiff (PAS)-reacting 70 kDa glycoprotein (Gp70) that was purified by elution from electrophoresis gels. The purified glycopeptide exhibited a pI of 4.1 and about 5.7% of its molecular mass was contributed by N-linked glycans with no evidence for O-linked oligosaccharides. Confocal analysis of immunofluorescence assays with polyclonal antibodies directed towards Gp70 revealed a rather uniform distribution of the antigen at the cell surface with no distinguishable differences among three different isolates. Localization of Gp70 at the cell surface was confirmed by immunogold staining. Gp70 seems specific for S. schenckii as no immunoreaction was observed in SDS-extracts from other pathogenic and non-pathogenic fungi. Yeast cells of the fungus abundantly adhered to the dermis of mouse tails and the anti-Gp70 serum reduced this process in a concentration-dependent manner. Results are discussed in terms of the potential role of Gp70 in the host-pathogen interaction.

Bursera fagaroides, effect of an ethanolic extract on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica.

The effect of an ethanolic extract from the stem bark of Bursera fagaroides on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica was evaluated. For this purpose, increasing concentrations of the extract, up to 8.0mg/mL, were added to amoeba cultures or ODC reaction mixtures, which were incubated at 37 degrees C. Metronidazole and G418 were added as controls. After 1.5 and 72 h, the ODC activity in vitro and growth, respectively, were determined. Results revealed a strong inhibition of growth with IC(50) values on the order of 0.05 mg/mL. ODC activity, on the other hand, was inhibited by 12% and 50% at concentrations of 4.0 and 8.0mg/mL, respectively.

Biosynthesis of glycoproteins in the human pathogenic fungus Sporothrix schenckii: synthesis of dolichol phosphate mannose and mannoproteins by membrane-bound and solubilized mannosyl transferases.

A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg2+ and Mn2+. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A-Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26-112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.

Entamoeba histolytica: biochemical and molecular insights into the activities within microsomal fractions.

One of the most fascinating aspects of the Entamoeba histolytica trophozoite ultrastructure is the lack of a typical secretory pathway, particularly of rough endoplasmic reticulum and Golgi system, in a cell with such a high secretory activity. Here, we describe the isolation of amoeba cell structures containing ER-typical activities. Following isopycnic centrifugation of plasma membrane-free extracts, microsomes enriched in enzymatic activities such as dolichol-P-mannose synthase (DPMS; EC 2.4.1.83), UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (NAGPT; EC 2.7.8.15), and UDP-D-GlcNAc:dolichol-PP GlcNAc (NAGT; EC 2.4.1.141) were resolved from phagolysosomal fractions. Sec61alpha-subunit, an ER-marker involved in the translocation of nascent proteins to the ER, was found to co-fractionate with DPMS activity indicating that they are contained in microsomes with a similar density. Further, we optimized conditions for trophozoite homogenization and differential centrifugation that resulted in the separation of a 57,000 g-sedimenting microsomal fraction containing EhSec61alpha-subunit, EhDPMS, and EhPDI (protein disulfide isomerase, a soluble marker of the lumen of the ER). A relevant observation was the lack of ER markers associated to the nuclear fraction. Large macromolecular structures such as Ehproteasome were sedimented at a higher speed. Our knowledge of the molecular machinery involved in the biosynthesis of dolichol-linked oligosaccharide was enriched with the identification of putative genes related to the stepwise assembly of the dolichol-PP-GlcNAc(2)Man(5) core. No evidence of genes supporting further assembly steps was obtained at this time.