The Recognition of N-Glycans by the Lectin ArtinM Mediates Cell Death of a Human Myeloid Leukemia Cell Line

ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus (jackfruit), interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6)Manβ1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC50 = 10 µg/mL), as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment

Lipidomic Analysis of Extracellular Vesicles from the Pathogenic Phase of Paracoccidioides brasiliensis

Background: Fungal extracellular vesicles are able to cross the cell wall and transport molecules that help in nutrient acquisition, cell defense, and modulation of the host defense machinery.Methodology/Principal Findings: Here we present a detailed lipidomic analysis of extracellular vesicles released by Paracoccidioides brasiliensis at the yeast pathogenic phase. We compared data of two representative isolates, Pb3 and Pb18, which have distinct virulence profiles and phylogenetic background. Vesicle lipids were fractionated into different classes and analyzed by either electrospray ionization- or gas chromatography-mass spectrometry. We found two species of monohexosylceramide and 33 phospholipid species, including phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. Among the phospholipid-bound fatty acids in extracellular vesicles, C181 predominated in Pb3, whereas C18:2 prevailed in Pb18. the prevalent sterol in Pb3 and Pb18 vesicles was brassicasterol, followed by ergosterol and lanosterol. Inter-isolate differences in sterol composition were observed, and also between extracellular vesicles and whole cells.Conclusions/Significance: the extensive lipidomic analysis of extracellular vesicles from two P. brasiliensis isolates will help to understand the composition of these fungal components/organelles and will hopefully be useful to study their biogenesis and role in host-pathogen interactions.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)National Institutes of Health (NIH)Universidade Federal de São Paulo, UNIFESP, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv Texas El Paso, Dept Biol Sci, Border Biomed Res Ctr, El Paso, TX 79968 USAUniversidade Federal de São Paulo, UNIFESP, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPESP: 06/05095-6FAPESP: 07/04757-8FAPESP: 07/59768-4CNPq: 301666/2010-5National Institutes of Health (NIH): 5G12RR008124-16A1National Institutes of Health (NIH): 5G12RR008124-16A1S1National Institutes of Health (NIH): G12MD007592Web of Scienc

Immunological Basis for the Gender Differences in Murine Paracoccidioides brasiliensis Infection

This study aimed to investigate the immunological mechanisms involved in the gender distinct incidence of paracoccidioidomycosis (pcm), an endemic systemic mycosis in Latin America, which is at least 10 times more frequent in men than in women. Then, we compared the immune response of male and female mice to Paracoccidioides brasiliensis infection, as well as the influence in the gender differences exerted by paracoccin, a P. brasiliensis component with carbohydrate recognition property. High production of Th1 cytokines and T-bet expression have been detected in the paracoccin stimulated cultures of spleen cells from infected female mice. In contrast, in similar experimental conditions, cells from infected males produced higher levels of the Th2 cytokines and expressed GATA-3. Macrophages from male and female mice when stimulated with paracoccin displayed similar phagocytic capability, while fungicidal activity was two times more efficiently performed by macrophages from female mice, a fact that was associated with 50% higher levels of nitric oxide production. In order to evaluate the role of sexual hormones in the observed gender distinction, we have utilized mice that have been submitted to gonadectomy followed by inverse hormonal reconstitution. Spleen cells derived from castrated males reconstituted with estradiol have produced higher levels of IFN-γ (1291±15 pg/mL) and lower levels of IL-10 (494±38 pg/mL), than normal male in response to paracoccin stimulus. In contrast, spleen cells from castrated female mice that had been treated with testosterone produced more IL-10 (1284±36 pg/mL) and less IFN-γ (587±14 pg/mL) than cells from normal female. In conclusion, our results reveal that the sexual hormones had a profound effect on the biology of immune cells, and estradiol favours protective responses to P. brasiliensis infection. In addition, fungal components, such as paracoccin, may provide additional support to the gender dimorphic immunity that marks P. brasiliensis infection

Lectin KM+-induced neutrophil haptotaxis involves binding to laminin

The lectin KM+ from Artocarpus integrifolia, also known as artocarpin, induces neutrophil migration by haptotaxis. the interactions of KM+ with both the extracellular matrix (ECM) and neutrophils depend on the lectin ability to recognize mannose-containing glycans. Here, we report the binding of KM+ to laminin and demonstrate that this interaction potentiates the KM+-induced neutrophil migration. Labeling of lung tissue by KM+ located its ligands on the endothelial cells, in the basement membrane, in the alveolus, and in the interstitial connective tissue. Such labeling was inhibited by 400 mM D-mannose, 10 mM Manalpha1-3[Manalpha1-6]Man or 10 muM peroxidase (a glycoprotein-containing mannosyl heptasaccharide). Laminin is a tissue ligand for KM+, since both KM+ and anti-laminin antibodies not only reacted with the same high molecular mass components of a lung extract, but also determined colocalized labeling in basement membranes of the lung tissue. the relevance of the KM+-laminin interaction to the KM+ property of inducing neutrophil migration was evaluated. the inability of low concentrations of soluble KM+ to induce human neutrophil migration was reversed by coating the microchamber filter with laminin. So, the interaction of KM+ with laminin promotes the formation of a substrate-bound KM+ gradient that is able to induce neutrophil haptotaxis. (C) 2004 Elsevier B.V. All rights reserved.Univ São Paulo, Fac Med Ribeirao Preto, Dept Biol Celular & Mol & Bioagentes Patogenicos, BR-14049900 Ribeirao Preto, SP, BrazilUniv São Paulo, Fac Med Ribeirao Preto, Dept Farmacol, BR-14049900 Ribeirao Preto, SP, BrazilUniversidade Federal de São Paulo, Ctr Electron Microscopy, BR-04023062 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Ctr Electron Microscopy, BR-04023062 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilWeb of Scienc

The novel lectin KM plus detects a specific subset of mannosyl-glycoconjugates in the rat cerebellum

KM+ is a D(+) mannose-specific lectin with a carbohydrate structure-affinity relationship different from those of most mannose-binding lectins. KM+ elicits carbohydrate-dependent biological effects in several mammalian cell types, but it has not yet been employed as a probe for the detection of its specific ligands. We show here for the first time the screening and partial identification of cerebellar mannosyl-glycoconjugates recognized by KM+, by means of lectin-histochemistry and lectin-blotting. Biotinylated KM+ stained most cellular structures in the adult rat cerebellum, particularly Purkinje cells bodies and the surface of granule cells, but not cellular processes. Capillaries in the choroid plexus were also strongly decorated, while blood vessels in the cerebellar parenchyma remained unstained. D(+) mannose, but not D(+) galactose, abolished the staining of all cerebellar structures. Higher inhibitory potencies were found for mannosyl-glycans such as mannotriose (man-alpha1,3-[man-alpha1,6]-man) and the biantennary heptasaccharide carried by the enzyme horseradish peroxidase. After separation of cerebellar proteins by SDS-PAGE, KM+ recognized three major unidentified mannosylglycoproteins of 132, 83 and 49 kDa. KM+ also detected high-Mw bands corresponding to the light and heavy chains of Type-I laminin, but not a 160-kDa cleavage product of laminin. We conclude that KM+ binds preferentially to a specific subset of mannose-containing glycoproteins in cerebellar tissue, thus being much more restricted than other mannose-specific lectins. KM+ can be used as a novel probe to screen the central nervous system for this specific subset of complex mannosyl-glycoconjugates.204185850150