The surface free energy of Leishmania mexicana amazonensis.

Surface charge of Leishmania mexicana amazonensis was investigated by direct zeta-potential determination and ultrastructural cytochemistry, and its surface tension was studied by measurements of the advancing contact angle formed by the parasite monolayers with drops of liquids of different polarities. Both virulent and avirulent promastigotes exhibited negatively charged surfaces with a zeta-potential of about -15 mV. Treatment of these cells with trypsin, alkaline phosphatase, or phospholipase C rendered their surfaces less negatively charged, whereas neuraminidase did not alter the parasite negativeness. Cytochemically, we could observe a reduction in the cationized ferritin binding after the parasite treatment with each of the former enzymes, but not with neuraminidase. The surface free energy of parasites was calculated by taken to account the London dispersion, the Keeson dipole-dipole, and the Debye dipole-induced forces, as well as the surface polarity of the parasites and their zeta-potentials, by considering their adhesion to polystyrene surfaces. The delta G values of -6.4 and -18.1 mJ.m-2 were obtained for avirulent and virulent promstigotes, respectively.     

Structures of the major oligosaccharides from a human rectal adenocarcinoma glycoprotein

Four oligosaccharides in the reduced form were isolated from RMG (a mucin-type glycoprotein from a human rectal adenocarcinoma). They were 1) Sia alpha s2 leads to 6GalNAc-ol; 2) Sia alpha 2 leads to 6(Gal beta 1 leads to 3)GalNAc-ol; 3) Sia alpha 2 leads to 6(GlcNAc beta 1 leads to 3)GalNAc-ol; and 4) Sia alpha 2 leads to 6(GalNAc alpha 1 leads to 3)GalNAc-ol. The amounts of oligosaccharides 1, 2, 3, and 4 corresponded to 27, 5, 11, and 8% of the total N-acetylgalactosaminitol produced on alkaline borohydride treatment of RMG. To determine the structures of oligosaccharides 2, 3, and 4, a mixture of the three was subjected to methylation analysis which revealed that the N-acetylgalactosaminitol was substituted at both C-3 and C-6 and other sugars at the nonreducing ends. Desialized oligosaccharides were prepared, and the structures were deduced by analysis of the permethylated sugars on gas-liquid chromatography/mass spectrometry. Anomeric configurations were determined by exoglycosidase digestions except for galactose which was analyzed by chromium trioxide oxidation.     

Inhibition of growth of Leishmania mexicana mexicana by Leishmania mexicana amazonensis during "in vitro" co-cultivation

Inhibition of one Leishmania subspecies by exometabolites of another subspecies, a phenomenon not previously reported, is suggested by our recent observations in cell cloning experiments with Leishmania mexicana mexicana and Leishmania mexicana amazonensis. Clones were identified using the technique of schizodeme analysis. The phenomenon observed is clearly relevant to studies of parasite isolation, leishmanial metabolism, cross-immunity and chemotherapy.     

Cell surface carbohydrate of Leishmania mexicana amazonensis: differences between infective and non-infective forms

The cell surface carbohydrates of Leishmania mexicana amazonensis (amastigotes and promastigotes, both infective and non-infective forms) were comparatively analyzed by agglutination assay employing 28 highly purified lectins, and by binding assay using 125I-labeled lectins. Among the D-GalNAc binding lectins, Bandeiraea simplicifolia-I, Dolichos biflorus, Phaseolus vulgaris and Glycine max were highly specific for the amastigotes, while that from Maclura aurantiaca selectively agglutinated promastigotes. The lectins from Wistaria floribunda, Phaseolus lunatus (D-GalNAc), Arachis hypogaea (D-Gal) and Triticum vulgaris (D-GlcNAc) were selective for the infective forms (both amastigotes and promastigotes), not reacting with the non-infective ones. Conversely, no parasite agglutination occurred with the L-fucose binding lectins Lotus tetragonolobus and Ulex europaeus-I. Binding studies with 125I-labeled lectins from Wistaria floribunda, Triticum vulgaris and Arachis hypogaea were performed to find whether unagglutinated non-infective promastigotes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the selectivity, demonstrated in the agglutination assays of these lectins for the infective promastigotes.     

Identification of a gp63 surface glycoprotein in Leishmania tarentolae

The promastigote stage of most if not all Leishmania species possesses an abundant surface glycoprotein of 63 kDa (gp63) that has protease activity. We show that the lizard parasite Leishmania tarentolae appears to lack the surface protease activity. L. tarentolae does, however, possess an approximately 63-kDa molecule that is antigenically cross-reactive with the L. major gp63. Additionally, the genome of L. tarentolae contains sequences that hybridise at high stringency to a L. major gp63 gene probe.     

The macrophage-attachment glycoprotein gp63 is the predominant C3-acceptor site on Leishmania mexicana promastigotes

The interaction between Leishmania promastigotes and their vertebrate host's complement system results not only in parasite lysis but also, due to surface-bound complement components, in increased macrophage binding potential. In this study we demonstrate, with the use of isolated complement components, that activation is via the alternative complement pathway, initiated by direct deposition of C3 onto the parasite surface. The predominant C3 acceptor site on the promastigotes was initially identified as the glycoprotein gp63 by anti-C3 antibody immunoprecipitation of radioiodinated promastigotes following incubation in the alternative pathway initiators C3, and factors B and D. The C3-binding properties of gp63 were confirmed and quantified, in relation to other surface antigens, by incubating parasites in iodinated C3 and immunoprecipitating bound C3 with antibodies directed against different promastigote surface antigens. The other abundant surface antigen, the glycolipid 'excreted factor', did not show any C3-binding activity. Further demonstration was provided by incubating liposomes containing either gp63 or excreted factor in iodinated C3 and factors B and D. Only gp63-containing liposomes bound C3. Considering that both gp63 and the excreted factor have recently been implicated in attachment and uptake by macrophage, these findings may have considerable bearing in the determination of which of the macrophage surface receptors identify which parasite ligand.     

The involvement of the major surface glycoprotein (gp63) of Leishmania promastigotes in attachment to macrophages

The interaction between the macrophage and the parasite plays a central role in the continued success of Leishmania infection. The promastigote surface ligand, and its complementary macrophage membrane receptor, involved in attachment and phagocytosis are likely to exert considerable influence over the outcome of a new infection. In this study, we report experiments pertaining to one such parasite membrane protein. Initial examination of promastigote surface proteins by radiolabeling and two-dimensional-polyacrylamide gel electrophoresis revealed an abundant polypeptide with an apparent m.w. of 63,000. Lectin-binding studies indicated that it was a glycoprotein containing mannose, N-acetyl glucosamine, and N-acetyl galactosamine residues. Monospecific antiserum raised against this glycoprotein, gp63, decorated the entire promastigote plasmalemma. Univalent antibody fragments from this antiserum blocked the interaction between promastigotes and macrophages by inhibiting attachment. Anti-gp63-inhibition reduced parasite/macrophage binding to 30 to 35% of the control binding level. Additional evidence of the involvement of gp63 in attachment to macrophages was provided by studies that made use of gp63-containing proteoliposomes. These vesicles were avidly phagocytosed by macrophages. Uptake of the gp63-containing liposomes was suppressed by greater than 90% by both anti-gp63 F(ab) fragments and the oligosaccharide mannan, indicating that their phagocytosis was receptor dependent. These results demonstrate that the abundant glycoprotein gp63 plays an important role in attachment of promastigotes to macrophages, and attachment via this parasite ligand is sufficient to trigger phagocytosis.     

The role of N-linked oligosaccharides in glycoprotein function.

N-linked glycoproteins include such biologically important molecules as cell-surface receptors, cell-adhesion molecules, immunoglobulins and other serum proteins, and tumor antigens. Investigating the role of carbohydrate in glycoprotein function has included the use of glycosylation inhibitors or site-directed mutagenesis of specific glycosylation sites to prevent the addition of carbohydrate, or glycosylation processing inhibitors or animal cell glycosylation mutants to alter carbohydrate structure. In some proteins, glycosylation plays an important role in recognition, while in others, it may stabilize and/or control the conformation of the protein. The cloning of genes in bacteria or lower eukaryotes--with the goal of producing biologically active proteins for biotechnological purposes--necessitates a better understanding of the role of specific carbohydrate structures.     

N-linked oligosaccharides as outfitters for glycoprotein folding, form and function

Glycosylation, particularly N-linked glycosylation, profoundly affects protein folding, oligomerization and stability. The increased efficiency of folding of glycosylated proteins could be due to the chaperone-like activity of glycans, which is observed even when the glycan is not attached to the protein. Covalently linked glycans could also facilitate oligomerization by mediating inter-subunit interactions in the protein or stabilizing the oligomer in other ways. Glycosylation also affects the rate of fibril formation in prion proteins: N-glycans reduce the rate of fibril formation, and O-glycans affect the rate either way depending on factors such as position and orientation. It has yet to be determined whether there is any correlation among the sites of glycosylation and the ensuing effect in multiply glycosylated proteins. It is also not apparent whether there is a common pattern in the conservation of glycans in a related family of glycoproteins, but it is evident that glycosylation is a multifaceted post-translational modification. Indeed, glycosylation serves to "outfit" proteins for fold-function balance.     

Granulocyte-macrophage colony-stimulating factor is a growth-factor for promastigotes of Leishmania mexicana amazonensis

In this paper we show that murine lung conditioned medium (LCM) displays, in addition to its already described colony-stimulating activity on bone marrow cells, a potent growth-stimulating activity on promastigotes of Leishmania mexicana amazonesis. Immunoprecipitation of LCM with an antibody specific for murine granulocyte-macrophage colony stimulating factor (GM-CSF) abrogates both activities, indicating that the leishmanial growth-promoting activity is due to the presence of GM-CSF on LCM. Furthermore, recombinant GM-CSF (rGM-CSF) added to the culture medium or to the immunoprecipitated LCM is able to respectively induce or to partially recover the growth-promoting activity of the LCM. Sequential in vitro passages of the parasite induces a progressive loss of sensitivity to the growth-factor. Parasite forms recently collected from lesions are significantly more responsive to the growth-factor than forms already adapted to grow in culture. Since it has been shown that several different microorganisms display receptors for vertebrate-like hormones and that GM-CSF is able to enhance a cutaneous leishmanial lesion, our results permit us to raise the hypothesis that a direct interaction between a host-derived hormone and a pathogenic microorganism can be of importance in defining the fate of an infection. The fact that GM-CSF is produced by cells that actively participate in a leishmanial infection (T-lymphocytes and macrophages) reinforces our hypothesis.     

Granulocyte-Macrophage Colony-Stimulating Factor is a Growth-Factor for Promastigotes of Leishmania mexicana amazonensis

ABSTRACT. In this paper we show that murine lung conditioned medium (LCM) displays, in addition to its already described colony-stimulating activity on bone marrow cells, a potent growth-stimulating activity on promastigotes of Leishmania mexicana amazonensis . Immunoprecipitation of LCM with an antibody specific for murine granulocyte-macrophage colony stimulating factor (GM-CSF) abrogates both activities, indicating that the leishmanial growth-promoting activity is due to the presence of GM-CSF on LCM. Furthermore, recombinant GM-CSF (rGM-CSF) added to the culture medium or to the immunoprecipitated LCM is able to respectively induce or to partially recover the growth-promoting activity of the LCM. Sequential in vitro passages of the parasite induces a progressive loss of sensitivity to the growth-factor. Parasite forms recently collected from lesions are significantly more responsive to the growth-factor than forms already adapted to grow in culture. Since it has been shown that several different microorganisms display receptors for vertebrate-like hormones and that GM-CSF is able to enhance a cutaneous leishmanial lesion, our results permit us to raise the hypothesis that a direct interaction between a host-derived hormone and a pathogenic microorganism can be of importance in defining the fate of an infection. The fact that GM-CSF is produced by cells that actively participate in a leishmanial infection (T-lymphocytes and macrophages) reinforces our hypothesis.     

Structures of N-linked oligosaccharides of glycoproteins from tobacco BY2 suspension cultured cells

The structures of N-linked sugar chains of glycoproteins expressed in tobacco BY2 cultured cells are reported. Five pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the glycoproteins by reversed-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were identified by two-dimensional PA-sugar chain mapping, ion-spray MS/MS analysis, and exoglycosidase digestions. The five structures fell into two categories; the major class (92.5% as molar ratio) was a xylose containing-type (Man3Fuc1 Xyl1GlcNAc2 (41.0%), GlcNAc2Man3Fuc1Xyl1GlcNAc2 (26.5%), GlcNAc1Man3Fuc1Xyl1GlcNAc2 (21.7%), Man3 Xyl1GlcNAc2 (3.3%)), and the minor class was a high-mannose type (Man5GlcNAc2 (7.5%)). This is the first report to show that alpha(1-->3) fucosylation of N-glycans does occur but beta(1-->4) galactosylation of the sugar chains does not in the tobacco cultured cells.     

Structures of N-linked oligosaccharides of microsomal glycoproteins from developing castor bean endosperms.

The structures of sugar chains of the glycoproteins from the microsomal fraction of developing castor bean endosperms have been analyzed. The structural analyses were done by a fluorescence method combined with component analysis, exoglycosidase digestions, partial acetolysis, Smith degradation, and 1H-NMR spectroscopy. The estimated structures fell into three categories; the first was oligomannose-type, the second xylomannose-type, the third complex-type. Among these oligosaccharides, Man3Fuc1Xyl1GlcNAc2 (M3FX) and Man6GlcNAc2 (M6B) were the major structures. The structures of Man4GlcNAc2 (M4C) and Man4Xyl1GlcNAc2 (M4X) have also been found in the microsomal glycoproteins of the developing bean endosperms. These results could indicate that the structures of M4C, M4X, and M3FX are formed in the stage of sugar chain processing in the microsomal fraction, in which oligomannose-type sugar chains are modified into complex-type ones by several kinds of processing enzymes.     

Structural and antigenic characterization of a species- and promastigote-specific Leishmania mexicana amazonensis membrane protein

A Leishmania mexicana amazonensis promastigote membrane glycoprotein (Mr 46,000) expressing the species-specific and promastigote-specific epitope of monoclonal antibody IX 2H7-E10(M-2) has been purified to homogeneity, and studies have been made to determine the minimum peptide fragment that retained antigenic activity. Peptide mapping experiments performed with the metabolically labeled or surface radioiodinated protein illustrated its highly folded nature and marked resistance to proteolytic digestion. The M-2 epitope was readily destroyed by limited proteolysis and/or reduction and alkylation, indicating disulfide bond involvement in its formation by at least the secondary protein structure. The stability of approximately half of the molecular mass of the protein (46kDa/M-2) was also dependent on disulfide bonding. Enzymic digests under various conditions generated a glycopolypeptide (Mr 22,000 to 27,000), extremely resistant to further enzymic digestion, that was the dominant immunogenic portion of the purified protein recognized by a specific rabbit heteroserum. No smaller or larger fragments were antigenic. Data obtained by using the radioiodinated hydrophobic probe 3-(trifluoromethyl)-3-([m-125]iodophenyl)-diazirine ([125I]TID) indicate that 46kDa/M-2 is an integral membrane protein with a component polypeptide (Mr 23,000 to 27,000), highly resistant to further enzymic cleavage and containing sequences within the external promastigote membrane. Data indicate that the [125I]TID-labeled fragment is identical to the immunodominant fragment. We suggest that hydrophobic interactions maintain the integrity of this fragment as amino acids within it fold through the parasite membrane.