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Biomedical subjects

V Nussenzweig

Publications and source records attributed to V Nussenzweig.

At least 55 records · Page 3Linked to original sources
PubMed1994

Structural and functional properties of Trypanosoma trans-sialidase.

Sialic acids and sialidases play important roles in cellular interactions and modulate the recognition of pathogenic microbes by mammalian host cells. Protozoan parasites of the genus Trypanosoma express a unique sialic acid-metabolizing enzyme. This enzyme, named trans-sialidase (TS), catalyzes the transfer of sialic acids from host glycoconjugates to acceptor molecules of the parasite plasma membrane. In African trypanosomes, the agents of sleeping sickness, TS is found only in forms developing within the insect vector, and the enzyme sialylates the major surface protein. In Trypanosoma cruzi, the causative agent of Chagas' disease in Central and South America, TS is expressed both in the insect and mammalian forms of the parasite. The T. cruzi enzyme has been biochemically characterized, and the gene encoding the enzyme has been cloned. The enzyme sialylates abundant mucin-like molecules present on the surface of the parasite. Several lines of evidence suggest that TS and sialic acid acceptors on the surface of T. cruzi participate in host-parasite interactions and mediate the initial stages of the trypanosomes' invasion of host cells.

Amino Acid Sequence↗
PubMed1994

Intrasplenic immunization with infected hepatocytes: a mouse model for studying protective immunity against malaria pre-erythrocytic stage.

Malaria liver forms are the target of antibody or T-cell-mediated immune mechanisms induced by previous or subsequent developmental stages of the parasite. The potential for vaccine development of antigens expressed exclusively in the liver stages has not been fully explored partly because of the lack of an experimental animal model. Here we show that protective immunity against sporozoite-induced infection with Plasmodium yoelii and P. berghei can be obtained by intrasplenic injection of a small number of liver stages of the parasites. The serum of the protected animals did not contain antibodies against sporozoites, liver or blood stage malaria parasites. Protective immunity was abolished by depletion of either CD4+ or CD8+ T cells from the vaccinated mice before challenge.

Animals↗
PubMed1993

Malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes.

During feeding by infected mosquitoes, malaria sporozoites are injected into the host's bloodstream and enter hepatocytes within minutes. The remarkable target cell specificity of this parasite may be explained by the presence of receptors for the region II-plus of the circumsporozoite protein (CS) on the basolateral domain of the plasma membrane of hepatocytes. We have now identified these receptors as heparan sulfate proteoglycans (HSPG). The binding of CS to the receptors is abolished by heparitinase treatment, indicating that the recognition of region II-plus is via the glycosaminoglycan chains. We have purified and partially characterized the CS-binding HSPGs from HepG2 cells. They have a molecular weight of 400,000-700,000, are tightly associated with the plasma membrane, and are released from the cell surface by very mild trypsinization, a property which the CS receptors share with the syndecan family of proteoglycans.

Amino Acid Sequence↗
PubMed1993

Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor.

Here we report the presence of a trans-sialidase on the surface of Trypanosoma brucei culture-derived procyclic trypomastigotes. The enzyme is not detected in lysates of bloodstream trypomastigotes enriched for either stumpy or slender forms. The trans-sialidase catalyzes the transfer of alpha(2-3)-linked sialic acid residues to lactose. beta-galactopyranosyl residues are at least 100 times better acceptors for sialic acid than alpha-galactopyranosyl residues. In the absence of efficient acceptors, the purified enzyme transfers sialic acid to water, i.e., it acts as a sialidase. Although the T. cruzi and T. brucei trans-sialidases have very similar donor and acceptor specificities, they are antigenically distinct. Sodium dodecyl sulfate-polyacramide gel electrophoresis under nonreducing conditions and silver staining of the purified trans-sialidase reveals a single band of 63 kD. When the surface membrane of live procyclic trypomastigotes is trans-sialylated, using radioactive sialyllactose as the donor substrate, it appears that the only sialylated surface molecule is procyclin. Pronase treatment of live parasites removes only part of the surface sialic acid, in agreement with recent data showing that the glycosylphosphatidylinositol anchor of procyclin is sialylated (Ferguson, M. A. J., M. Murray, H. Rutherford, and M. J. McConville. 1993. Biochem. J. In press).

Animals↗
PubMed1993

Cells lacking glycan phosphatidylinositol-linked proteins have impaired ability to vesiculate.

Erythrocytes shed membrane vesicles in response to many stimuli. It has been previously demonstrated that glycan phosphatidylinositol-linked (GPI-linked) proteins such as decay accelerating factor and acetylcholinesterase are concentrated in these vesicles relative to the erythrocyte membrane. We have examined the requirement for GPI-linked proteins for the process of vesiculation. Erythrocytes that do not express GPI-linked proteins, obtained from patients with paroxysmal nocturnal hemoglobinuria (PNH), release between 10% and 50% of the quantity of vesicles as normal cells in response to the Ca2+ ionophore A23187. Platelets from the same patients produced 10% to 20% of the amount of vesicles as normal platelets. In addition, a mutant B-lymphoblastoid cell line that lacks GPI-linked molecules produces about half of the number of vesicles as compared with the wild-type cell line in response to the Ca2+ ionophore. Prior findings indicate that vesiculation is one of the mechanisms that the cell uses to remodel the plasma membrane, as well as protect itself from membrane-damaging agents such as the terminal complement components C5b-9. On the basis of the present results, we conclude that GPI-linked proteins play an important role in membrane vesiculation.

Acetylcholinesterase↗
PubMed1993

Trypanosoma rangeli sialidase lacks trans-sialidase activity.

Extracts and tissue culture supernatants of axenic forms of T. rangeli were assayed for the presence of sialidase and trans-sialidase activities. Using sialyl(alpha 2-3)lactose, sialyl(alpha 2-6)lactose, poly(alpha 2-8)N-acetylneuraminic acid, fetuin and 4-methylumbelliferyl-N-acetylneuraminic acid as sialic acid donors, and lactose as a sialic acid acceptor, no trans-sialidase activity was detected. Nevertheless, T. rangeli lysates and culture supernatants contain a sialidase that hydrolyzes sialyl(alpha 2-3)lactose, and much less efficiently sialyl(alpha 2-6)lactose, but not poly(alpha 2-8)N-acetylneuraminic acid. T. cruzi trans-sialidase hydrolyzed only sialyl(alpha 2-3)lactose under the same conditions. The T. rangeli and the T. cruzi enzymes differ antigenically and in their pH optimum for hydrolase activity.

Animals↗
PubMed1993

Transgenic mice expressing C-reactive protein are susceptible to infection with Plasmodium yoelii sporozoites.

Human and rat C-reactive proteins, major acute-phase reactants, bind to sporozoites and inhibit their in vitro development in hepatocytes (A. Nussler, S. Pied, M. Pontet, F. Miltgen, L. Renia, M. Gentilini, and D. Mazier, Exp. Parasitol. 72:1-7, 1991, and S. Pied, A. Nussler, M. Pontet, F. Miltgen, H. Matile, P.-H. Lambert, and D. Mazier, Infect. Immun. 57:278-282, 1989). We show here that rabbit C-reactive protein has identical properties. Nevertheless, infection by Plasmodium yoelii sporozoites was not prevented in transgenic mice engineered to express rabbit C-reactive protein following induction of gluconeogenesis.

Animals↗
PubMed1992

The basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of Plasmodium falciparum sporozoites.

Minutes after injection into the circulation, malaria sporozoites enter hepatocytes. The speed and specificity of the invasion process suggest that it is receptor mediated. We show here that recombinant Plasmodium falciparum circumsporozoite protein (CS) binds specifically to regions of the plasma membrane of hepatocytes exposed to circulating blood in the Disse space. No binding has been detected in other organs, or even in other regions of the hepatocyte membrane. The interaction of CS with hepatocytes, as well as sporozoite invasion of HepG2 cells, is inhibited by synthetic peptides representing the evolutionarily conserved region II of CS. We conclude that region II is a sporozoite ligand for hepatocyte receptors localized to the basolateral domain of the plasma membrane. Our findings provide a rational explanation for the target cell specificity of malaria sporozoites.

Amino Acid Sequence↗
PubMed1992

Evidence for the participation of the Ssp-3 antigen in the invasion of nonphagocytic mammalian cells by Trypanosoma cruzi.

Trypomastigotes of Trypanosoma cruzi have to invade mammalian cells in order to multiply. They bear on their plasma membrane a sialic acid-containing epitope (Ssp-3) defined by a series of monoclonal antibodies (mAbs). Previous investigations have shown that Fab fragments of these mAbs inhibit the attachment of trypomastigotes to 3T3 fibroblasts. To further define the role of Ssp-3 in invasion, here we use, as targets for infection, L cells and CHO cells stably transfected with cDNA coding for the mouse Fc receptors genes. When the trypomastigotes are incubated with small, nonagglutinating amounts of antibodies to Ssp-3, their attachment to the transfected cells is greatly enhanced, without a parallel increase in invasion. The enhancement in attachment is Fc mediated, since it is abolished by treatment of the transfected cells with mAbs to Fc receptors. In contrast, both attachment to, and invasion of, the transfected cells are increased if the parasites are incubated with polyclonal or monoclonal antibodies against T. cruzi surface membrane antigens other than Ssp-3. If, however, antibodies to Ssp-3 are added to the incubation mixtures containing any of the other anti-T. cruzi antibodies, the enhancement of invasion (but not of attachment) is reversed. These results suggest that Ssp-3-bearing molecules participate in the process of parasite internalization.

3T3 Cells↗
PubMed1992

Trypanosoma cruzi trans-sialidase and neuraminidase activities can be mediated by the same enzymes.

Trans-sialidase and neuraminidase activities have been detected on the surface membrane of trypomastigotes of Trypanosoma cruzi, and both have been implicated in the parasite's invasion of host cells. We show here that these enzymes are structurally related. They are recognized by two independently derived monoclonal antibodies, are anchored to the membrane by glycosylphosphatidylinositol, copurify by ion exchange, molecular sieving, and hydrophobic chromatography, have maximal activities between pH 6.5 and 7.5, and are inactivated by heating at 56 degrees C. Furthermore, the neuraminidase and trans-sialidase reactions are coupled. An increase of the concentration of acceptors of the transfer reaction decreases the amount of free sialic acid released through the neuraminidase reaction. We conclude that a single enzyme can catalyze the transfer or the hydrolysis of macromolecular-bound sialic acid. The predominant direction of the reaction will depend on the availability of appropriate oligosaccharide acceptors of sialic acid.

Animals↗
PubMed1992

Only some members of a gene family in Trypanosoma cruzi encode proteins that express both trans-sialidase and neuraminidase activities.

Trypomastigotes, the blood stage form of the human parasite Trypanosoma cruzi, contain an enzyme on their surface, trans-sialidase, which catalyses the transfer of sialic acid from host glycoconjugates to acceptors on its own cell surface. At least a subset of the sialic acid-bearing acceptor molecules are involved in parasite invasion of host cells, an essential step in the life cycle of the parasite. Another trypomastigote surface enzyme that affects host cell invasion is neuraminidase and recent evidence suggests that both trans-sialidase and neuraminidase activities may be expressed by the same proteins on the parasite surface. We describe here the isolation and expression of several members of a trans-sialidase--neuraminidase gene family from T.cruzi. One of the isolated genes does indeed encode a protein with both trans-sialidase and neuraminidase activities, while other members of the gene family encode closely related proteins that express neither enzymatic activity. Chimeric protein constructs combining different portions of active and inactive genes identified a region of the gene necessary for enzymatic activity. Sequence analysis of this portion of the gene revealed a limited number of amino acid differences between the predicted active and inactive gene products.

Amino Acid Sequence↗
PubMed1992

Binding of malarial circumsporozoite protein to sulfatides [Gal(3-SO4)beta 1-Cer] and cholesterol-3-sulfate and its dependence on disulfide bond formation between cysteines in region II.

Region II of the malaria circumsporozoite (CS) protein is highly conserved between the CS proteins of different species of malaria. Amino acid sequences homologous to that of region II are found in thrombospondin, properdin, von Willebrand factor and a few other proteins. We show here that the native CS protein from the rodent parasite Plasmodium berghei, and recombinant Plasmodium vivax and Plasmodium falciparum CS proteins containing region II, but not recombinant proteins lacking region II, specifically bind to sulfatides and cholesterol-3-sulfate. The binding is abolished following reduction and alkylation of the proteins. Region II contains 2 cysteines separated by only 3 amino acids, S(N), V, T, and these are the only cysteines present in our recombinant proteins. Therefore, our findings strongly suggest that the region II cysteines are linked by a disulfide bond forming a small peptide loop. We also present evidence that the recognition of sulfatides, cholesterol-3-sulfate, or other cross-reactive sulfated macromolecules by region II may be required during sporozoite invasion of liver cells. Antibodies to a peptide representing region II react with live sporozoites and with sporozoites fixed with glutaraldehyde, indicating that this region is exposed on the surface of the parasites. Furthermore, we have found that the sulfatide and cholesterol-3-sulfate recognition by the CS proteins, and the invasion of hepatocytes by P. berghei sporozoites, are specifically inhibited by dextran sulfate.

Amino Acid Sequence↗
PubMed1992

Substrate specificity of the Trypanosoma cruzi trans-sialidase.

Trypanosoma cruzi trypomastigotes acquire sialic acid (SA) from host glycoconjugates by means of a plasma membrane-associated trans-sialidase (TS). Here we study the substrate specificity of TS, which differs from all known sialyltransferases in that it does not require cytidine monophosphate (CMP)-SA as donor. The T. cruzi TS reversibly transfers SA to saccharides with terminal beta-Gal (but not alpha-Gal) residues. Donors are saccharides with SA linked to terminal beta-Gal residues by (alpha 2-3), but not (alpha 2-6) bonds. The type of beta-linkage of the terminal Gal residue is of minor importance (beta 1-4 and beta 1-6 are slightly better than beta 1-3), whereas chain length and the structure of additional vicinal sugar residues are not relevant. SA on the surface of living trypomastigotes of T. cruzi is transferred back and forth between the parasite surface and acceptor molecules with terminal beta-Gal, either in solution or on the surface of neighbouring mammalian cells. Addition of fucose residue on or close to the terminal galactose impairs TS activity. As a consequence, the enzyme acts poorly on the E-selectin ligand sialyl-Lewisx and its precursor Lewisx, and in vitro adhesion of TS-treated neutrophils to L-cells expressing L-selectin is not affected. Modifications in the structure of the (alpha 2-3)-linked N-acetyl-neuraminic acid (Neu5Ac) (deoxy or methoxy) of the donor molecules do not impair transfer if the changes are at C9, whereas changes at C4, C7 and C8 impair the ability to donate the modified SA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Resialylation of sialidase-treated sheep and human erythrocytes by Trypanosoma cruzi trans-sialidase: restoration of complement resistance of desialylated sheep erythrocytes.

Trypanosoma cruzi trans-sialidase (TS) is a recently described enzyme which transfers alpha(2-3)-linked sialic acid from host-derived sialylated glycoconjugates to parasite surface molecules [Schenkman et al. (1991) Cell, 65, 1117]. We report here on the ability of TS to transfer sialic acid from donor sialyl-alpha(2-3)lactose to sialidase-treated sheep and human erythrocytes. Up to approximately 50% resialylation of both desialylated red cells could be attained. Resialylation of desialylated sheep erythrocytes restores their resistance to lysis by human complement. This ascribes a possible biological role for T. cruzi TS and demonstrates directly that sialic acid is solely responsible for preventing alternative pathway activation of human complement by sheep erythrocytes.

Animals↗
PubMed1992

Stage-specific expression and intracellular shedding of the cell surface trans-sialidase of Trypanosoma cruzi.

We have used antibodies to the Trypanosoma cruzi trans-sialidase and to its product, the host cell invasion-related Ssp-3 epitope, to study the expression of the corresponding antigens during the intracellular development of the parasite and in the extracellular trypomastigotes. As soon as 2 h after host cell invasion, trans-sialidase was no longer detected, whereas the Ssp-3 epitope was still present on intracellular parasites. The amastigotes which subsequently developed remained nonreactive with the antibodies. Expression of enzymatically active T. cruzi trans-sialidase started again only after transformation of the amastigotes into trypomastigotes 72 h after host cell invasion. trans-Sialidase was shed from the trypanosomes into the host cell cytoplasm, where the enzyme accumulated until release of the parasites. All released trypomastigotes expressed trans-sialidase on their surfaces and in the flagellar pockets, but stumpy trypomastigotes were stained more intensely than slender trypomastigotes. Ssp-3, the sialylated reaction product of trans-sialidase, was assembled only after rupture of the host cell membrane and was detected on the plasma membranes and in the flagellar pockets of all trypomastigotes.

Animals↗
PubMed1992

An improved polymerase chain reaction assay to detect Trypanosoma cruzi in blood.

Amplification by the polymerase chain reaction of Trypanosoma cruzi satellite DNA was used to enhance sensitivity in the detection of the parasite in blood, with the ultimate goal of improving diagnosis of the chronic phase of Chagas' disease. Two contiguous oligonucleotides were synthesized corresponding to the most conserved region of the 195-basepair repeated sequence and used as primers for the amplification reaction. Nineteen femtograms of parasite DNA that was amplified in the presence of 15 micrograms of human or mouse DNA produced a visible band upon electrophoresis in agarose gels and staining with ethidium bromide. In reconstitution experiments, one parasite in 10 ml of blood could be unambiguously determined when the DNA was isolated from nuclei after the blood was treated with NP40 and centrifuged. Polymerase chain reaction assays were carried out to detect T. cruzi in chronically infected mice. Most mice were parasite-positive when organs or tissues were tested, but all were negative when total blood was tested.

Animals↗
PubMed1991

Membrane vesiculation protects erythrocytes from destruction by complement.

Nucleated cells can resist attack by C by exocytosis or endocytosis of the terminal C components C5b-9 (membrane attack complex) (MAC), but it is generally accepted that formation of a single MAC channel on E leads to lysis (one-hit theory). We find that human and guinea pig E, but not SRBC, can eliminate the MAC from the membrane in the form of microvesicles and escape destruction. When guinea pig or human E are incubated with C5b-9, vesiculation proceeds without a lag and is detected at nonlytic doses of C9. Continuous Ca2+ influx is required for vesiculation. The amount of released vesicles is in direct relation to Ca2+ concentration, and the increase in vesiculation is associated with a parallel decrease in lysis. SRBC, which do not vesiculate when Ca2+ loaded, are lysed by C5b-9 with the same efficiency in the presence or absence of Ca2+. Vesicles released from guinea pig RBC under C5b-9 attack are enriched in C9 by a factor of 10, compared with the unlysed cells, and by a factor of 3 to 4, compared with ghosts. We conclude that E are protected from lysis not only by CD59 and C8bp/HRF, which prevent MAC assembly, but also by selective elimination of the MAC.

Animals↗