2 Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, K7L3N6, Canada; 3 Fundamental and Applied Molecular Biology, Ghent University, Flanders Interuniversity Institute for Biotechnology (V.I.B.) Technologiepark 927 B-9052 Gent-Zwijnaarde Belgium. 4 Laboratory of Neural Signaling, Cell Biology Group, Robarts Research Institute, London, Ontario, N6A 5K8, Canada; and 5 Department of Physiology, Queen's University, Kingston, Ontario, K7L3N6, Canada
Received on May 4, 2004; revised on June 28, 2004; accepted on June 29, 2004
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Abstract |
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Key words: cell signaling / receptor activation / sialic acid / TrkA tyrosine kinase receptor / trypanosome trans-sialidase
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Introduction |
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Using the rat pheochromocytoma cell line PC12 and Schwann cells, Chuenkova et al. (2001; Chuenkova and Pereira, 2000
) discovered that a highly purified trans-sialidase (TS) derived from T. cruzi parasites induced neurite outgrowth in these cells as well as mediated neuroprotection from apoptotic death caused by growth factor deprivation. These observations provided an important clue to a possible clinical significance of TS-mediated neuroprotection during the indeterminate phase in patients infected with T. cruz (Genovese et al., 1996
; Koberle, 1968
). Indeed, most individuals infected with T. cruzi survive the acute infection to enter a subclinical, asymptomatic, indeterminate phase that lasts for decades. These patients showed no signs of peripheral neuropathy (Genovese et al., 1996
). Earlier studies of autopsied Chagas' disease cases revealed relatively few lesions in the autonomic nervous system of the heart and gastrointestinal tract (Koberle, 1968
). The average number of neurons actually increased with the age of the patient, in contrast to that found in the age-matched disease-free individuals (Koberle, 1968
). Furthermore, rodents infected with T. cruzi also showed signs of neurite development, axon regeneration, and sprouting in sympathetic nerve fibers of the heart and colon (Losavio et al., 1989
; Machado et al., 1987
). Whether T. cruzi TS plays a neuroprotective role in these patients remains speculative. It is interesting to note here that T. cruzi TS has two unique catalytic properties (Colman and Smith, 2002
; Lee and Kim, 2001
), one of which is the sialyltransferase activity that catalyzes the transfer of
2,3-linked sialic acids to terminal ß-D-galactose acceptors, and the other is the sialidase activity that hydrolyzes these sialic acids from sialyl-oligosaccharides. It is intriguing to speculate that TS might target glycosylated Trk expressing neurons, hydrolyzing
2,3-linked sialic acids and inducing receptor internalization and activation.
In the present study, the rat pheochromocytoma cell line PC12 and its mutant Trk derivatives (MacDonald et al., 2000; Meakin et al., 1997
, 1999a
; Meakin and MacDonald, 1998
) were used as well-characterized models to further study the neuronal receptor involved in TS-mediated receptor activation and cell differentiation. We demonstrate that TS targets TrkA receptors and colocalizes with TrkA internalization and activation, which subsequently leads to neurite outgrowth in TrkA-expressing PC12 cells.
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Results |
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To confirm that TrkA receptors may be involved in TS-induced neurite outgrowth, neurite extensions were examined in TS-treated PC12nnr5 cells stably transfected with rat TrkA receptors (B2 and B5 cells) (Meakin et al. 1997; Meakin and MacDonald, 1998
). TS-treated B5 cells that overly express TrkA receptors by 100-fold compared to PC12 cells extended neurites similar to those observed for NGF-treated cells (Figure 2). TS-treated B2 cells which overly express TrkA receptors by fourfold compared to PC12 cells extended neurites, but these were also much shorter and fewer than those observed extending from NGF-treated cells. These findings suggest that TS-induced neurite outgrowth in PC12 cells requires TrkA receptors.
When PC12 cells were pretreated with K252a, an inhibitor of Trk tyrosine kinase or PD98059, a MAP/MEK protein kinase inhibitor, the effects of NGF- and TS-induced neurite outgrowth in these inhibitor-treated cells were both blocked (Figure 3). These results support the previous observations that TS-induced cell differentiation of naive PC12 cells involves TrkA tyrosine kinase as the signal generating receptor kinase and that the signal is transduced through the MAP/MEK protein kinase pathways for cell differentiation.
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TS induces phosphorylation (pTyr490) of TrkA receptors
If TS binds to TrkA receptors, we asked whether TS would induce phosphorylation of TrkA (pTrkA). We analyzed this question in fixed and permeabilized TrkA-PC12 cells using phospho-specific anti-pTyr490 Trk antibody and immunofluorescence microscopy. Following TS treatment of cells for 15, 30, 60, and 120 min, the immunostained cells revealed pTrkA after 15 min of treatment (Figure 5A). In contrast, these effects were not observed in TrkA-deficient PC12nnr5 cells but were reestablished in PC12nnr5 cells stably transfected with TrkA (Figure 5A).
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TS colocalizes with pTrkA
If TS induces TrkA activation by binding to the receptor and catalyzing the hydrolysis of sialyl -2,3-linked ß-galactosyl residues, then TS should colocalize with phosphorylated TrkA (pTyr490). To test this, TrkA-PC12 cells were treated with TS for 5-, 15-, 30-, or 60-min intervals or were left untreated as controls. Cells were fixed, permeabilized, and immunostained simultaneously with monoclonal mouse anti-E tag antibody specific for TS and polyclonal rabbit anti-pTyr490 antibody specific for pTrkA followed by anti-mouse Alexa Fluor 488 (green) and anti-rabbit Alexa Fluor 568 (red). Cells were then visualized using a confocal inverted microscope (Leica TCS SP2 MP inverted Confocal Microscope) using a 100x objective (oil). TS colocalized with pTrkA 15 min after treatment, and the amount of colocalization in the cell increased over a 60 min incubation with TS (Figure 5D). Considered together with the mutant TS data, these results show that TS targets TrkA receptors and colocalizes with receptor internalization and activation. During the 60-min period, the catalytic activity of TS hydrolyzes
2,3 linked sialic acids and this enzymatic reaction colocalizes with TrkA phosphorylation in the cytoplasm.
Lectin block of TS binding to TrkA-PC12 cells with subsequent inhibition of TS colocalization with TrkA activation
The specificity of TS binding to TrkA-PC12 cells was examined in a lectin inhibition of wild-type TS colocalization with phosphorylated TrkA receptors. TrkA-PC12 cells were first treated with MAL-II (Maackia amurensis lectin II, which binds preferentially to sialic acid in an -2,3 linkage) or SNA (Sambucus nigra lectin, which binds to sialic acids attached to terminal galactose in an
-2,6 linkage and to a lesser degree
-2,3 linkage) for 30 min followed by TS for an additional 30 min. Cells were fixed, permeabilized, and immunostained simultaneously with monoclonal mouse anti-E tag antibody specific for TS and polyclonal rabbit anti-pTyr490 antibody specific for pTrkA followed by anti-mouse Alexa Fluor 488 (green) and anti-rabbit Alexa Fluor 568 (red). Cells were visualized using a confocal inverted microscope (Leica TCS SP2 MP inverted confocal microscope) using a 100x objective (oil). Both MAL-II and SNA blocked TS binding to TrkA-PC12 cells in a dose-dependent manner with subsequent inhibition of TS colocalization with pTrkA (Figure 6). Cells treated with lectins alone did not express pTrkA.
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Discussion |
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A couple of models have been proposed to explain the enzymatic reactions of T. cruzi TS (Buschiazzo et al., 2000; Schenkman et al., 1994
). The substrate used by TS is the sialyl
-2,3-linked ß-galactosyl residues that are common on glycoproteins, glycolipids, and oligosaccharides. TS does not transfer free sialic acid (Frasch, 2000
; Schenkman et al., 1994
), and the sialyl
-2,6-,
-2,8-, and
-2,9-linked sialic acids are known to be poor donor substrates (Schenkman et al., 1994
). Structural analyses have shown that TS folds into two distinct domains: a catalytic ß-propeller fold (
395 amino acids) tightly associated with a C-terminal (
247 amino acids) lectin-like domain with repeats of 12 amino acids (SAPA repeats, 100500 amino acids) in tandem (Buschiazzo et al., 2000
, 2002
; Frasch, 2000
; Schenkman et al., 1994
). The lectin domain does not have any part in the catalytic activity of the enzyme, and there are no galactose binding sites present (Buschiazzo et al., 2002
). Using TS in combination with substrates and sialidase inhibitors, Buschiazzo et al. (2002)
have found that a sialic acidinduced conformational switch activates the inactive free enzyme and concurrently modulates the binding affinity for the acceptor substrate. Both substrate binding sites are close to each other in the catalytic domain. According to this model of TS activity, the transfer reaction is preferred over the sialidase action because the sialic acidinduced conformational change makes the catalytic center more hydrophobic.
Site-directed mutagenesis experiments and the crystal structure of TS clearly demonstrated the crucial role of a few amino acid residues within the substrate-binding cleft in modulating the sialyltransferase activity (Buschiazzo et al., 2000, 2002
). The equivalent aspartic acid (Asp96) residue in bacterial and eukaryotic sialidases may indeed be important for sialic acid binding, but this interaction was found not to be the same in T. cruzi TS (Buschiazzo et al., 2002
). The Asp96 carboxylate in TS was shown to interact with the glycerol moiety of sialic acid substrate instead of the pyranose ring. Furthermore, Todeschini et al. (2004)
have demonstrated that the inactive trans-sialidase has a ß-galactoside recognition site formed following a conformational switch induced by sialoside binding, and this prior positioning of a sialyl residue is required for the ß-galactoside interaction. The mutant TS
Asp98-Glu used in our studies has one additional amino acid (Ala24), and the start codon (Met) was included in our amino acid sequence, which may explain the difference in the amino acid number (Laroy and Contreras, 2000
). Although Chuenkova and Pereira (2000)
reported that TS-induced neurite outgrowth in PC12 cells was independent of the enzyme catalytic activity, the mutant TS that they used to demonstrate this had no transferase activity but still retained
68% of its sialidase activity (Chuenkova et al., 1999
). The mutant TS
Asp98-Glu used in our studies had a point mutation in the catalytic domain of TS reducing both the sialyltransferase and sialidase activities to
4.5% and 3.6%, respectively, of the wild-type TS. The mutant TS
Asp98-Glu bound to TrkA-expressing cells similarly like the wild-type TS and was subsequently internalized into the cytoplasm after 15 min. However, TS
Asp98-Glu did not induce TrkA Tyr490 phosphorylation 60 min after treatment. It was also unable to mediate neurite outgrowth in TrkA-PC12 cells.
Considered together, the results indicate that the sialidase and not the sialyltransferase activity of wild-type TS was involved in activating TrkA receptors. Similar findings were observed using a highly purified recombinant 2,3-neuraminidase (S. pneumoniae). It is important to note that the actual physical binding of wild-type TS, mutant TS
Asp98-Glu, or sialic acidspecific lectins to TrkA-expressing cells is not sufficient to induce TrkA receptor activation. In support of this, Todeschini et al. (2002a)
have shown that their inactive mutant of trans-sialidase (irTS) can physically interact with CD4+ T cells and that this binding of irTS to T cells was abrogated by the prior incubation of irTS with
2-3-sialyllactose but not with
2-6-sialyllactose, demonstrating the specificity of the interaction of irTS with
2-3-linked sialic acids. In addition, Todeschini et al. (2002b)
demonstrated that their native and recombinant TSs alone failed to induce T cell mitogenesis but were able to costimulate CD4+ T cell activation in vitro only in the presence of phorbol 12-myristate 13-acetate as the costimulus.
Here we propose a model of TrkA activation based on the glycosylation state of the receptors. TS binding to TrkA-expressing cells colocalizes with TrkA phosphorylation in cytoplasmic vesicles after 15 min. This interaction is specifically abrogated in a dose-dependent manner with MAL-II and SNA lectins, which bind preferentially to sialic acids attached to terminal galactose in -2,3 and
-2,6 linkages, respectively. It is interesting to note that although TS interacts with the glycerol moiety of sialic acid substrate instead of the pyranose ring, both lectins were still able to block TS binding to TrkA-PC12 cells with subsequent inhibition of TrkA activation. A possible explanation for this may be due to the large size of the lectins (MAL-II, a molecular weight of 140,000 Da and SNA 150,000 Da). Following TS binding to TrkA receptors, it is the hydrolysis of sialyl
-2,3-linked ß-galactosyl residues of TrkA receptors by TS that may be important in facilitating the dimerization of Trks and subsequent phosphorylation of Tyr490 residues. For Trk receptors, glycosylation is required to localize the receptors to the cell surface where it prevents ligand-independent activation of receptors (Watson et al., 1999
). Our results suggest that sialyl
-2,3-linked ß-galactosyl residues of TrkA receptors may be the important sugar property required for cell surface Trk expression, and the hydrolysis of these residues would subsequently facilitate Trk receptor internalization, dimerization, and phosphorylation. Phosphorylated TrkA receptors would then lead to the activation of downstream signaling molecules, such as MAP/MEK protein kinases. Indeed, phosphorylation of Trk Tyr490 has been shown to be a requirement for Shc association and recruitment of FRS2/Crk (Meakin et al., 1999b
), both of which might be the likely source for long-term mitogen-activated protein kinase (MAPK) activation (phospho-MAPK) (Encinas et al., 1999
; Yoon et al., 1997
). Phosphorylation of MAPK would suggest internalization of phosphorylated Trk receptors (Watson et al., 2001
). In support of this concept we have shown, using inhibitors of tyrosine kinase and MAP/MEK protein kinase, that both NGF- and TS-induced neurite outgrowth in PC12 cells were completely blocked.
There is an extensive body of literature on the cellular role of sialic acids (Brockhausen and Kuhns, 1997; Schauer et al., 1997
; Schauer, 1982
, 2000
; Varki, 1993
), but there are only few examples of a role of sugars in signal transduction. One is the addition of a single O-linked carbohydrate modification, which is essential for Notch signaling (Moloney et al., 2000
). Furthermore, transfection of PC12 cells with core 2 GlcNAc transferase resulted in increased Trk receptor levels and phosphorylation as well as increased PC12 differentiation in the absence of NGF (Koya et al., 1999
). Trk activity was also modified when PC12 cells were transfected with GlcNAc-transferase III (Ihara et al., 1997
), an enzyme in the N-glycan pathway, or with ganglioside GD3 synthase gene (GD3 synthase is a ganglioside-specific sialyltransferase) (Fukumoto et al., 2000
).
TrkA receptors are distributed at the plasma membrane as a type I membrane protein with the N-terminus located extracellularly. The total number of potential N-glycosylation sites ranges from 11 to 15 in the various TrkA, TrkB, and TrkC sequences (Watson et al., 1999). The structures of N-glycans on Trk receptors are unknown. It has been suggested that TrkA might be O-glycosylated (Koya et al., 1999
). Watson et al. (1999)
have shown that unglycosylated TrkA core protein is fully activated as a signal-generating receptor tyrosine kinase, but it is mislocalized or trapped intracellularly and is not transduced through the Ras/MAP/MEK and Erk kinase pathways, and the cells do not display features of cellular differentiation. This property of glycosylation has been shown for a number of other cell surface receptors (Brockhausen and Kuhns, 1997
). However, the exact role of Trk receptor glycosylation for the neurotrophin NGF binding, receptor dimerization, kinase activation, and signal transduction has not been defined yet.
The findings in these studies suggest for the first time an important sugar property of TrkA tyrosine kinase receptors playing a role in their activation. They suggest that the hydrolysis of sialyl -2,3-linked ß-galactosyl residues on Trk receptors by TS is sufficient to trigger receptor internalization and activation, signal transduction, and cell differentiation.
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Material and methods |
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The optimal dose of 200 ng TS/ml was determined for neurite outgrowth in PC12 cells in a 3 day culture.
NGF and inhibitors
NGF (Sigma, St. Louis, MO) was used at a predetermined optimal dosage of 50 ng/ml. K-252a (Calbiochem, San Diego, CA), an inhibitor of Trk tyrosine kinase, and PD98059 (Calbiochem), an inhibitor of MAP/MEK protein kinase, were used at predetermined optimal dosages of 200 nM for K-252a and 20 mM for PD98059.
Cell lines
The NGF-responsive PC12 rat pheochromocytoma cell line, PC12nnr5 (tyrosine kinasedeficient mutant, NGF nonresponsive) (Kaplan and Miller, 1997, 2000
), TrkA-PC12 cell line (overly expressing human TrkA receptors) (Hempstead et al., 1992
), and the PC12nnr5 cell lines stably transfected with rat TrkA receptors (B5 and B2) (Meakin and MacDonald, 1998
) were used in these studies. All cell lines were grown at 37°C in 5% CO2 in culture media containing RPMI 1640 (Gibco, Rockville, MD) supplemented with 5% horse serum and 5% fetal calf serum (Gibco).
Scoring of neurite outgrowth
Cells (5x 106/glass slide) were grown in 24-well tissue culture plates on 12-mm circular glass slides coated with poly-D-lysine (Sigma) for 24 h prior to experimental treatment. After 23 days of treatment, the cells were fixed in 3.7% paraformaldehyde for 30 min, permeabilized with 0.2% Triton X-100 in phosphate buffered saline on ice for 5 min, followed with a blocking solution of 3% bovine serum albumin on ice for 20 min. The cells were then stained with phalloidin-rhodamine (Sigma) for 1 h at 37°C. The stained cells were air-dried and mounted on microscope slides. Cell staining was analyzed qualitatively using fluorescence microscopy (Aviovert 100A DIC imaging) and quantitatively by counting the number of cells with multiple neurite extensions (
200 of the total cells; one or more extensions greater than 2 cell bodies in length).
pTyr490 and internalization in TrkA-PC12 cells
Cells were grown in 24-well tissue culture plates on 12-mm circular glass slides coated with poly-D-lysine for 24 h prior to experimental treatment. Cells were treated with 200 ng/ml TS for 15-, 30-, or 60-min intervals or with 50 ng/ml NGF for 15 min. They were then fixed in 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100 in phosphate buffered saline on ice for 5 min, and immunostained with the polyclonal rabbit anti-phospho-Trk (anti-pY490) antibody (anti-pY490; kind gift from Dr. Rosalind Segal, Dana Farber) followed by anti-rabbit Alexa Fluor 488 (green) and phalloidin-rhodamine (red) for 1 h at 37°C. Cell staining was analyzed qualitatively using either fluorescence microscopy (Aviovert 100A DIC imaging) or a confocal inverted microscope (Leica TCS SP2 MP inverted Confocal Microscope) using a 100x objective (oil). Anti-rabbit Alexa Fluor 488 was excited at 488 nm, and the emission was measured at 500535 nm. Phalloidin- rhodamine was excited at 555 nm, and the emission was measured at 556618 nm. In the colocalization experiments, cells were fixed, permeabilized, and immunostained simultaneously with monoclonal mouse anti-E tag antibody specific for TS and polyclonal rabbit anti-pTyr490 antibody specific for pTrkA followed by anti-mouse Alexa Fluor 488 (green) and anti-rabbit Alexa Fluor 568 (red). Images were captured using a z-stage of 810 images per cell at 0.5 µm steps and were processed and merged using LCS Lite software.
Lectin inhibition of TS colocalization with pTrkA
TrkA-PC12 cells were first treated with 10, 1, or 0.1 µg/ml MAL-II or with 10, 1, or 0.1 µg/ml SNA for 30 min followed by 200 ng/ml of wild-type TS for an additional 30 min. Control cells were either untreated, TS-treated alone, or lectin-treated alone at a dose of 10 µg/ml. As previously described, cells were then fixed, permeabilized, and immunostained simultaneously with monoclonal mouse anti-E tag antibody specific for TS and polyclonal rabbit anti-pTyr490 antibody specific for pTrkA followed by anti-mouse Alexa Fluor 488 (green) and anti-rabbit Alexa Fluor 568 (red). Cells were visualized using a confocal inverted microscope (Leica TCS SP2 MP inverted) using a 100x objective (oil).
pTyr490 TrkA immunoblot analysis
TrkA-PC12 cells were treated with 200 ng/ml TS for 60 min, 50 ng/ml NGF for 15 min, or were left untreated as controls (Ctrl). Cell preparations were homogenized in lysis buffer containing Tris-buffered saline, 1% v/v NP40, 10% v/v glycerol, 2 µl/ml aprotinin, 2 µl/ml leupeptin, and 5 µl/ml phenylmethanesulfonyl fluoride, and further microcentrifuged to remove nuclei. The lysates were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) on 8% gels, transferred to polyvinylidene difluoride Immobilon membranes, and processed for blotting against anti-phospho-Tyr490 antibodies (anti-pY490), horseradish peroxidaseconjugated secondary antibody and Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer, Boston, MA).
Statistics
Comparisons between two groups were made by one-way analysis of variance at 95% confidence using Bonferroni's multiple comparison test, or Dunnett's multiple comparison test for comparisons among more than two groups.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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Buschiazzo, A., Tavares, G.A., Campetella, O., Spinelli, S., Cremona, M.L., Paris, G., Amaya, M.F., Frasch, A.C., and Alzari, P.M. (2000) Structural basis of sialyltransferase activity in trypanosomal sialidases. EMBO J., 19, 1624.
Buschiazzo, A., Amaya, M.F., Cremona, M.L., Frasch, A.C., and Alzari, P.M. (2002) The crystal structure and mode of action of trans-sialidase, a key enzyme in Trypanosoma cruzi pathogenesis. Mol. Cell, 10, 757768.[ISI][Medline]
Callewaert, N., Geysens, S., Molemans, F., and Contreras, R. (2001) Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment. Glycobiology, 11, 275281.
Chuenkova, M.V. and Pereira, M.A. (2000) A trypanosomal protein synergizes with the cytokines ciliary neurotrophic factor and leukemia inhibitory factor to prevent apoptosis of neuronal cells. Mol. Biol. Cell, 11, 14871498.
Chuenkova, M., Pereira, M., and Taylor, G. (1999) Trans-sialidase of Trypanosoma cruzi: location of galactose-binding site(s). Biochem. Biophys. Res. Commun., 262, 549556.[CrossRef][ISI][Medline]
Chuenkova, M.V., Furnari, F.B., Cavenee, W.K., and Pereira, M.A. (2001) Trypanosoma cruzi trans-sialidase: a potent and specific survival factor for human Schwann cells by means of phosphatidylinositol 3-kinase/Akt signaling. Proc. Natl Acad. Sci. USA, 98, 99369941.
Colman, P.M. and Smith, B.J. (2002) The trypanosomal trans-sialidase: two catalytic functions associated with one catalytic site. Structure. (Camb.), 10, 14661468.[Medline]
Encinas, M., Iglesias, M., Llecha, N., and Comella, J.X. (1999) Extracellular-regulated kinases and phosphatidylinositol 3-kinase are involved in brain-derived neurotrophic factor-mediated survival and neuritogenesis of the neuroblastoma cell line SH-SY5Y. J. Neurochem., 73, 14091421.[CrossRef][ISI][Medline]
Frasch, A.C. (2000) Functional diversity in the trans-sialidase and mucin families in Trypanosoma cruzi. Parasitol. Today, 16, 282286.[CrossRef][ISI][Medline]
Fukumoto, S., Mutoh, T., Hasegawa, T., Miyazaki, H., Okada, M., Goto, G., Furukawa, K., Urano, T., and Furukawa, K. (2000) GD3 synthase gene expression in PC12 cells results in the continuous activation of TrkA and ERK1/2 and enhanced proliferation. J. Biol. Chem., 275, 58325838.
Genovese, O., Ballario, C., Storino, R., Segura, E., and Sica, R.E. (1996) Clinical manifestations of peripheral nervous system involvement in human chronic Chagas disease. Arq. Neuropsiquiatr., 54, 190196.[ISI][Medline]
Hempstead, B.L., Rabin, S.J., Kaplan, L., Reid, S., Parada, L.F., and Kaplan, D.R. (1992) Overexpression of the trk tyrosine kinase rapidly accelerates nerve growth factor-induced differentiation. Neuron, 9, 883896.[ISI][Medline]
Ihara, Y., Sakamoto, Y., Mihara, M., Shimizu, K., and Taniguchi, N. (1997) Overexpression of N-acetylglucosaminyltransferase III disrupts the tyrosine phosphorylation of Trk with resultant signaling dysfunction in PC12 cells treated with nerve growth factor. J. Biol. Chem., 272, 96299634.
Kaplan, D.R. and Miller, F.D. (1997) Signal transduction by the neurotrophin receptors. Curr. Opin. Cell Biol., 9, 213221.[CrossRef][ISI][Medline]
Kaplan, D.R. and Miller, F.D. (2000) Neurotrophin signal transduction in the nervous system. Curr. Opin. Neurobiol., 10, 381391.[CrossRef][ISI][Medline]
Koberle, F. (1968) Chagas' disease and Chagas' syndromes: the pathology of American trypanosomiasis. Adv. Parasitol., 6, 63116.[Medline]
Koya, D., Dennis, J.W., Warren, C.E., Takahara, N., Schoen, F.J., Nishio, Y., Nakajima, T., Lipes, M.A., and King, G.L. (1999) Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice. FASEB J., 13, 23292337.
Laroy, W. and Contreras, R. (2000) Cloning of Trypanosoma cruzi trans-sialidase and expression in Pichia pastoris. Protein Expr. Purif., 20, 389393.[CrossRef][ISI][Medline]
Lee, S. and Kim, B. (2001) Trans-sialidase catalyzed sialylation of beta-galactosyldisaccharide with an introduction of beta-galactosidase. Enzyme Microb. Technol., 28, 161167.[CrossRef][ISI][Medline]
Losavio, A., Jones, M.C., Sanz, O.P., Mirkin, G., Gonzalez Cappa, S.M., Muchnik, S., and Sica, R.E. (1989) A sequential study of the peripheral nervous system involvement in experimental Chagas' disease. Am. J. Trop. Med. Hyg., 41, 539547.[ISI][Medline]
MacDonald, J.I., Gryz, E.A., Kubu, C.J., Verdi, J.M., and Meakin, S.O. (2000) Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA. J. Biol. Chem., 275, 1822518233.
Machado, C.R., Gomez, M.V., and Machado, A.B. (1987) Changes in choline acetyltransferase activity of rat tissues during Chagas' disease. Braz. J. Med. Biol. Res., 20, 697702.[ISI][Medline]
Meakin, S.O. and MacDonald, J.I. (1998) A novel juxtamembrane deletion in rat TrkA blocks differentiative but not mitogenic cell signaling in response to nerve growth factor. J. Neurochem., 71, 18751888.[ISI][Medline]
Meakin, S.O. and Shooter, E.M. (1992) The nerve growth factor family of receptors. Trends Neurosci., 15, 323331.[CrossRef][ISI][Medline]
Meakin, S.O., Gryz, E.A., and MacDonald, J.I. (1997) A kinase insert isoform of rat TrkA supports nerve growth factor-dependent cell survival but not neurite outgrowth. J. Neurochem., 69, 954967.[ISI][Medline]
Meakin, S.O., MacDonald, J.I., Gryz, E.A., Kubu, C.J., and Verdi, J.M. (1999a) The signaling adapter FRS-2 competes with Shc for binding to the nerve growth factor receptor TrkA. A model for discriminating proliferation and differentiation. J. Biol. Chem., 274, 98619870.
Meakin, S.O., MacDonald, J.I.S., Gryz, E.A., Kubu, C.J., and Verdi, J.M. (1999b) The signaling adapter FRS-2 competes with Shc for binding to the nerve growth factor receptor TrkAa model for discriminating proliferation and differentiation. J. Biol. Chem., 274, 98619870.
Moloney, D.J., Panin, V.M., Johnson, S.H., Chen, J.H., Shao, L., Wilson, R., Stanley, P., Irvine, K.D., Vogt, T.F., and Haltiwanger, R.S. (2000) Modulation of signal transduction by differential receptor glycosylation: the role of O-linked fucose in notch signaling. Glycobiology, 10, 10811082.
Schauer, R. (1982) Chemistry, metabolism, and biological functions of sialic acids. Adv. Carbohydr. Chem. Biochem., 40, 131234.[ISI][Medline]
Schauer, R. (2000) Achievements and challenges of sialic acid research. Glycoconj. J., 17, 485499.[CrossRef][ISI][Medline]
Schauer, R., de Freese, A., Gollub, M., Iwersen, M., Kelm, S., Reuter, G., Schlenzka, W., Vandamme-Feldhaus, V., and Shaw, L. (1997) Functional and biosynthetic aspects of sialic acid diversity. Indian J. Biochem. Biophys., 34, 131141.[Medline]
Schenkman, S., Eichinger, D., Pereira, M.E., and Nussenzweig, V. (1994) Structural and functional properties of Trypanosoma trans-sialidase. Annu. Rev. Microbiol., 48, 499523.[CrossRef][ISI][Medline]
Todeschini, A.R., Girard, M.F., Wieruszeski, J.M., Nunes, M.P., DosReis, G.A., Mendonca-Previato, L., and Previato, J.O. (2002a) Trans-sialidase from Trypanosoma cruzi binds host T-lymphocytes in a lectin manner. J. Biol. Chem., 277, 4596245968.
Todeschini, A.R., Nunes, M.P., Pires, R.S., Lopes, M.F., Previato, J.O., Mendonca-Previato, L., and DosReis, G.A. (2002b) Costimulation of host T lymphocytes by a trypanosomal trans-sialidase: Involvement of CD43 signaling. J. Immunol., 168, 51925198.
Todeschini, A.R., Dias, W.B., Girard, M.F., Wieruszeski, J.M., Mendonca-Previato, L., and Previato, J.O. (2004) Enzymatically inactive trans-sialidase from Trypanosoma cruzi binds sialyl and beta-galactopyranosyl residues in a sequential ordered mechanism. J. Biol. Chem., 279, 53235328.
Varki, A. (1993) Biological roles of oligosaccharidesall of the theories are gorrect. Glycobiology, 3, 97130.[Abstract]
Watson, F.L., Porcionatto, M.A., Bhattacharyya, A., Stiles, C.D., and Segal, R.A. (1999) TrkA glycosylation regulates receptor localization and activity. J. Neurobiol., 39, 323336.[CrossRef][ISI][Medline]
Watson, F.L., Heerssen, H.M., Bhattacharyya, A., Klesse, L., Lin, M.Z., and Segal, R.A. (2001) Neurotrophins use the Erk5 pathway to mediate a retrograde survival response. Nat. Neurosci., 4, 981988.[CrossRef][ISI][Medline]
Yoon, S.O., Soltoff, S.P., and Chao, M.V. (1997) A dominant role of the juxtamembrane region of the TrkA nerve growth factor receptor during neuronal cell differentiation. J. Biol. Chem., 272, 2323123238.
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