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Glycosylation and Lewis X motif in neuronal tissue

Instituto de Tecnologia Química e Biológica
Project classification

Scientific area

3.3 Health sciences

Discipline(s)

Infectious diseases

Project description

Project title

Glycosylation and Lewis X motif in neuronal tissue

Scientific Coordinator's name:

Julia Costa

Scientific Coordinator's e-mail:

jcosta@itqb.unl.pt

Principal R&D Unit:

Instituto de Tecnologia Química e Biológica (ITQB/UNL)

Other R&D Units involved in the project:

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Project keyword(s)

Lewis X; neuron adhesion; fucosyltransferase 9; neurite outgrowth

Short abstract and comments

Carbohydrates from mammalian cells play several functional roles in cell adhesion and recognition, cell development, glycoprotein folding among others. The fucosylated carbohydrate structure Lewis X (Le X, Galbeta4[Fucalpha3]GlcNAc) is abundant in the brain where the major neutral complex-type N-linked oligosaccharides contain the monofucosylated Le X structure in one antenna. Le X expression in neurons is temporally and spacially regulated and it seems to be involved in neuron adhesion and neurite outgrowth, however, the molecules that participate in the process have not been identified and the mechanisms underlying these roles have not been elucidated. In the central nervous system, Le X expression also identifies stem cells and specific progenitor cells. In brain, Le X is synthesized by fucosyltransferase 9 (FUT9). This is an alpha3 fucosyltransferase expressed at higher levels in brain tissue. The knock-out mouse FUT9-/- showed disappearance of Le X in the brain, concomitant with behavior alterations. Previous work from the laboratory has identified the expression of Le X specifically in differentiated human NT2N neurons in vitro most likely synthesized by FUT9. A specific neuron glycoprotein or proteoglycan Le X-carrier of 460 kDa was also identified. Furthermore, incubation with antibody anti-Le X led to the inhibition of neurite outgrowth and impairment of neuron adhesion. Le X was found in the exocytotic compartment defined by the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP), which is involved in neurite outgrowth, of NT2N neurons and also of rat hippocampus neurons in culture. In the present project, our major aim is to elucidate the functional relevance of the Le X motif in neurite outgrowth and neuron adhesion. In the first phase of the project, the Le X-carrier from NT2N neurons will be purified by chromatographic or electrophoretic techniques, and will be identified by peptide mass fingerprinting. Specific antibodies raised against the polypeptide part will be used to confirm the localization of this potential novel component of the TI-VAMP compartment. Their effect on neurite outgrowth and neuron adhesion will also be evaluated. The carrier will also be characterized with respect to its N- and O-glycosylation to investigate if it contains the monofucosylated Le X-containing oligosaccharides that are characteristic of neuronal tissue. The detailed structure of the oligosaccharides will be elucidated after high performance anion exchange chromatography with pulsed amperometric detection and mass spectrometry. Secondly, the effect of small oligosaccharide molecules and N-linked oligosaccharides containing the Le X structure synthesized enzymaticaly with recombinant FUT9, will be tested on neuron adhesion and neurite outgrowth. With this purpose a library of oligosaccharides enzymaticaly fucosylated with recombinant FUT9 produced in the laboratory will be constructed. In the third phase of the project, FUT9 expression will be silenced in neurons using the siRNA technique. Consequences for Le X expression, cellular localization, neuron adhesion and neurite outgrowth will be examined. In the fourth phase of the project, putative Le X-binding lectin(s) will be identified in neuronal tissue and further characterized. Several approaches may be used and include: i) cDNA microarray analysis using the Glycov4 chip human gene from the Consortium of Functional Glycomics; ii) binding of exogenously supplied recombinant lectins (for example, DC-SIGN); iii) Western blot analysis with specific antibodies against different lectins; iv) affinity isolation of putative Le X-receptors using resins with immobilized monofucosylated Le X-containing N-linked glycopeptides synthesized with recombinant FUT9; v) oligosaccharide microarray analysis. With this project we expect that by characterizing molecules that are associated with the Le X motif and identifying the corresponding lectin receptors, we contribute to elucidate mechanisms that underlie the functional role of Le X in neurite outgrowth and neuron adhesion. The results might have impact in the improvement of brain plasticity and brain repair.

Potential uses/indications

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Status

Ongoing

Partner Status: Seeking Partners?

No

Project weblink

http://Not applicable

Grant number (QREN, FP7, Eureka, etc)

Project FCT: PTDC/SAU-NEU/100724/2008

Last edited on

2012-10-04 14:06:09

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