Expression of glycogenes in differentiating human NT2N neurons. Downregulation of fucosyltransferase 9 leads to decreased Lewisx levels and impaired neurite outgrowth

• Published in: Biochimica et biophysica acta. General subjects Vol. 1820; no. 12; pp. 2007 - 2019
• Main Authors: Gouveia, Ricardo, Schaffer, Lana, Papp, Suzanne, Grammel, Nicolas, Kandzia, Sebastian, Head, Steven R., Kleene, Ralf, Schachner, Melitta, Conradt, Harald S., Costa, Júlia
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.12.2012
• Subjects: carbohydrates; cell adhesion; cells; Central nervous system; epitopes; fluorescence microscopy; Fucosyltransferase 9; genes; glycoproteins; glycosylation; high performance liquid chromatography; HPLC; humans; immunofluorescence; Lewisx; messenger RNA; microarray technology; microscopy; Neurite outgrowth; Neuron glycosylation; Neuronal differentiation; neurons; peptides; precipitin tests; proteins; regulations; RNA interference; PBS; TBS; HRP; Lewisx; PNGase F; HPAEC-PAD; EDTA; EGTA; BSA; MALDI-TOF MS; SDS-PAGE; HNK-1; Gal; Fuc; HexNAc; Neuronal differentiation; dHex; Fucosyltransferase 9; GlcNAc; Hex; Man; Neuron glycosylation; Lex; Neurite outgrowth
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2012.09.004

Several glycan structures are functionally relevant in biological events associated with differentiation and regeneration which occur in the central nervous system. Here we have analysed the glycogene expression and glycosylation patterns during human NT2N neuron differentiation. We have further studied the impact of downregulating fucosyltransferase 9 (FUT9) on neurite outgrowth. The expression of glycogenes in human NT2N neurons differentiating from teratocarcinoma NTERA-2/cl.D1 cells has been analysed using the GlycoV4 GeneChip expression microarray. Changes in glycosylation have been monitored by immunoblot, immunofluorescence microscopy, HPLC and MALDI-TOF MS. Peptide mass fingerprinting and immunoprecipitation have been used for protein identification. FUT9 was downregulated using silencing RNA. One hundred twelve mRNA transcripts showed statistically significant up-regulation, including the genes coding for proteins involved in the synthesis of the Lewisx motif (FUT9), polysialic acid (ST8SIA2 and ST8SIA4) and HNK-1 (B3GAT2). Accordingly, increased levels of the corresponding carbohydrate epitopes have been observed. The Lewisx structure was found in a carrier glycoprotein that was identified as the CRA-a isoform of human neural cell adhesion molecule 1. Downregulation of FUT9 caused significant decreases in the levels of Lewisx, as well as GAP-43, a marker of neurite outgrowth. Concomitantly, a reduction in neurite formation and outgrowth has been observed that was reversed by FUT9 overexpression. These results provided information about the regulation of glycogenes during neuron differentiation and they showed that the Lewisx motif plays a functional role in neurite outgrowth from human neurons. ► Changes in glycogene expression were found during NT2N neuron differentiation. ► Increase in FUT9 expression that encodes the enzyme synthesizing Lewisx was observed. ► NCAM is the predominant glycoprotein carrier of Lewisx in NT2N neurons. ► Downregulation of FUT9 caused decreased Lewisx levels and impaired neurite outgrowth.