title: Expression of glycogenes in differentiating human NT2N neurons. Downregulation of fucosyltransferase 9 leads to decreased Lewis(x) levels and impaired neurite outgrowth.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  One hundred twelve mRNA transcripts showed statistically significant up-regulation, including the genes coding for proteins involved in the synthesis of the Lewis(x) motif (FUT9), polysialic acid (ST8SIA2 and ST8SIA4) and HNK-1 (B3GAT2). Accordingly, increased levels of the corresponding carbohydrate epitopes have been observed. The Lewis(x) 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 Lewis(x), 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.
  
  
  CONCLUSIONS
  These results provided information about the regulation of glycogenes during neuron differentiation and they showed that the Lewis(x) motif plays a functional role in neurite outgrowth from human neurons.
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Oeiras, Portugal.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Gouveia
authors.forename: Lana
authors.initials: L
authors.lastname: Schaffer
authors.forename: Suzanne
authors.initials: S
authors.lastname: Papp
authors.forename: Nicolas
authors.initials: N
authors.lastname: Grammel
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kandzia
authors.forename: Steven R
authors.initials: SR
authors.lastname: Head
authors.forename: Ralf
authors.initials: R
authors.lastname: Kleene
authors.forename: Melitta
authors.initials: M
authors.lastname: Schachner
authors.forename: Harald S
authors.initials: HS
authors.lastname: Conradt
authors.forename: Júlia
authors.initials: J
authors.lastname: Costa
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002454:Cell Differentiation; D002478:Cells, Cultured; D015536:Down-Regulation; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D047468:Immunoprecipitation; D016256:Lewis X Antigen / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism; D016501:Neurites / Q000378:metabolism / Q000473:pathology*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D011134:Polysaccharides / Q000378:metabolism*; D012794:Sialic Acids / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006023:Glycoproteins; D016256:Lewis X Antigen; D019006:Neural Cell Adhesion Molecules; D010446:Peptide Fragments; D011134:Polysaccharides; D012794:Sialic Acids; C021319:polysialic acid; C519500:FUT9 protein, human; D005647:Fucosyltransferases