Recombinant fish neurotrophin-6 is a heparin-binding glycoprotein: implications for a role in axonal guidance.
Neurotrophin-6 (NT-6) was identified in the teleost fish Xiphophorus as a new member of the neurotrophin gene family. NT-6 binds specifically the glycosaminoglycan heparin. In this study NT-6 was expressed in a stably transfected mammalian cell line, and in insect cells via a recombinant baculovirus. It was purified to homogeneity and characterized by MS and N-terminal sequencing. NT-6 from both expression systems was proteolytically processed at one of two protease cleavage motifs and was found to be glycosylated. It supported the survival of embryonic chick sensory neurons; half-maximal survival was observed at 100 ng/ml. Furthermore, NT-6 elicited neurite outgrowth in explanted embryonic dorsal root ganglia. Addition of heparin into the medium did not potentiate the activity of NT-6 in survival assays. However, when a sensory ganglion explant was cultured in a collagen gel matrix assay adjacent to a heparin bead coated with NT-6, neurite outgrowth directed towards the bead was observed. This indicated that NT-6 was slowly released from the heparin bead generating a concentration gradient of NT-6 instrumental for axonal guidance in vitro. Thus the interaction of NT-6 with heparin might not be required for the activation of the cellular receptor for NT-6 on responsive cells but rather may serve to control, in vivo, the distribution of NT-6.
Li
X
X
Clinical Research Unit, Department of Neurology, University of Würzburg, D-97080 Würzburg, Germany.
Franz
J
J
Lottspeich
F
F
Götz
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001369:Axons / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003532:Cyprinodontiformes / Q000235:genetics / Q000378:metabolism*; D005727:Ganglia, Spinal / Q000166:cytology; D006031:Glycosylation; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D005810:Multigene Family; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology / Q000502:physiology; D017924:Nucleopolyhedroviruses; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology; D018411:Spodoptera; D014162:Transfection
D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; C075936:neurotrophin 6; D006493:Heparin