Structural and electron microscopic analysis of neurocan and recombinant neurocan fragments.
Neurocan, a nervous tissue-specific chondroitin sulfate proteoglycan of the aggrecan family which has been shown to interact with neural cell adhesion molecules and tenascin, could be visualized by rotary shadowing electron microscopy as two globular domains interconnected by an extended flexible filament of 60-90 nm. Several recombinant neurocan fragments generated in the human embryonic kidney cell line 293 represent as observed by electron microscopy the expected parts of this structure, which indicates a correct folding of these molecules. Biological activity of the recombinant N-terminal globular domain could be demonstrated by its coelution with hyaluronan in gel permeation chromatography. In addition, the modification of the recombinant fragments with certain carbohydrate structures was analyzed. High mannose oligosaccharides could be mapped to the N-terminal globular domain of the brain-derived molecule. Only recombinant fragments containing parts of the central region of the molecule were modified with chondroitin sulfate chains and with the HNK-1 epitope, and could be considerably altered in their migratory behavior on SDS-polyacrylamide gel electrophoresis by neuraminidase treatment. These findings and the electron microscopic shape indicate a mucin-like character for the central neurocan region.
Retzler
C
C
Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany.
Wiedemann
H
H
Kulbe
G
G
Rauch
U
U
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis / Q000737:chemistry* / Q000648:ultrastructure*; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D007668:Kidney; D037181:Lectins, C-Type; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000648:ultrastructure*; D058580:Neurocan; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D000911:Antibodies, Monoclonal; D011508:Chondroitin Sulfate Proteoglycans; D018076:DNA, Complementary; D037181:Lectins, C-Type; D009419:Nerve Tissue Proteins; D058580:Neurocan; D010446:Peptide Fragments; D011994:Recombinant Proteins; C076817:NCAN protein, human; D006820:Hyaluronic Acid