title: Cloning and primary structure of neurocan, a developmentally regulated, aggregating chondroitin sulfate proteoglycan of brain.
abstract: We have obtained the complete coding sequence of neurocan, a chondroitin sulfate proteoglycan of rat brain which is developmentally regulated with respect to its molecular size, concentration, carbohydrate composition, sulfation, and immunocytochemical localization. Two degenerate oligonucleotides, based on amino acid sequence data from the proteoglycan isolated from adult brain by immunoaffinity chromatography with the 1D1 monoclonal antibody, were used as sense and antisense primers in the polymerase chain reaction with a brain cDNA library as template to generate an unambiguous cDNA probe. A second probe for the N-terminal portion of the early postnatal form of the proteoglycan was obtained by reverse transcription/polymerase chain reaction. The composite sequence of overlapping cDNA clones is 5.2-kilobases (kb) long, including 1.3 kb of 3'-untranslated sequence and 76 base pairs of 5'-untranslated sequence. An open reading frame of 1257 amino acids encodes a protein with a molecular mass of 136 kDa containing 10 peptide sequences present in the adult and/or early postnatal brain proteoglycans. The deduced amino acid sequence revealed a 22-amino acid signal peptide followed by an immunoglobulin domain, tandem repeats characteristic of the hyaluronic acid-binding region of aggregating proteoglycans, and an RGDS sequence. The C-terminal portion (amino acids 951-1215) has approximately 60% identity to regions in the C termini of the fibroblast and cartilage proteoglycans, versican and aggrecan, including two epidermal growth factor-like domains, a lectin-like domain, and a complement regulatory protein-like sequence. The central 595-amino acid portion of neurocan has no homology with other reported protein sequences. The proteoglycan contains six potential N-glycosylation sites and 25 potential threonine O-glycosylation sites. In the adult form of the proteoglycan (which represents the C-terminal half of neurocan) a single 32-kDa chondroitin 4-sulfate chain is linked at serin-944, whereas three additional potential chondroitin sulfate attachment sites (only two of which are utilized) are present in the larger proteoglycan species. A probe corresponding to a region of neurocan having no homology with versican or aggrecan hybridized with a single band at approximately 7.5 kb on Northern blots of mRNA from both 4-day and adult rat brain (but not with muscle, kidney, liver, or lung mRNA), indicating that the 1D1 proteoglycan of adult brain, containing a 68-kDa core protein, is generated by a developmentally regulated in vivo proteolytic processing of the 136-kDa species which is predominant in early postnatal brain.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pharmacology, New York University Medical Center, New York 10016.
authors.forename: U
authors.initials: U
authors.lastname: Rauch
authors.forename: L
authors.initials: L
authors.lastname: Karthikeyan
authors.forename: P
authors.initials: P
authors.lastname: Maurel
authors.forename: R U
authors.initials: RU
authors.lastname: Margolis
authors.forename: R K
authors.initials: RK
authors.lastname: Margolis
mesh_terms: D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry; D011508:Chondroitin Sulfate Proteoglycans / Q000737:chemistry / Q000235:genetics*; D002809:Chondroitin Sulfates / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D016326:Extracellular Matrix Proteins; D018260:Gelsolin; D015870:Gene Expression; D037181:Lectins, C-Type; D008840:Microfilament Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics*; D058580:Neurocan; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011509:Proteoglycans / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D016415:Sequence Alignment; D014018:Tissue Distribution; D053675:Versicans
chemical_list: C508321:Acan protein, rat; D053674:Aggrecans; D002135:Calcium-Binding Proteins; D011508:Chondroitin Sulfate Proteoglycans; D004220:Disulfides; D016326:Extracellular Matrix Proteins; D018260:Gelsolin; D037181:Lectins, C-Type; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D058580:Neurocan; D009838:Oligodeoxyribonucleotides; D011509:Proteoglycans; D012333:RNA, Messenger; C508322:VCAN protein, human; C508324:Vcan protein, rat; D053675:Versicans; C076817:NCAN protein, human; D002809:Chondroitin Sulfates; D004247:DNA