Molecular Cloning and Characterization of N-Syndecan, a Novel Transmembrane Heparan Sulfate Proteoglycan
A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reacti...
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Published in | The Journal of cell biology Vol. 117; no. 1; pp. 191 - 201 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
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New York, NY
Rockefeller University Press
01.04.1992
The Rockefeller University Press |
Subjects | |
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Abstract | A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5′ protein coding sequences. The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain. The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues. Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence. On Northern blots the cDNA hybridized to a single 5.6-kb mRNA that was present in Schwann cells, neonatal rat brain, rat heart, and rat smooth muscle cells. A 16-kD protein fragment encoded by the cDNA was expressed in bacteria and used to immunize rabbits. The resulting antibodies reacted on immunoblots with the core protein of a detergent extracted heparan sulfate proteoglycan. The core protein had an apparent mass of 120 kD. When the anti-core protein antibodies were used to stain tissue sections immunoreactivity was present in peripheral nerve, newborn rat brain, heart, aorta, and other neonatal tissues. A ribonuclease protection assay was used to quantitate levels of the core protein mRNA. High levels were found in neonatal rat brain, heart, and Schwann cells. The mRNA was barely detectable in neonatal or adult liver, or adult brain. |
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AbstractList | A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5' protein coding sequences. The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain. The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues. Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence. On Northern blots the cDNA hybridized to a single 5.6-kb mRNA that was present in Schwann cells, neonatal rat brain, rat heart, and rat smooth muscle cells. A 16-kD protein fragment encoded by the cDNA was expressed in bacteria and used to immunize rabbits. The resulting antibodies reacted on immunoblots with the core protein of a detergent extracted heparan sulfate proteoglycan. The core protein had an apparent mass of 120 kD. When the anti-core protein antibodies were used to stain tissue sections immunoreactivity was present in peripheral nerve, newborn rat brain, heart, aorta, and other neonatal tissues. A ribonuclease protection assay was used to quantitate levels of the core protein mRNA. High levels were found in neonatal rat brain, heart, and Schwann cells. The mRNA was barely detectable in neonatal or adult liver, or adult brain. |
Author | Stahl, Richard C. Asundi, Vinod K. Cizmeci-Smith, Gunay Carey, David J. Evans, Diane M. Conner, Kimberly J. Garbes, Peter |
Author_xml | – sequence: 1 givenname: David J. surname: Carey fullname: Carey, David J. – sequence: 2 givenname: Diane M. surname: Evans fullname: Evans, Diane M. – sequence: 3 givenname: Richard C. surname: Stahl fullname: Stahl, Richard C. – sequence: 4 givenname: Vinod K. surname: Asundi fullname: Asundi, Vinod K. – sequence: 5 givenname: Kimberly J. surname: Conner fullname: Conner, Kimberly J. – sequence: 6 givenname: Peter surname: Garbes fullname: Garbes, Peter – sequence: 7 givenname: Gunay surname: Cizmeci-Smith fullname: Cizmeci-Smith, Gunay |
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Keywords | Transmembrane protein Proteoglycan Rat Nucleotide sequence Rodentia Central nervous system Homology Tissue Vertebrata Mammalia Complementary DNA Schwann cell Blotting assay Heparitin sulfate Molecular cloning Immunofluorescence Aminoacid sequence |
Language | English |
License | CC BY 4.0 This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
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SubjectTerms | Aging Amino Acid Sequence Amino acids Analytical, structural and metabolic biochemistry Animals Animals, Newborn Antibodies Base Sequence Biological and medical sciences Blotting, Northern Brain - growth & development Brain - physiology Cells Cloning, Molecular - methods Complementary DNA DNA - genetics DNA - isolation & purification Embryo, Mammalian Fluorescent Antibody Technique Fundamental and applied biological sciences. Psychology Gene Library Glycoproteins Heparan Sulfate Proteoglycans Heparitin Sulfate - genetics Humans Liver - growth & development Liver - physiology Membrane Glycoproteins - analysis Membrane Glycoproteins - genetics Messenger RNA Molecular Sequence Data Neurons Oligonucleotide Probes Proteins Proteoglycans Proteoglycans - analysis Proteoglycans - genetics Rats RNA RNA, Messenger - genetics Schwann cells Schwann Cells - physiology Sequence Homology, Nucleic Acid Syndecans Transcription, Genetic |
Title | Molecular Cloning and Characterization of N-Syndecan, a Novel Transmembrane Heparan Sulfate Proteoglycan |
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