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 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.
Carey
D J
DJ
Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
Evans
D M
DM
Stahl
R C
RC
Asundi
V K
VK
Conner
K J
KJ
Garbes
P
P
Cizmeci-Smith
G
G
D000375:Aging; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D015152:Blotting, Northern; D001921:Brain / Q000254:growth & development / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004622:Embryo, Mammalian; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000254:growth & development / Q000502:physiology; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011509:Proteoglycans / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012583:Schwann Cells / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D053667:Syndecans; D014158:Transcription, Genetic
D004247:DNA; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D008562:Membrane Glycoproteins; D015345:Oligonucleotide Probes; D011509:Proteoglycans; D012333:RNA, Messenger; D053667:Syndecans