Molecular cloning and expression of two distinct cDNA-encoding heparan sulfate proteoglycan core proteins from a rat endothelial cell line.
The cloned rat fat pad endothelial cell (RFP-EC) line synthesizes anticoagulantly active heparan sulfate proteoglycans (HSPGact) and anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact), both of which exhibit 25-, 30-, and 50-kDa core proteins of extremely similar structure. The primary sequences of internal peptides obtained from HSPGinact core proteins and the NH2-terminal sequence analyses of the 25-kDa component from the HSPGinact core proteins demonstrate that the 30-kDa component is a previously unidentified species, designated as ryudocan, with the 25-kDa component representing a proteolytic degradation product, while the 50-kDa component is the rat homolog of syndecan (Saunders, S. Jalkanen, M., O'Farrell, S., and Bernfield, M. (1989) J. Cell Biol. 108, 1547-1556). Specific oligonucleotide probes were obtained for ryudocan and syndecan by polymerase chain reaction, and the corresponding cDNAs were isolated from a RFP-EC library. The cDNAs encode type I integral membrane proteins of 202 and 313 amino acids, respectively, which have homologous transmembrane and intracellular domains but very distinct extracellular regions. In particular, ryudocan exhibits only three potential glycosaminoglycan attachment sites within the extracellular region while syndecan has five glycosaminoglycan attachment sites within the same domain. Both species are expressed in RFP-EC lines, primary rat aortic smooth muscle cells and primary rat skin fibroblast cells. The levels of ryudocan and syndecan mRNA were measured by quantitative polymerase chain reaction in primary microvascular endothelial cells and closely associated non-endothelial cells isolated by cell sorting. Ryudocan and syndecan mRNAs were abundantly expressed in both populations representing about 0.1-0.5% of mRNA.
Kojima
T
T
Department of Biology, Massachusetts, Institute of Technology, Cambridge 02139.
Shworak
N W
NW
Rosenberg
R D
RD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics; D011509:Proteoglycans / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D053671:Syndecan-4; D053667:Syndecans
D004247:DNA; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D008562:Membrane Glycoproteins; D011506:Proteins; D011509:Proteoglycans; D012333:RNA, Messenger; D053671:Syndecan-4; D053667:Syndecans; C508307:Sdc4 protein, rat