Isolation and characterization of ryudocan and syndecan heparan sulfate proteoglycans, core proteins, and cDNAs from a rat endothelial cell line

Abstract

We have isolated heparan sulfate proteoglycans (HSPGs) from cloned rat microvascular endothelial cells using a combination of ion-exchange chromatography, affinity fractionation with antithrombin III (AT III), and gel filtration in denaturing solvents. The anticoagulantly active heparan sulfate proteoglycans (HSPGact) which bind tightly to AT III bear mainly anticoagulantly active heparan sulfate (HSact) whereas the anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact) possess mainly anticoagulantly inactive heparan sulfate (HSinact). The core proteins of HSPGact and HSPGinact were isolated by treatment with Flavobacterium heparitinase and purification by ion-exchange chromatography. SDS-PAGE showed that both sets of core proteins exhibited three major components with M(r) of 25-, 30-, and 50-kD, respectively. Peptide mapping revealed that HSPGact and HSPGinact possess extremely similar core proteins. The primary sequences of internal peptides obtained from HSPGinact core proteins and the NH2-terminal sequence analyses of the 25-kD component from the HSPGinact core proteins demonstrate that the 30-kD component is a previously unidentified species--designated as ryudocan--with the 25-kD component representing a proteolytic degradation product; while the 50-kD component is the rat homolog of syndecan [Saunders S, Jalkanen M, O'Farrell S, Bernfield M: J Cell Biol 1989; 108:1547-1556]. Specific oligonucleotide probes were obtained for ryudocan and syndecan by PCR, 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 3 potential glycosaminoglycan (GAG) attachment sites within the extracellular region while syndecan has 5 GAG attachment sites within the same domain. The levels of ryudocan and syndecan mRNA were measured by quantitative PCR in primary microvascular endothelial cells and 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.