Heparin sequences in the heparan sulfate chains of an endothelial cell proteoglycan.

Abstract

The structure of the glycosaminoglycan chain of a heparan sulfate proteoglycan isolated from the conditioned medium of an endothelial cell line has been analyzed by using various degradative enzymes (heparitinase I, heparitinase II, heparinase, glycuronidase, sulfatases) from Flavobacterium heparinum. This proteoglycan inhibits the thromboplastinactivated pathway of coagulation; as a consequence, the catalytic conversion of prothrombin to thrombin is arrested. Heparitinase I (EC 4.2.2.8), an enzyme with specificity restricted to the heparan sulfate portion of the polysaccharide, releases fragments with the electrophoretic mobility and the structure of heparin. Conversely, an assessment of the size and distribution of the heparan sulfate regions has been provided by the use of heparinase (EC 4.2.2.7), which, by degrading the heparin sections of the chain, releases two segments that exhibit the structure of heparan sulfate. One of these segments is attached to the protein core. On the basis of these findings, the heparan sulfate chain can be defined as a copolymer containing heparin regions in its structure. The combined use of these enzymes has made it possible to establish the disaccharide sequence of parts of the glycosaminoglycan moiety of this proteoglycan.Heparan sulfate proteoglycans are complex macromolecules that consist of a protein backbone to which heparan sulfate chains are covalently linked (1). They are ubiquitous compounds found in a wide variety of vertebrate and invertebrate tissues (2) and are actively synthesized by cells in culture (3). These proteoglycans have been found to be present on the plasma membrane and in the extracellular matrix (4, 5) and exhibit a peculiar structural variability according to the tissue and species of origin (2,6). Despite their wide occurrence, little is known of their biological function. They have been implicated in several biological processes such as cell-cell recognition (7), tissue differentiation (8), organization of extracellular matrices (9), and cell-matrix and cell-substrate adhesion (10).The availability of two heparitinases (11, 12) and a heparinase (EC 4.2.2.7) from Flavobacterium heparinum (12, 13), which can be used in conjunction to elucidate the distribution and grouping in the polymeric chain of disaccharides with various degrees of sulfation and with different hexuronic acid moieties, has enabled us to undertake the structural study of a proteoglycan isolated from the conditioned medium of endothelial cell cultures that appears to be highly characteristic of this cell type. Using these enzymes, we have determined that the glycosaminoglycan chain of this proteoglycan contains heparin segments and have developed a strategy for the elucidation of the sequence of the disaccharide repeating units that may be applicable to the study of other structurally related compounds. MATERIALS AND METHODSSubstrates, Enzymes, and Materials. Heparin from bovine intestinal mucosa and heparan sulfate from bovine pancreas were gifts from P. Bianchini (Opocri...

The Putative Heparin-specific N-AcetylglucosaminylN-Deacetylase/N-Sulfotransferase Also Occurs in Non-heparin-producing Cells

“…The results obtained with trachea heparan sulfate imply that the iduronic acid residues are located in different regions along the molecule; this pattern of distribution differs from known standards. The same type of profile of heparinase products was found previously in rabbit aorta endothelial cells (36). DISCUSSION We have unexpectedly found in cells from a tissue that does not synthesize heparin a very abundant message encoding a protein that has 95% sequence identity with the putative heparin N-acetylglucosaminyl N-deacetylase/N-sulfotransferase.…”

Cell biology of arterial proteoglycans.

“…In fact, a recent study demonstrates heparin sequences in the heparan sulfate chains of an endothelial cell proteoglycan. 189 Anticoagulant HS has been isolated from vascular tissue that was shown to be free of mast cells. 38 However, Imamura and Mitsui 190 recently demonstrated that heparan sulfate and heparin exhibited opposing activities on the growth of human umbilical vein endothelial cells in the presence of acidic and basic FGF.…”

Inhibitory Peptides of the Sulfotransferase Domain of the Heparan Sulfate Enzyme, N-Deacetylase-N-sulfotransferase-1

“…Endogenous (30) and by enzymatic digestion with chondroitinase ABC (Sigma) and heparitinases I and II (31).…”

“…After electrophoresis heparan sulfate was precipitated in the gel with 0.1% cetyltrimethyl ammonium bromide (Sigma). The dried gel was stained with 0.1% toluidine blue as described to visualize the heparan sulfate, and the radioactive heparan sulfate was detected by autoradiography (30).…”