Preparation of Completely 6-O-Desulfated Heparin and Its Ability to Enhance Activity of Basic Fibroblast Growth Factor

Abstract

Although regioselective removal of 6-O-sulfate groups of heparin has been undertaken by several researchers, complete 6-O-desulfation with little side reaction has not been attained successfully. In this work, a modified method with a certain silylating reagent, N-methyl-N-(trimethylsilyl)trifluoroacetamide, has been established to produce completely 6-O-desulfated heparin with few other chemical changes. The degrees of 6-O-desulfation were estimated by means of chemical disaccharide analyses and/or 13 C NMR spectra. Although the completely 6-O-desulfated heparin lost about 20% of 2-O-sulfate groups, any other chemical changes and depolymerization were not detected. The completely 6-O-desulfated heparin displayed strong inhibition of COS-1 cell adhesion to basic fibroblast growth factor (bFGF)-coated well in a dose-dependent manner, as was clarified by the competitive cell-adhesion assay. Furthermore, the completely 6-O-desulfated heparin was shown to promote in vitro A31 fibroblast proliferation in a dose-dependent manner in the presence of bFGF. These results suggest that signal transduction through bFGF/bFGF receptor in A31 cells occurs in the absence of 6-O-sulfate groups in heparin. The involvement of 6-O-sulfate group(s) of heparin/heparan sulfate in the promotion of bFGF mitogenic activity was reported by several groups. This discrepancy between our results and those of other groups would be due to the differences in molecular size of heparin/heparan sulfate derivatives and/or cell species used for the assay. Heparin and heparan sulfate (HS)1 are known as glycosaminoglycan (GAG) components of extracellular matrix-forming connective tissues of animals. The former GAG, heparin, is exclusively distributed in mast cells, and the latter GAG, HS, is widely distributed in animal tissues. With the accumulation of the information concerning biological roles of heparin and HS, it has been revealed that their biological functions mostly depend upon interaction between polysaccharides and physiologically active molecules, although the biological roles of heparin and HS are highly diverged. For instance, they interact with lipoprotein lipase (1, 2), anti-thrombin III (3, 4), basic fibroblast growth factor (bFGF) (5-8), etc. Furthermore, minimum structures of heparin and/or HS necessary for binding with antithrombin III and/or bFGF have been determined (9, 12). Chemical modification of heparin has been undertaken by several researchers, focusing on the elucidation of the mechanism underlying interaction between heparin and the physiologically active molecules as described above. Specific removal of major sulfate groups of heparin such as 2-O-sulfate, 6-Osulfate, and N-sulfate groups would be useful in order to clarify the backbone structures of oligosaccharides bearing specific array of sulfate groups responsible for the interactions with physiologically active molecules. For instance, selective removal of 6-O-sulfate groups from glucosamine residues of heparin is of great importance in order to evaluate the involvemen...

Cell Type-specific Differences in Glycosaminoglycans Modulate the Biological Activity of a Heparin-binding Peptide (RKRLQVQLSIRT) from the G Domain of the Laminin α1 Chain

“…Thr 12 3 Ala and AG73T both lack the biological activity of AG73; they do not inhibit branching morphogenesis of submandibular glands, do not inhibit B16F10 network formation on Matrigel, and are unable to compete attachment of HSG cells to AG73 (Table I). Many substitutions altered the k a by a few fold, which in this type of assay is not considered significant, but two substitutions, Ile 10 3 Leu and Arg 11 3 Lys, decreased the k a by greater than 5-fold. Ile 10 3 Leu partly inhibited HSG cell attachment to AG73, and Arg 11 3 Lys partly inhibited branching morphogenesis of submandibular glands; however, both peptides were unable to inhibit B16F10 network formation on Matrigel.…”

“…Biological Activity-Peptides with a series of alanine substitutions, conserved and non-conserved substitutions in residues Ile 10 and Arg 11 , and N-terminal truncations were synthesized and tested in various assays (Table I). Cell attachment assays to these peptides with both HSG and B16F10 cells showed similar results; no single amino acid substitution inhibited cell attachment.…”

“…Alanine substitutions that resulted in the peptide with decreased ability to compete HSG cell attachment to AG73 were Arg 3 3 Ala, Leu 4 3 Ala, Val 6 3 Ala, Leu 8 3 Ala, Ile 10 3 Ala, and Thr 12 3 Ala. Truncated peptides that were unable to compete HSG cell attachment were AG73D and AG73G, which are both shorter than 8 amino acids, and AG73H, which was only missing the N-terminal Arg 1 and the C-terminal Thr 12 . Biotinylated heparin binding assays identified four peptides with alanine substitutions that resulted in decreased heparin binding: Leu 4 3 Ala, Val 6 3 Ala, Leu 8 3 Ala, and Ile 10 3 Ala (Table I).…”

“…In addition, surface plasmon resonance analysis identified the C-terminal IRT of the sequence to be important for heparin binding. Structure-based sequence alignment predicts AG73 in a ␤-sheet with the N-terminal K (Lys 2 ) and the C-terminal R (Arg 10 ) on the surface of the G domain. In conclusion, we have determined that differences in cell surface glycosaminoglycans and differences in the amino acids in AG73 recognized by cells modulate the biological activity of the peptide and provide a mechanism to explain its cell-specific activities.…”

Modified Heparin Inhibits P-selectin-mediated Cell Adhesion of Human Colon Carcinoma Cells to Immobilized Platelets under Dynamic Flow Conditions

“…N-Desulfated/2-O, 3-O-desulfated heparin (N/2/3DS-heparin) was typically prepared by 2-O, 3-O-desulfation of N-desulfated heparin as described for 2-O, 3-O-desulfated heparin. 6-O-Desulfated heparin (6ODS-heparin) and N-desulfated 6-O-desulfated heparin (N/6DS-heparin) was prepared as recently described (28,29). The preparation contained about 10% fewer 2-O-sulfate groups.…”