Deciphering mode of action of heparanase using structurally defined oligosaccharides

Abstract

Heparan sulfate (HS) is a highly sulfated polysaccharide that serves many biological functions, including regulating cell growth and inflammatory responses as well as the blood coagulation process. Heparanase is an enzyme that cleaves HS and is known to display a variety of pathophysiological effects in cancer, diabetes, and Alzheimer disease. The link between heparanase and diseases is a result of its selective cleavage of HS, which releases smaller HS fragments to enhance cell proliferation, migration, and invasion. Despite its importance in pathological diseases, the structural cues in HS that direct heparanase cleavage and the steps of HS depolymerization remain unknown. Here, we sought to probe the substrate specificity of heparanase using a series of structurally defined oligosaccharide substrates. The sites of heparanase cleavage on the oligosaccharide substrates were determined by mass spectrometry and gel permeation chromatography. We discovered that heparanase cleaves the linkage of glucuronic acid linked to glucosamine carrying 6-O-sulfo groups. Furthermore, our findings suggest that heparanase displays different cleavage modes by recognizing the structures of the nonreducing ends of the substrates. Our results deepen the understanding of the action mode of heparanase.

FIGURE 1.

Scheme for synthesis of oligosaccharide substrates. Synthesis was initiated from GlcUA-pNP. The starting monosaccharide was elongated by glycosyltransferases to the desired size. The resultant oligosaccharides were then N-sulfated and 6-O-sulfated by N-sulfotransferase (NST) and 6-O-sulfotransferases 1 and 3 (6OSTs), respectively. A total of eight oligosaccharides were synthesized, and structural analysis of each oligosaccharide was conducted by ESI-MS (supplemental Fig. S1). The purity was assessed by DEAE-HPLC (supplemental Fig. S2). KfiA is the N-acetylglucosaminyltransferase from E. coli strain K5. pmHS2, P. multocida heparosan synthase 2; GlcA, GlcUA; PAPS, 3′-phosphoadenosine 5′-phosphosulfate.

FIGURE 2.

Analysis of heparanase-digested Nona-3. A, Bio-Gel P-2 elution profiles of ³⁵S-labeled Nona-3 without (upper panel) and with (lower panel) heparanase digestion. B, ESI-MS spectra of heparanase-digested Nona-3 from fractions eluted from the P-2 column. Left panel, spectrum of Tri-12 from fractions 51–57. Middle panel, spectrum of Tetra-17 from fractions 43–49. Right panel, spectrum of Di(pNP)-9 from fractions 71–75. C, chemical reaction involved in the digestion of Nona-3 with heparanase.

FIGURE 3.

Schematic representation of heparanase cleavage of different nonasaccharide substrates. A, consecutive cleavage pattern. When GlcNAc6S is present at residue 2, heparanase displays the consecutive cleavage pattern, namely cleaving the linkages between residues 3 and 4 and residues 5 and 6. B, gapped cleavage pattern. When GlcNS6S is present at residue 2, heparanase displays the gapped cleavage pattern, namely cleaving the linkages between residues 3 and 4 and residues 7 and 8 and skipping the bond between residues 5 and 6. GlcA, GlcUA.

FIGURE 4.

DEAE-HPLC chromatograms of ³⁵S-labeled Nona-3 and Nona-7 digested with heparanase at different times. Heparanase was incubated with ³⁵S-labeled Nona-3 and Nona-7 at 0, 5, 15, 25, 60, 120, 240, and 480 min. The reaction was stopped, and the cleavage products were analyzed and separated by high resolution DEAE-HPLC. A, degradation of ³⁵S-labeled Nona-3 and Nona-7 at 0–480 min by heparanase. B, upper panel, schematic identifying the cleavage products from the chemical reaction involved in the digestion of Nona-3 with heparanase. Lower panel, chemical reaction involved in the digestion of Nona-7 with heparanase. GlcA, GlcUA.