Unraveling the Specificity of Heparanase Utilizing Synthetic Substrates

Heparanase is a promising anticancer target because of its involvement in cancer invasion and metastasis. Heparanase cleaves heparan sulfate (HS), a sulfated polysaccharide, and activates a series of HS-mediated cell proliferation and angiogenesis processes. Understanding the substrate specificity of heparanase will aid the discovery of heparanase inhibitors. Here, we sought to determine the specificity of heparanase using synthetic polysaccharide substrates. The substrates were prepared using purified HS biosynthetic enzymes. Using these substrates, we were able to dissect the structural moieties required for heparanase. Our data suggest that heparanase cleaves the linkage between a GlcA unit and an N-sulfo glucosamine unit carrying either a 3-O-sulfo or a 6-O-sulfo group. In addition, heparanase cleaves the linkage of a GlcA unit and N-sulfo glucosamine unit with a 2-O-sulfated GlcA residue, not a 2-O-sulfated IdoA residue, in proximity. We also discovered that the polysaccharide with repeating disaccharide units of IdoA2S-GlcNS inhibits the activity of heparanase. Our findings advance the understanding of the substrate specificity of heparanase and identify a lead compound for developing polysaccharide-based heparanase inhibitors

OGT (O-GlcNAC transferase) selectively modifies multiple residues unique to Lamin A

The LMNA gene encodes lamins A and C with key roles in nuclear structure, signaling, gene regulation, and genome integrity. Mutations in LMNA cause over 12 diseases (‘laminopathies’). Lamins A and C are identical for their first 566 residues. However, they form separate filaments in vivo, with apparently distinct roles. We report that lamin A is β-O-linked N-acetylglucosamine-(O-GlcNAc)-modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays with purified O-GlcNAc transferase (OGT) enzyme showed robust O-GlcNAcylation of recombinant mature lamin A tails (residues 385–646), with no detectable modification of lamin B1, lamin C, or ‘progerin’ (Δ50) tails. Using mass spectrometry, we identified 11 O-GlcNAc sites in a ‘sweet spot’ unique to lamin A, with up to seven sugars per peptide. Most sites were unpredicted by current algorithms. Double-mutant (S612A/T643A) lamin A tails were still robustly O-GlcNAc-modified at seven sites. By contrast, O-GlcNAcylation was undetectable on tails bearing deletion Δ50, which causes Hutchinson–Gilford progeria syndrome, and greatly reduced by deletion Δ35. We conclude that residues deleted in progeria are required for substrate recognition and/or modification by OGT in vitro. Interestingly, deletion Δ35, which does not remove the majority of identified O-GlcNAc sites, does remove potential OGT-association motifs (lamin A residues 622–625 and 639–645) homologous to that in mouse Tet1. These biochemical results are significant because they identify a novel molecular pathway that may profoundly influence lamin A function. The hypothesis that lamin A is selectively regulated by OGT warrants future testing in vivo, along with two predictions: genetic variants may contribute to disease by perturbing OGT-dependent regulation, and nutrient or other stresses might cause OGT to misregulate wildtype lamin A