Exploring Bacterial Heparinase II Activities with Defined Substrates

Abstract

Bacterial heparinases that cleave heparan sulfate (HS) and heparin are widely used to generate low-molecular-weight heparins (LMWHs) and to structurally and functionally characterise heparin and HS biomolecules. We provide novel insights into the substrate specificity of heparinase II from two different bacteria: Pedobacter heparinus (formerly Flavobacterium heparinum) and Bacteroides eggerthii. The activity towards various well-defined HS oligosaccharides was investigated by (1) H NMR spectroscopy; this revealed distinct specificities for the two heparinases. Heparinase II from P. heparinus appears to be more active and displays a broader substrate specificity than B. eggerthii heparinase II. Furthermore, HS di- and tetrasaccharides inhibited B. eggerthii heparinase II activity. A better understanding of heparinase substrate specificity will contribute to the production of homogenous LMWHs, provide better characterisation of heparin and HS and assist therapeutic applications.

Antiangiogenic platinum through glycan targeting

“…22–24 The course of FPX hydrolysis can conveniently be determined by 1 H NMR spectroscopy as the anomeric protons are sensitive reporters of the cleavage reaction by both heparanase and the bacterial heparinases. 22,23,25 Monitoring the hydrolysis of the native substrates of HPSE, heparan sulfate or heparin, is complicated by the heterogeneous nature of the substrate and by severe signal overlap in the 1 H NMR spectra. In addition, HPSE can cleave the native substrate in several locations on the polysaccharide chain resulting in multiple HS/heparin fragments which may themselves be either further substrates or inhibitors of the enzyme.…”

“…42 Fondaparinux (GlcNS6S-GlcA-GlcNS3,6S-IdoA2S-GlcNS6S-OMe) and PI-88 (Muparfostat) were sourced as previously. 3,23 The unsulfated 5-mer (GlcA–GlcNAc–GlcA–GlcNAc–GlcA) was purchased from Polysciences, Inc.…”

“…23 Briefly, FPX (0.5 mM) was pre-incubated with Pt inhibitor (0.5 mM TriplatinNC or Triplatin) and then was incubated with 1 μg human heparanase at 37 °C in 40 mM deuterated sodium acetate buffer, pH 5. 1 H NMR spectroscopy was performed in 200 μL Shigemi tubes in an Avance 600 MHz Ultrashield NMR spectrometer (Bruker) with a 1 H/ 13 C/ 15 N gradient cryoprobe system, data being collected and analysed with the TOPSPIN software (Bruker).…”

Substitution‐Inert Polynuclear Platinum Complexes as Metalloshielding Agents for Heparan Sulfate

“…FPX is a well‐defined, highly sulfated synthetic glycosaminoglycan‐based fragment that has been used clinically as an antithrombotic agent since the 1940s . It is a substrate for both bacterial heparinases and human heparanase and is useful in screening the efficiency and reaction kinetics of potential heparanase inhibitors . Fondaparinux is also an ideal substrate for mechanistic studies because it is homogeneous, has low molecular weight, and represents a single point of cleavage for both mammalian and bacterial enzymes .…”

“…Fondaparinux is also an ideal substrate for mechanistic studies because it is homogeneous, has low molecular weight, and represents a single point of cleavage for both mammalian and bacterial enzymes . The course of FPX hydrolysis can conveniently be determined by 1 H NMR spectroscopy because the anomeric protons are sensitive reporters of the cleavage reaction by both heparanase and the bacterial heparinases …”