Structural basis of heparan sulfate-specific degradation by heparinase III

Abstract

Heparinase III (HepIII) is a 73-kDa polysaccharide lyase (PL) that degrades the heparan sulfate (HS) polysaccharides at sulfate-rare regions, which are important co-factors for a vast array of functional distinct proteins including the well-characterized antithrombin and the FGF/FGFR signal transduction system. It functions in cleaving metazoan heparan sulfate (HS) and providing carbon, nitrogen and sulfate sources for host microorganisms. It has long been used to deduce the structure of HS and heparin motifs; however, the structure of its own is unknown. Here we report the crystal structure of the HepIII from Bacteroides thetaiotaomicron at a resolution of 1.6 Å. The overall architecture of HepIII belongs to the (α/α)5 toroid subclass with an N-terminal toroid-like domain and a C-terminal β-sandwich domain. Analysis of this high-resolution structure allows us to identify a potential HS substrate binding site in a tunnel between the two domains. A tetrasaccharide substrate bound model suggests an elimination mechanism in the HS degradation. Asn260 and His464 neutralize the carboxylic group, whereas Tyr314 serves both as a general base in C-5 proton abstraction, and a general acid in a proton donation to reconstitute the terminal hydroxyl group, respectively. The structure of HepIII and the proposed reaction model provide a molecular basis for its potential practical utilization and the mechanism of its eliminative degradation for HS polysaccarides.

Syndecan-4 regulates extravillous trophoblast migration by coordinating protein kinase C activation

“…To determine whether HSPGs are necessary for EVT invasion and migration, Matrigel-based invasion assays and scratch-wound migration assays were performed using a well-established EVT line, HTR8/SVneo (henceforth referred to as HTR8 EVTs). Cells were treated with different concentrations of unfractionated heparin (which competitively inhibits heparan sulfate chains interacting with ligands 24 ) or heparinase III (which cleaves heparan sulfate chains from HSPGs 25 ) during assays. Compared to controls, approximately 35% less cells invaded through Matrigel following exposure to 10 µg/ml or 100 µg/ml unfractionated heparin (Fig.…”

Discovery of exolytic heparinases and their catalytic mechanism and potential application

“…The active centers of exoHep and Hepase II partially overlap, indicating that their catalytic mechanism might have some features in common. As shown in this study, the residues His 337 , Tyr 390 and His 555 of exoHep corresponding to the three key residues His 202 , Tyr 257 and His 406 of Hepase II 22 play key roles in the catalytic mechanism of exoHep, indicating that similar to other lyases, including Hepases I, II and III, exolytic Hepases catalyze the degradation of HP via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid 19,22,23 . However, unlike in the case of the His 337 to Ala mutation that completely destroyed the ability of exoHep to cleave the tetrasaccharide ΔUA2S1-4GlcNS6S1-4IdoA2S1-4GlcNS6S ( Supplementary Fig.…”

“…Hepase II adopts a topology similar to Hepase III and has no selectivity for certain GlcA/IdoA or IdoA2S/GlcA2S structures 18,21,22 . All Hepases share similar catalytic mechanisms in which Tyr-His acts as a Brønsted base and acid 19,22,23 .…”

Uronic polysaccharide degrading enzymes

“…Their catalytic residues and global architecture of their active sites are similar, suggesting similarity of their catalytic mechanisms ( Figure 4, Table S2). The catalytic tyrosine is strictly conserved, Tyr314 in PL12 (PDB code 4FNV [27 ]), Tyr258 in PL17 (PDB code 4OJZ [25 ]) and Tyr299 in PL23 (PDB code 3VSM [28 ]) (Figure 5b,c). Histidine, which plays the role of a Brønsted base during anti elimination, is present in some PL12 enzymes (e.g.…”

“…Heparinase III from P. heparinus (PDB code 4MMH) has an open conformation with the neutralizer Asn240 positioned 10 Å from the catalytic Tyr294, an arrangement clearly incompatible with catalysis [29]. B. thetaiotaomicron heparinase III (PDB code 4FNV) has a more compact conformation [27 ]. The first PL23 structure also displays an open conformation where the neutralizer Asn236 is apparently too far from the catalytic residues [28 ] (Figure 5c).…”