Abstract
Heparan sulfate (HS) is a glycosaminoglycan that forms a key component of the extracellular matrix (ECM). Breakdown of HS is carried out by heparanase (HPSE), an endo-β-glucuronidase of the glycoside hydrolase 79 (GH79) family. Overexpression of HPSE results in breakdown of extracellular HS and release of stored growth factors and hence is strongly linked to cancer metastasis. Here we present crystal structures of human HPSE at 1.6-Å to 1.9-Å resolution that reveal how an endo-acting binding cleft is exposed by proteolytic activation of latent proHPSE. We used oligosaccharide complexes to map the substrate-binding and sulfate-recognition motifs. These data shed light on the structure and interactions of a key enzyme involved in ECM maintenance and provide a starting point for the design of HPSE inhibitors for use as biochemical tools and anticancer therapeutics.
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Change history
08 December 2015
In the version of this article initially published, there were errors in two figures. The schematic in Figure 4a was mistakenly drawn with a β1→3 linkage between the –1 GlcUA and the +1 GlcNS(6S), rather than the correct β1→4 linkage. Supplementary Figure 1 incorrectly gave the names of the GlcNX monomers as N-acetyl-α-D-glucuronic acid and N-sulfo-α-D-glucuronic acid, rather than N-acetyl-α-D-glucosamine and N-sulfo- α-D-glucosamine. These errors have been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank Diamond Light Source for access to beamlines I02, I03 and I04 (proposal mx-9948), which contributed to the results presented here. DH10EMBacY cells used to generate recombinant bacmids were a gift from I. Berger (University of Bristol). The full HPSE gene was obtained from a baculoviral construct from D. Jarvis (University of Wyoming; US National Institutes of Health grant RR005351). G.J.D. and L.W. acknowledge support from the European Research Council through Advanced Grant Glycopoise (AdG 322942 (G.J.D.)). A.M.B. and C.M.V. thank the UK Medical Research Council for funding (MR/K000179/1 (A.M.B.)).
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L.W. and G.J.D. designed and interpreted the experiments. L.W. and C.M.V. cloned, expressed and purified proteins with help from A.M.B. in eukaryotic protein expression facilities. L.W. carried out kinetics experiments and protein crystallizations, and solved the structures of protein and ligand complexes. L.W. and G.J.D. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Molecular and domain composition of HS.
HS is a structurally heterogeneous linear polysaccharide composed of repeating HexUA-(1,4)-GlcNX-(1,4) disaccharide units. (a) Chemical structures of the constituent HS sugars. HexUA is either GlcUA or IdoUA, which differ only in stereochemistry of the 5 position. GlcNX is either GlcNAc or its N-deacetylated-N-sulfated congener GlcNS. IdoUA, GlcNAc and GlcNS are subject to varying degrees of O-sulfation. Also shown is the structure of ΔHexUA, an unsaturated uronic acid formed at the non-reducing end of heparins depolymerized by bacterial heparin lyases, such as the dp4 tetrasaccharide used in this study. (b) Macromolecular 'domain' like organization of mature HS. NA domains are rich in GlcUA and GlcNAc, and show low levels of O-sulfation. NS domains are enriched in IdoUA and GlcNS, with a higher degree of O-sulfation. NA and NS domains are bounded by mixed NA/NS domains, which show intermediate characteristics.
Supplementary Figure 2 Supplemental crystal-structure illustrations.
(a) Ribbon representations of HPSE (blue and gold) superposed against the bacterial exoglucuronidase AcaGH79 (dark red; PDB accession code 3VNY), and bacterial endoglucuronidase BpHPSE (coral; PDB accession code 5BWI). The three GH79 proteins possess a high degree of similarity in their overall fold topology. (b) M09 S05a −2 subsite GlcNS(6S) with density contoured to 1σ (0.25 electrons/Å3) and 2σ (0.51 electrons/Å3). The relative weakness of 6O sulfate density compared to N-sulfate can be seen. Electron densities are REFMAC maximum-likelihood/σÅ weighted 2Fo−Fc syntheses. (c) View of HPSE (colored by secondary structure) along the active site cleft towards the 'positive' end, showing the presence of a symmetry molecule (ice blue) at the opening. This symmetry molecule prevents HPSE interactions in crystallo which involve substrates protruding too far out of the 'positive' end of the cleft.
Supplementary Figure 3 Michaelis-Menten kinetics for HPSE hydrolysis of the HepMers.
Reaction was measured using the reducing end detection dye WST1. Baseline subtractions using a no enzyme control were carried out for all reactions, to control for non-enzymatic paranitrophenol autohydrolysis. Error bars are standard errors of the mean for technical replicates (n=3 for all points). N.d. stands for not determinable.
Supplementary Figure 4 Relationship of HPSE active site residues to those of other GH79 enzymes.
(a) Clustalω (Sievers, F. et al. Mol Syst Biol 7 (2011)) alignment of 4 eukaryotic GH79 sequences, AcaGH79, and BpHPSE. Residues corresponding to those which interact with substrates in human HPSE (as outlined in Figure 4) are highlighted: green where identical with the human sequence, or orange where a non-conserved residue can interact in a similar fashion. Residues of the −1 subsite are hyperconserved, illustrating their requirement for interacting with GlcUA. In contrast, residues of the −2 and +1 subsites are only well conserved amongst the mammalian heparanases. (b) Active site view of the dp4-HPSE complex, with residues colored by conservation to bacterial enzymes AcaGH79 and BpHPSE (green – identical, yellow – partially conserved, red – not conserved). Dp4 ligand is shown in grey.
Supplementary Figure 5 Relationship of HSPE proenzyme linker sequence to those of other GH79 enzymes.
Alignment of 4 eukaryotic GH79 sequences, AcaGH79, and BpHPSE, across the region corresponding to the proenzyme linker of human HPSE (dashed box). Eukaryotic GH79s show an extended sequence at this position, likely corresponding to a proteolytically cleavable linker as in human HPSE. The corresponding AcaGH79 sequence is shorter, and forms a loop which creates part of its exo- binding pocket. The corresponding BpHPSE sequence is effectively absent, and corresponds to a very short loop which reveals the endo- acting binding cleft of this enzyme.
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Wu, L., Viola, C., Brzozowski, A. et al. Structural characterization of human heparanase reveals insights into substrate recognition. Nat Struct Mol Biol 22, 1016–1022 (2015). https://doi.org/10.1038/nsmb.3136
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DOI: https://doi.org/10.1038/nsmb.3136
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