Identification of Active-Site Residues of the Pro-Metastatic Endoglycosidase Heparanase

Hulett, Mark D.; Hornby, June; Ohms, Stephen; Zuegg, Johannes; Freeman, Craig; Gready, Jill E.; Parish, Christopher R.

doi:10.1021/bi002080p

Cited by 148 publications

(162 citation statements)

References 52 publications

(85 reference statements)

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“…This study identified heparanase a binding mode of aspirin that was beyond heparin-binding sites. We have shown that Lys159 and Glu225 of heparanase are critical for aspirin binding, consistent with previous findings that Glu225 and Glu343 are key residuals essential for the activity of heparanase (46,47). Hence, we proposed a mechanism for aspirin that inhibiting the activity of heparanase by binding to Glu150 (Q9Y251: Glu 225), similar to two types of small-molecular inhibitors (48,49).…”

Section: Discussionsupporting

confidence: 89%

Aspirin Inhibits Cancer Metastasis and Angiogenesis via Targeting Heparanase

Dai

Yan

et al. 2017

Clinical Cancer Research

100

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Purpose: Recent epidemiological and clinical studies have suggested the benefit of aspirin for patients with cancer, which inspired increasing efforts to demonstrate the anticancer ability of aspirin and reveal the molecular mechanisms behind. Nevertheless, the anticancer activity and related mechanisms of aspirin remain largely unknown. This study aimed to confirm this observation, and more importantly, to investigate the potential target contributed to the anticancer of aspirin.Experimental Design: A homogeneous time-resolved fluorescence (HTRF) assay was used to examine the impact of aspirin on heparanase. Streptavidin pull-down, surface plasmon resonance (SPR) assay, and molecular docking were performed to identify heparanase as an aspirin-binding protein. Transwell, rat aortic rings, and chicken chorioallantoic membrane model were used to evaluate the antimetastasis and anti-angiogenesis effects of aspirin, and these phenotypes were tested in a B16F10 metastatic model, MDA-MB-231 metastatic model, and MDA-MB-435 xenograft model.Results: This study identified heparanase, an oncogenic extracellular matrix enzyme involved in cancer metastasis and angiogenesis, as a potential target of aspirin. We had discovered that aspirin directly binds to Glu225 region of heparanase and inhibits the enzymatic activity. Aspirin impeded tumor metastasis, angiogenesis, and growth in heparanase-dependent manner.Conclusions: In summary, this study has illustrated heparanase as a target of aspirin for the first time. It provides insights for a better understanding of the mechanisms of aspirin in anticancer effects, and offers a direction for the development of smallmolecule inhibitors of heparanase.

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Section: Discussionsupporting

confidence: 89%

Aspirin Inhibits Cancer Metastasis and Angiogenesis via Targeting Heparanase

Dai

Yan

et al. 2017

Clinical Cancer Research

100

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“…The three-dimensional structure of heparanase is not yet known in detail. Translation of the primary structure of heparanase over another endo-␤-glycosidase (␤-xylanase) shows clusters of basic amino acid residues at least one of which is conceivably implicated in binding to sulfate groups of the substrate (55). Our preliminary studies, applying point mutations and deletions as well as synthetic peptides, identified amino acid residues 158 -171 as the predominant HS binding domain of the heparanase molecule.…”

Section: Discussionmentioning

confidence: 82%

Modulation of the Heparanase-inhibiting Activity of Heparin through Selective Desulfation, Graded N-Acetylation, and Glycol Splitting

Naggi

Casu

Perez

et al. 2005

Journal of Biological Chemistry

204

147

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Heparanase is an endo-␤-glucuronidase that cleaves heparan sulfate (HS) chains of heparan sulfate proteoglycans on cell surfaces and in the extracellular matrix (ECM). Heparanase, overexpressed by most cancer cells, facilitates extravasation of blood-borne tumor cells and causes release of growth factors sequestered by HS chains, thus accelerating tumor growth and metastasis. Inhibition of heparanase with HS mimics is a promising target for a novel strategy in cancer therapy. In this study, in vitro inhibition of recombinant heparanase was determined for heparin derivatives differing in degrees of 2-O-and 6-O-sulfation, N-acetylation, and glycol splitting of nonsulfated uronic acid residues. The contemporaneous presence of sulfate groups at O-2 of IdoA and at O-6 of GlcN was found to be non-essential for effective inhibition of heparanase activity provided that one of the two positions retains a high degree of sulfation. N-Desulfation/ N-acetylation involved a marked decrease in the inhibitory activity for degrees of N-acetylation higher than 50%, suggesting that at least one NSO 3 group per disaccharide unit is involved in interaction with the enzyme. On the other hand, glycol splitting of preexisting or of both preexisting and chemically generated nonsulfated uronic acids dramatically increased the heparanase-inhibiting activity irrespective of the degree of N-acetylation. Indeed N-acetylated heparins in their glycol-split forms inhibited heparanase as effectively as the corresponding N-sulfated derivatives. Whereas heparin and N-acetylheparins containing unmodified D-glucuronic acid residues inhibited heparanase by acting, at least in part, as substrates, their glycol-split derivatives were no more susceptible to cleavage by heparanase. Glycol-split N-acetylheparins did not release basic fibroblast growth factor from ECM and failed to stimulate its mitogenic activity. The combination of high inhibition of heparanase and low release/potentiation of ECM-bound growth factor indicates that N-acetylated, glycol-split heparins are potential antiangiogenic and antimetastatic agents that are more effective than their counterparts with unmodified backbones.

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“…1A) suggests that the N terminus of the 50 kDa protein undergoes conformational changes upon heparanase processing, exposing an epitope that is not present in the 65 kDa heparanase precursor. Although this region is not considered to be part of the heparanase active site (Hulett et al, 2000), it may well be involved in a three-dimensional organization assumed by the 50 kDa heparanase upon processing, and that is necessary for enzymatic activity. To test this hypothesis, recombinant purified heparanase was incubated with affinity-purified antibody 733 or control rabbit IgG, and enzymatic activity was determined.…”

Section: Antibody 733 Inhibits Heparanase Enzymatic Activitymentioning

confidence: 99%

Processing and activation of latent heparanase occurs in lysosomes

Zetser¹,

Levy-Adam²,

Kaplan³

et al. 2004

Journal of Cell Science

207

259

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Heparanase is a heparan sulfate degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Heparanase is synthesized as a 65 kDa non-active precursor that subsequently undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. The protease responsible for heparanase processing is currently unknown, as is the sub-cellular processing site. In this study, we characterize an antibody (733) that preferentially recognizes the active 50 kDa heparanase form as compared to the non-active 65 kDa heparanase precursor. We have utilized this and other anti-heparanase antibodies to study the cellular localization of the latent 65 kDa and active 50 kDa heparanase forms during uptake and processing of exogenously added heparanase. Interestingly, not only the processed 50 kDa, but also the 65 kDa heparanase precursor was localized to perinuclear vesicles, suggesting that heparanase processing occurs in lysosomes. Indeed, heparanase processing was completely inhibited by chloroquine and bafilomycin A1, inhibitors of lysosome proteases. Similarly, processing of membrane-targeted heparanase was also chloroquine-sensitive, further ruling out the plasma membrane as the heparanase processing site. Finally, we provide evidence that antibody 733 partially neutralizes the enzymatic activity of heparanase, suggesting that the N-terminal region of the molecule is involved in assuming an active conformation. Monoclonal antibodies directed to this region are likely to provide specific heparanase inhibitors and hence assist in resolving heparanase functions under normal and pathological conditions.

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Identification of Active-Site Residues of the Pro-Metastatic Endoglycosidase Heparanase

Cited by 148 publications

References 52 publications

Aspirin Inhibits Cancer Metastasis and Angiogenesis via Targeting Heparanase

Aspirin Inhibits Cancer Metastasis and Angiogenesis via Targeting Heparanase

Modulation of the Heparanase-inhibiting Activity of Heparin through Selective Desulfation, Graded N-Acetylation, and Glycol Splitting

Processing and activation of latent heparanase occurs in lysosomes

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