Expression, purification and characterization of a biologically active and thermally stable human lysyl oxidase

Lysyl oxidase (LOX), a promising therapeutic target for the progression of cancer and fibrosis, has not been well characterized yet. A major difficulty faced in LOX characterization is its lack of solubility in common buffers. In this study, mature LOX (mLOX) was cloned, purified and its purity was ascertained by mass spectroscopy. Through screening various buffers, 0.2 M glycine-NaOH buffer with 10% glycerol pH 8.0 was identified to maintain mLOX in its soluble state. About 67% of the refolded mLOX was found to be in copper bound state after His-tag removal. Catalytic properties Km and kcat were found to be 3.72 × 10−4 M and 7.29 ×103s−1. In addition, collagen cross-linking in ARPE-19 cells was augmented on exposure to mLOX, endorsing its biological activity. Circular Dichroism revealed that mLOX comprises 8.43% of α-helix and 22% of β-strand and it was thermally stable up to 90°C. Disulfide linkage imparts the structural stability in LOX which was experimentally ascertained with intrinsic and extrinsic fluorescence studies

Cell surface nucleolin is a novel ADAMTS5 receptor mediating endothelial cell apoptosis

A Disintegrin and Metalloproteinase with ThromboSpondin motif (ADAMTS) 5 functions as an anti-angiogenic and anti-cancer protein independent of its metalloproteinase activity. Both full-length ADAMTS5 and TS5-p45, the autocatalytically cleaved C-terminal 45 kDa truncate of ADAMTS5, inhibits angiogenesis, and induces endothelial cell (EC) apoptosis. However, how ADAMTS5 triggers EC apoptosis remains unclear. This work shows that caspase-8 (Cas-8) and caspase-9 (Cas-9) are involved in TS5-p45-induced EC apoptosis. We identify cell surface nucleolin (NCL) as a novel high-affinity receptor for TS5-p45 in ECs, mediating TS5-p45's cell surface binding and pro-apoptotic function. We show that the central RNA-binding domain (RBD) of NCL is essential and sufficient for its binding to TS5-p45. Upon interacting with EC surface NCL, TS5-p45 is internalized through clathrin- and caveolin-dependent endocytosis and trafficked to the nucleus via late endosomes (LEs). We demonstrate that the nuclear trafficking of TS5-p45 is important for its pro-apoptotic activity as disruption of LE membrane integrity with an endosomolytic peptide suppressed both nuclear trafficking and pro-apoptotic activity of TS5-p45. Through cell surface biotinylation, we revealed that cell surface NCL shuttles extracellular TS5-p45 to the nucleus to mediate apoptosis. Furthermore, blocking the importin α1/ß1 receptor hindered the nuclear trafficking of TS5-p45, suggesting the involvement of the nuclear importing machinery for this nuclear translocation. RNA-seq identified many apoptosis-related genes that are differentially expressed at least two-fold in TS5-p45-treated ECs, with 10 of them qRT-PCR-validated and at least 5 of these genes potentially contributing to TS5-p45-NCL-induced apoptosis. Altogether, our work identifies NCL as a novel cell surface receptor for ADAMTS5 and demonstrates the critical role of NCL-mediated internalization and nuclear trafficking for ADAMTS5-induced EC apoptosis. These findings reveal novel mechanistic insights of the secreted metalloproteinase ADAMTS5 in angiogenesis inhibition.Published versionThis work was supported by a grant awarded from the Singapore Ministry of Education to Ruowen Ge (MOE2014-T2-2-150). DCK was supported by the Singapore International Graduate Award from the Agency for Science, Technology & Research and the National University of Singapore

Expression, purification and characterization of a biologically active and thermally stable human lysyl oxidase

105-116Lysyl oxidase (LOX), a promising therapeutic target for the progression of cancer and fibrosis, has not been well characterized yet. A major difficulty faced in LOX characterization is its lack of solubility in common buffers. In this study, mature LOX (mLOX) was cloned, purified and its purity was ascertained by mass spectroscopy. Through screening various buffers, 0.2 M glycine-NaOH buffer with 10% glycerol pH 8.0 was identified to maintain mLOX in its soluble state. About 67% of the refolded mLOX was found to be in copper bound state after His-tag removal. Catalytic properties Km and kcat were found to be 3.72 × 10−4 M and 7.29 ×103s−1. In addition, collagen cross-linking in ARPE-19 cells was augmented on exposure to mLOX, endorsing its biological activity. Circular Dichroism revealed that mLOX comprises 8.43% of α-helix and 22% of β-strand and it was thermally stable up to 90°C. Disulfide linkage imparts the structural stability in LOX which was experimentally ascertained with intrinsic and extrinsic fluorescence studies

Recombinant TSR1 of ADAMTS5 Suppresses Melanoma Growth in Mice via an Anti-angiogenic Mechanism

Inhibiting tumor angiogenesis is a well-established approach for anticancer therapeutic development. A Disintegrin-like and Metalloproteinase with ThromboSpondin Motifs 5 (ADAMTS5) is a secreted matrix metalloproteinase in the ADAMTS family that also functions as an anti-angiogenic/anti-tumorigenic molecule. Its anti-angiogenic/anti-tumorigenic function is independent from its proteinase activity, but requires its first thrombospondin type 1 repeat (TSR1). However, it is not known if recombinant TSR1 (rTSR1) can function as an anticancer therapeutic. In this report, we expressed and purified a 75-residue recombinant TSR1 polypeptide from E. coli and investigated its ability to function as an anticancer therapeutic in mice. We demonstrate that rTSR1 is present in the blood circulation as well as in the tumor tissue at 15 min post intraperitoneal injection. Intraperitoneal delivery of rTSR1 potently suppressed subcutaneous B16F10 melanoma growth as a single agent, accompanied by diminished tumor angiogenesis, increased apoptosis, and reduced cell proliferation in the tumor tissue. Consistently, rTSR1 dose-dependently induced the apoptosis of cultured human umbilical vein endothelial cells (HUVECs) in a caspase-dependent manner. This work indicates that rTSR1 of ADAMTS5 can function as a potent anticancer therapy in mice. It thus has the potential to be further developed into an anticancer drug