Modulation of Lysyl Oxidase-like 2 Enzymatic Activity by an Allosteric Antibody Inhibitor

Rodrı́guez, Héctor M.; Vaysberg, Maria; Mikels, Amanda; McCauley, Scott; Velayo, Arleene C.; García, Carlos; Smith, Victoria

doi:10.1074/jbc.m109.094136

Cited by 128 publications

(121 citation statements)

References 28 publications

Supporting

Mentioning

116

Contrasting

Order By: Relevance

“…When the fourth SRCR domain was absent, K m was not affected, but k cat was reduced by ϳ65%, suggesting that the fourth SRCR domain plays an important role in optimizing the catalytic efficacy of the LOX catalytic domain of hLOXL2. This supports the observation that AB0023, a specific antibody against the fourth SRCR domain, allosterically inhibits hLOXL2 (21,22).…”

Section: Resultssupporting

confidence: 76%

Post-translational Modifications of Recombinant Human Lysyl Oxidase-like 2 (rhLOXL2) Secreted from Drosophila S2 Cells*

Xu¹,

Finney

et al. 2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: hLOXL2 induces metastasis/invasion of breast cancer cells. Results:The N-glycans and lysine tyrosylquinone (LTQ) cofactor of rhLOXL2 are determined. Conclusion: N-Glycans are essential for proper folding and secretion of hLOXL2 from S2 cells. Significance: This is the first determination of 1) LTQ in the hLOX family of proteins and 2) N-glycosylation in the LOX catalytic domain in the LOX family of proteins.Human lysyl oxidase-like 2 (hLOXL2) is highly up-regulated in metastatic breast cancer cells and tissues and induces epithelial-to-mesenchymal transition, the first step of metastasis/invasion. hloxl2 encodes four N-terminal scavenger receptor cysteine-rich domains and the highly conserved C-terminal lysyl oxidase (LOX) catalytic domain. Here, we assessed the extent of the post-translational modifications of hLOXL2 using truncated recombinant proteins produced in Drosophila S2 cells. The recombinant proteins are soluble, in contrast to LOX, which is consistently reported to require 2-6 M urea for solubilization. The recombinant proteins also show activity in tropoelastin oxidation. After phenylhydrazine derivatization and trypsin digestion, we used mass spectrometry to identify peptides containing the derivatized lysine tyrosylquinone cross-link at Lys-653 and Tyr-689, as well as N-linked glycans at Asn-455 and Asn-644. Disruption of N-glycosylation by site-directed mutagenesis or tunicamycin treatment completely inhibited secretion so that only small quantities of inclusion bodies were detected. The N-glycosylation site at Asn-644 in the LOX catalytic domain is not conserved in human LOX (hLOX), although the LOX catalytic domain of hLOX shares ϳ50% identity and ϳ70% homology with hLOXL2. The catalytic domain of hLOX was not secreted from S2 cells using the same expression system. These results suggest that the N-glycan at Asn-644 of hLOXL2 enhances the solubility and stability of the LOX catalytic domain.Human lysyl oxidase (hLOX) 4 and human lysyl oxidase-like 2 (hLOXL2) represent relatively new therapeutic targets for metastatic/invasive breast cancer (1-3). Recently, the extracellular matrix (ECM) stiffening caused by hLOX and hLOXL2 has been linked to activation of the focal adhesion kinase (FAK)/Src signaling pathway and up-regulation of tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase-9 (MMP-9), thereby increasing degradation/remodeling of the ECM and enabling subsequent metastatic dissemination (1,4,5). hLOXL2 is generally considered to play an equivalent role to hLOX in the ECM. However, the extent of the post-translational modifications (PTMs) of hLOXL2 and its subcellular localization have not been characterized. Ultimately, the function of hLOXL2 at the molecular level remains unclear.hLOXL2 belongs to the LOX family of proteins, but differs from hLOX by an additional four N-terminal scavenger receptor cysteine-rich (SRCR) domains, as shown in Fig. 1A (6). LOX is a copper-and lysine tyrosylquinone (LTQ)-dependent amine oxidase that is traditionally known ...

show abstract

Section: Resultssupporting

confidence: 76%

Post-translational Modifications of Recombinant Human Lysyl Oxidase-like 2 (rhLOXL2) Secreted from Drosophila S2 Cells*

Xu¹,

Finney

et al. 2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…2a). One potential caveat in these studies is that BAPN can block other amine oxidases and it can suppress the activity of LOXL1-4 isoforms in addition to inhibiting LOX [33][34][35] . To more directly modify this pathway, we treated mice with a LOX-specific antibody (Ab) 13,36 , which has been previously shown to inhibit LOX activity and disrupt collagen organization in vitro and in vivo 13,36,37 .…”

Section: Resultsmentioning

confidence: 99%

Control of lung vascular permeability and endotoxin-induced pulmonary oedema by changes in extracellular matrix mechanics

et al. 2013

View full text Add to dashboard Cite

Increased vascular permeability contributes to many diseases, including acute respiratory distress syndrome, cancer and inflammation. Most past work on vascular barrier function has focused on soluble regulators, such as tumour-necrosis factor-a. Here we show that lung vascular permeability is controlled mechanically by changes in extracellular matrix structure. Our studies reveal that pulmonary vascular leakage can be increased by altering extracellular matrix compliance in vitro and by manipulating lysyl oxidase-mediated collagen crosslinking in vivo. Either decreasing or increasing extracellular matrix stiffness relative to normal levels disrupts junctional integrity and increases vascular leakage. Importantly, endotoxin-induced increases of vascular permeability are accompanied by concomitant increases in extracellular matrix rigidity and lysyl oxidase activity, which can be prevented by inhibiting lysyl oxidase activity. The identification of lysyl oxidase and the extracellular matrix as critical regulators of lung vascular leakage might lead to the development of new therapeutic approaches for the treatment of pulmonary oedema and other diseases caused by abnormal vascular permeability.

show abstract

“…Lysyl oxidases, consisting in lysyl oxidase (LOX) and 4 lysyl oxidase-like proteins (LOXL 1 to 4), catalyze the deamination of lysines and hydroxylysines, generating aldehydes that spontaneously react to form covalent cross-links. 6,7 These secreted enzymes are characterized by a conserved C-terminal catalytic domain that contains the lysyl tyrosyl quinone cofactor and the copper-binding site. The N-terminal part is specific to each member and is thought to confer partner and/or substrate specificity.…”

mentioning

confidence: 99%

Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane

Bignon

Pichol-Thievend

Hardouin

et al. 2011

Blood

171

173

View full text Add to dashboard Cite

Sprouting angiogenesis is associated with extensive extracellular matrix (ECM) remodeling. The molecular mechanisms involved in building the vascular microenvironment and its impact on capillary formation remain elusive. We therefore performed a proteomic analysis of ECM from endothelial cells maintained in hypoxia, a major stimulator of angiogenesis. Here, we report the characterization of lysyl oxidase-like protein-2 (LOXL2) as a hypoxia-target expressed in neovessels and accumulated in the endothelial ECM. IntroductionAngiogenesis occurs during development and tissue remodeling, and in the pathologic context of cardiovascular ischemic diseases or tumor growth. Sprouting of new vessels is initiated by stimulation of endothelial cells (ECs) by a combination of signals from the microenvironment that includes oxygen tension and growth factors. Cells from not yet vascularized tissue and ECs that invade this microenvironment are both hypoxia targets through the Hypoxia Inducible Factor (HIF) pathway. HIF activates transcription of genes coding for autocrine/paracrine factors like vascular endothelial growth factor (VEGF) and extracellular matrix (ECM) components. 1 Synergy between these responses is assumed through local concentration of growth factors in the ECM where they function as attractant for ECs. Specialized ECs, called tip cells, lead vascular growth by sending out filopodia to explore the hypoxic microenvironment. 2 Tip cells are thus continuously exposed to low oxygen concentration. 3 Stalk cells, located behind the tip cells, serve to vessel growth by proliferation, lumen formation and junction establishment. 4 Vascular ECM undergoes major remodeling during angiogenesis, consisting in ECM/basement membrane degradation, provisional ECM generation and assembly of a new basement membrane. ECM-mediated mechanotransductive signaling regulates 3D multicellular organization, including lumen formation and tubulogenesis. Thus, in addition to storing angiogenic factors and providing structural features, ECM is a dynamic promoter of angiogenesis. 5 Subendothelial basement membrane is composed of nonfibrillar collagen IV, laminin, perlecan and nidogens. Other collagens (VIII, XV, and XVIII), fibronectin and matricellular proteins (thrombospondin-1, Cyr61) are associated with the basement membrane. There is only little data concerning the assembly of the vascular microenvironment and its impact on angiogenesis. Genes encoding ECM components and enzymes involved in their assembly and stabilization are targets of hypoxia in ECs. 1 To identify key regulators of the angiogenesis-associated ECM remodeling, we performed a proteomic analysis of endothelial ECM generated in vitro under hypoxic conditions. We found that the ECM cross-linking enzyme, lysyl oxidase-like protein-2 (LOXL2) is a major target of hypoxia. Lysyl oxidases, consisting in lysyl oxidase (LOX) and 4 lysyl oxidase-like proteins (LOXL 1 to 4), catalyze the deamination of lysines and hydroxylysines, generating aldehydes that spontaneously react to form co...

show abstract

Modulation of Lysyl Oxidase-like 2 Enzymatic Activity by an Allosteric Antibody Inhibitor

Cited by 128 publications

References 28 publications

Post-translational Modifications of Recombinant Human Lysyl Oxidase-like 2 (rhLOXL2) Secreted from Drosophila S2 Cells*

Post-translational Modifications of Recombinant Human Lysyl Oxidase-like 2 (rhLOXL2) Secreted from Drosophila S2 Cells*

Control of lung vascular permeability and endotoxin-induced pulmonary oedema by changes in extracellular matrix mechanics

Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane

Contact Info

Product

Resources

About