Role of Lysyl Oxidase Propeptide in Secretion and Enzyme Activity

Grimsby, Jessica L.; Lucero, Héctor A.; Trackman, Philip C.; Ravid, Katya; Kagan, Herbert M.

doi:10.1002/jcb.22845

Cited by 51 publications

(44 citation statements)

References 47 publications

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“…3F). These results suggest that the propeptide of LOX is essential for proper folding and secretion of hLOX, supporting the recent study wherein the propeptide of hLOX was shown to be essential for secretion in CHO cells (30). In that study, N-linked glycosylation of the propeptide of hLOX was shown to be neither required for secretion nor required for extracellular proteolytic processing of pro-hLOX in CHO cells; however, N-linked glycosylation was required to achieve optimal enzymatic activity of mature hLOX.…”

Section: Resultssupporting

confidence: 88%

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

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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 ...

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Section: Resultssupporting

confidence: 88%

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

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“…As a result, LOX may play an important role in the pathogenesis of IPF, as it is essential for the insolubilization and stabilization of extracellular matrix proteins [17], i.e. the hallmark of IPF.…”

Section: Discussionmentioning

confidence: 99%

“…Lysyl oxidase (LOX), a copper-dependent enzyme, oxidizes specific lysine residues in collagen and elastin leading to the formation of inter- or intramolecular crosslinks essential for stabilization of the extracellular matrix [17]. Strong evidence supports an association of pulmonary fibrosis with elevated LOX activity [18,19].…”

Section: Introductionmentioning

confidence: 99%

N-Acetylcysteine Downregulation of Lysyl Oxidase Activity Alleviating Bleomycin-Induced Pulmonary Fibrosis in Rats

Yang

et al. 2012

Respiration

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Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease without beneficial therapy, except for lung transplantation. A high oral dose of N-acetylcysteine (NAC) added to prednisone and azathioprine has been found to improve lung function in IPF patients, though the mechanism of action remains poorly understood. Objective: Based on our previous findings showing elevation of glutathione (GSH) content associated with downregulation of lysyl oxidase (LOX) activity, which is essential for collagen deposition, the aim of the present study was to test the hypothesis that NAC alleviates IPF by regulating LOX function. Methods: We firstly analyzed the time course of collagen deposition in lung tissue, hydroxyproline content, LOX activity, GSH levels, and transforming growth factor-β₁ (TGF-β₁) and α-smooth muscle actin (α-SMA) expression in bleomycin (BLM)-induced pulmonary fibrosis in a rat model. Then, we focused our studies on NAC modulation of LOX activity. Results: LOX activity was increased on day 9 and peaked 14 days after BLM administration, while TGF-β₁ protein peaked on day 9. Interestingly, NAC treatment for 14 days from day 0 reversed LOX activity to normal levels and increased GSH levels in the lung of BLM-dosed rats. Consistently, NAC partially attenuated pulmonary fibrosis and inhibited TGF-β₁ and α-SMA expression in this model. Conclusions: Our study supports a novel mechanism of NAC alleviating IPF by inhibition of LOX activity via elevation of lung GSH in BLM-induced pulmonary fibrosis. The TGF-β₁/α-SMA pathway may also play an important role in modulation of LOX activity.

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“…LOX is catalytically activated in the ECM when bone morphogenetic protein-1 (BMP-1) cleaves the LOX pro-domain between Gly168 and Asp169 ( Fig. 1A) (16)(17)(18)(19). The BMP-1 cleavage site is not conserved in LOXL2 nor other SRCR domaincontaining LOXLs (i.e.…”

mentioning

confidence: 99%

WITHDRAWN: PACE4 Proteolytically Processes LOXL2 with little impact on its catalytic activity

Okada

Moon

Finney³

et al. 2017

J. Biol. Chem.

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Role of Lysyl Oxidase Propeptide in Secretion and Enzyme Activity

Cited by 51 publications

References 47 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*

N-Acetylcysteine Downregulation of Lysyl Oxidase Activity Alleviating Bleomycin-Induced Pulmonary Fibrosis in Rats

WITHDRAWN: PACE4 Proteolytically Processes LOXL2 with little impact on its catalytic activity

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