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

Abstract

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 ...

MCF-7 Cells Expressing Nuclear Associated Lysyl Oxidase-like 2 (LOXL2) Exhibit an Epithelial-to-Mesenchymal Transition (EMT) Phenotype and Are Highly Invasive in Vitro

“…We previously confirmed by mass spectrometry that Lys-653 and Tyr-689 are the precursor residues for the LTQ cofactor in LOXL2 (19). Biogenesis of the LTQ cofactor is proposed to be autocatalytic, requiring only Cu 2ϩ and molecular oxygen (3,29).…”

“…These results confirm that the N-linked glycans at Asn-455 and Asn-644 are independently essential for secretion from breast cancer cells as disruption of N-glycosylation at either site results in the loss of the N-linked glycan at the other site and total inhibition of secretion, a phenomenon for which there is some precedent (23,24). Interestingly, the N455Q and N644Q LOXL2s produced in MCF-7 cells were soluble, in contrast to the corresponding truncated forms of recombinant LOXL2 expressed in S2 cells, which were detected in minute quantities only in the insoluble S2 cell lysate, implicating rapid endoplasmic reticulum-associated degradation of nonglycosylated LOXL2 (19). The stark difference suggests that in MCF-7 cells, the N-linked glycans are essential neither for the folding nor for the stability of LOXL2.…”

“…The primer pair used to generate the N288Q mutant was: forward (5Ј-GGACCCCATGAAGCAAGTCACCTGCGAG-3Ј), reverse (5Ј-CTCGCAGGTGACTTGCTTCATGGGGTCC-3Ј). The sets of primers for generating N455Q and N644Q LOXL2 are described elsewhere (19). The lysine residue at 653 was mutated to either arginine (K653R) or serine (K653S) using the following primer pairs: K653R forward (5Ј-CAGAGGGCCACAGGGCCA-GCTTCTG-3Ј), K653R reverse (5Ј-CAGAAGCTGGCCCTGT-GGCCCTCTG-3Ј); K653S forward (5Ј-CAGAGGGCCACAGT-GCCAGCTTCTGCT-3Ј), K653S reverse (5Ј-AGCAGAAGCTG-GCACTGTGGCCCTCTG-3Ј Immunoblot Analysis-The primary antibodies used in this study were as follows: mouse StrepMAB-Classic (IBA); rabbit anti-LOXL2 and mouse anti-vimentin (Sigma-Aldrich); rabbit antiSnail1, rabbit anti-claudin-1, rabbit anti-GSK3␤, rabbit anti-estrogen receptor ␣, rabbit anti-MMP-2, and rabbit anti-MMP-9 (Cell Signaling Technology); rabbit anti-␤-actin, rabbit anti-fibronectin, rabbit anti-lamin B1, mouse anti-E-cadherin, rabbit anti-occludin, mouse anti-proliferating cell nuclear antigen, and rabbit anti-Snail1 (Abcam); and rabbit anti-MT1-MMP (Santa Cruz Biotechnology).…”

“…1A). Using truncated LOXL2s produced in Drosophila Schneider 2 (S2) cells, we recently demonstrated that secreted LOXL2 is N-glycosylated at least at Asn-455 and Asn-644 and that the N-linked glycans at these sites are independently important for proper protein folding and secretion from S2 cells (19,22). In addition to Asn-455 and Asn-644, NetNGlyc 1.0 server predicts that Asn-288 is also an N-glycosylation site (i.e.…”

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

“…We have worked to define the posttranslational modifications of LOXL2 and their impact on LOXL2 subcellular localization, enzyme activity and biological functions. We recently reported that N-glycosylation, but not LTQ cofactor formation, is essential for LOXL2 secretion (24)(25)(26). Here, we focused on rLOXL2 secreted from HEK293 cells to define the mode of proteolytic processing of LOXL2 and assess the importance of the processing on the LOX amine oxidase activity of LOXL2 in vitro.…”