Post-translational modifications of recombinant human lysyl oxidase-like 2 (rhLOXL2) secreted from Drosophila S2 cells

Abstract

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 ∼50% identity and ∼70% 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.

FIGURE 1.

A, schematic diagram of full-length hLOXL2 and two truncated recombinant hLOXL2s used in this study. B, SDS-PAGE (lanes 1 and 2) and Western blot (lanes 3 and 4) analyses of affinity-purified Δ1-4SRCR-hLOXL2 (lanes 1 and 3) and Δ1-3SRCR-hLOXL2 (lanes 2 and 4). C, Western blot analyses of PNGase F-treated purified Δ1-3SRCR-hLOXL2 (lanes 1 and 2) and Δ1-4SRCR-hLOXL2 (lanes 3 and 4). D, DIG-labeled lectin analyses of Δ1-3SRCR-hLOXL2 and Δ1-4SRCR-hLOXL2. Protein samples were fractionated by SDS-PAGE and transferred to PVDF membranes. Each membrane strip was incubated sequentially with a DIG-labeled lectin (peanut agglutinin (PNA), Sambucus nigra agglutinin (SNA), Datura stramonium agglutinin (DSA), G. nivalis agglutinin (GNA), or Maackia amurensis agglutinin (MAA)), alkaline phosphatase-conjugated anti-DIG antibody, and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Lane 1, positive control glycoprotein (0.3 mg) supplied with the kit; lane 2, PNGase F-treated recombinant hLOXL2 (0.1 μg); lane 3, recombinant hLOXL2 (0.1 μg). Arrows indicate detection of high mannose type glycoconjugates. M, protein molecular mass marker.

FIGURE 2.

MS analyses of the PTMs of Δ1-3SRCR-hLOXL2. A, glycopeptide fragment 1 (455–478) detected by mass spectrometry. B, glycopeptide fragment 2 (628–647) detected by mass spectrometry. C, ESI-MS/MS analysis of the peptide fragment containing phenylhydrazine-derivatized LTQ after tryptic digestion. The derivatized LTQ-containing cross-link, shown as an inset as the azo tautomer, is circumscribed by a green oval at the top.

FIGURE 3.

Effect of N-linked glycosylation on the secretion of recombinant hLOXL2s and hLOX from S2 cells. A and B, Western blot analyses of the expression profiles of the glycosylation site mutants of Δ1-3SRCR-hLOXL2 (A)(nonadjacent lanes from the same blot are digitally juxtaposed) and Δ1-4SRCR-hLOXL2 (B) from induced transiently transfected cells. M, cell culture medium; S, soluble cell lysate; I, insoluble cell lysate (cell pellet). C, PCR confirmation of the stable integration of the N455Q and N644Q mutants of Δ1-3SRCR-hLOXL2 into the S2 genome. Stably integrated WT Δ1-3SRCR-hLOXL2 was used as a positive control. D and E, Western blot analyses of the expression profiles of unglycosylated WT Δ1-3SRCR-hLOXL2 (D) and WT Δ1-4SRCR-hLOXL2 (E) from tunicamycin-treated induced stable cells. Panels D and E are γ-adjusted to enhance minimally visible bands. Tunica., tunicamycin. F, Western blot analysis of expression of the LOX catalytic domain of hLOX in stable S2 cells.