Extracellular Processing of Lysyl Oxidase-like 2 and Its Effect on Amine Oxidase Activity

Abstract

Overexpression of lysyl oxidase-like 2 (LOXL2) is associated with several hepatic and vascular fibrotic diseases and tumor progression in some aggressive cancers. Secreted LOXL2 promotes extracellular matrix cross-linking by catalyzing the oxidative deamination of peptidyl lysine. A great deal remains to be learned about the post-translational modifications of LOXL2, including whether such modifications modulate enzymatic and disease-promoting activities; such knowledge would inform the development of potential therapies. We discovered that upon secretion in cell culture, LOXL2 undergoes proteolytic processing of the first two of four scavenger receptor cysteine-rich domains at the N-terminus. A similar pattern of processing was also evident in tissue extracts from an invasive ductal carcinoma patient. Processing occurred at ³¹⁴Arg-³¹⁵Phe-³¹⁶Arg-³¹⁷Lys↓-³¹⁸Ala-, implicating proprotein convertases. siRNA-mediated knockdown of proprotein convertases (furin, PACE4, and PC5/6), as well as incubation with their recombinant forms, showed that PACE4 is the major protease that acts on extracellular LOXL2. Unlike LOX, which requires cleavage of its propeptide for catalytic activation, cleavage of LOXL2 was not essential for tropoelastin oxidation or for cross-linking of collagen type IV in vitro. However, in the latter case, processing enhanced the extent of collagen cross-linking ∼2-fold at ≤10 nM LOXL2. These results demonstrate an important difference in the regulatory mechanisms for LOX and LOXL2 catalytic activity. Moreover, they pave the way for further studies of potential differential functions of LOXL2 isoforms in fibrosis and tumor progression.

Figure 1.. A schematic representation of human LOX and LOXL2.

The N-linked glycosylation sites identified in LOXL2 are Asn288, Asn455 and Asn644. The proposed Cu²⁺ binding site for LOXL2 is His626-X-His628-X-His630.

Figure 2.. Secreted LOXL2 undergoes proteolytic processing.

A, Time-dependent processing of WT-LOXL2 detected in the culture media of HEK-WT-LOXL2. B, Time-dependent processing of endogenous LOXL2 secreted from MDA-MB-231 cells C, Detection of the two forms (~100-kDa and ~60-kDa) of LOXL2 in tissue extracts prepared from biopsy samples of breast cancer patients. Western blot analysis of total extracts prepared from a sample of IDC (invasive ductual carcinoma) and two samples of DCIS (ductural carcinoma in situ) tissues dissected from breast cancer patients. β-Actin staining was included as a loading control (left panel). Signal relative intensity was normalized to β-actin (right panel). BG: background intensity (mean ± S.D.) from three independent areas from the same X-ray film. D, N-terminal sequence of 60-kDa LOXL2 revealed that the processing occurs in between the second and the third SRCR domains at ³¹⁷Lys-³¹⁸Ala. Left, N-terminal sequence of the 60-kDa LOXL2 by Edman degradation. Right, Schematic representation of the proteolytic cleavage site of LOXL2 identified in this study.

Figure 3.. Effect of protease inhibitors and site-directed mutagenesis on the processing of LOXL2.

A, Effect of protease inhibitors on the proteolytic processing of LOXL2. B, Time-dependent processing of LOXL2 point mutants in the conditioned media of the corresponding HEK293 stable cells. C, No processing was observed for triple mutants,AAA-LOXL2 and AAQ-LOXL2 in cell culture at day 3. D, Effect of proprotein convertase inhibitors on WT-LOXL2 processing in conditioned media of HEK-WT-LOXL2 (day 2).

Figure 4.. PACE4 catalyzes the proteolytic processing of WT-LOXL2.

A, Furin, PACE4 or PC5/6 were knocked down by specific siRNAs, and the knockdown efficacy was determined by probing whole cell lysates with anti-furin, anti-PACE4 or anti-PC5/6 antibodies, respectively (left panel). The effect of PC-specific knockdown on WT-LOXL2 processing was determined by probing the conditioned media of siRNA-treated HEK-WT-LOXL2 cells with anti-LOXL2 antibody (right panel). B, Furin, PACE4, or PC5/6 were knocked down by specific siRNAs, then the knockdown efficacy was determined (left panel) as described in Fig. 4A. The impact of PC-specific knockdown on K317R-LOXL2 processing was determined in the conditioned media of HEK-K317R-LOXL2 cells (right panel). C, SDS-PAGE analysis of purified rLOXL2s (2 μg) used for in vitro assays in this study. The 100-kDa form of K317R used in this study was further isolated from the mixture of 100-kDa and 60-kDa (C) by size-exclusion chromatography (Fig. S3). D, Cleavage of 100-kDa rLOXL2s by recombinant PCs.

Figure 5.. The -Arg(P4)-X-Arg(P2)-Lys(P1)↓-site is unique to LOXL2.

A, Sequence alignment of the processing site of LOXL2 (underline + arrow) with the corresponding sequences of LOXL3 and LOXL4 (paired basic residues are underlined). B, Detection of recombinant LOXL3, endogenous LOXL4 and R314P-LOXL2 in the day 3 culture media of rLOXL3-transfected HEK293 cells, MDA-MB-231 cells and HEK-R314P-LOXL2, respectively.

Figure 6.. LOXL2 catalyzes the crosslinking of collagen type IV in vitro.

A, Detection of collagen type IV 7S domains in the collagenase-resistant fraction of ECM overlaid with MDA-MB-231, HEK-WT-LOXL2, and HEK-K317R-LOXL2 cells. None: no cells were overlaid (negative control). The ECM overlaid with empty vector-transfected cells (HEK-pcDNA) and HEK-WT-LOXL2 incubated with BAPN (1 mM) also served as negative controls. M: monomer, D: dimer, T: trimer, TT: tetramer, P: pentamer, H: hexamer of collagen type IV 7S domains. B, Crosslinking of collagen type IV by purified WT-LOXL2 (20 nM) was dependent on LOX amine oxidase activity. Neither BAPN (1 mM)-inhibited rLOXL2 nor phenylhydrazine-derivatized rLOXL2 (PH-WT-LOXL2) could catalyze crosslinking of collagen type IV.

Figure 7.. Proteolytic processing of LOXL2 does not significantly impact collagen type IV-crosslinking activity in vitro.

A, Accumulation of crosslinked 7S domains (H, P) was observed in a concentration-dependent manner when ECM was incubated in 20 mM sodium phosphate (pH 8.0) containing purified WT-, AAQ-, or Δ1–2SRCR-LOXL2. B, The purity of rLOXL2 being added to PFHR-9-ECM as determined by Western blot analysis. Top panel: Immediately after addition to ECM. Bottom panel: After 5 days incubation with ECM. C, Quantitation of crosslinked 7S domains (H, P) formed by incubating ECM with AAQ-or Δ1–2SRCR-LOXL2. Error bars indicate ± SEM.