Proteolytic processing of lysyl oxidase–like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV

Lysyl oxidase–like-2 (LOXL2) is an enzyme secreted into the extracellular matrix that crosslinks collagens by mediating oxidative deamination of lysine residues. Our previous work demonstrated that this enzyme crosslinks the 7S domain, a structural domain that stabilizes collagen IV scaffolds in the basement membrane. Despite its relevant role in extracellular matrix biosynthesis, little is known about the structural requirements of LOXL2 that enable collagen IV crosslinking. In this study, we demonstrate that LOXL2 is processed extracellularly by serine proteases, generating a 65-kDa form lacking the first two scavenger receptor cysteine-rich domains. Site-specific mutagenesis to prevent proteolytic processing generated a full-length enzyme that is active in vitro toward a soluble substrate, but fails to crosslink insoluble collagen IV within the extracellular matrix. In contrast, the processed form of LOXL2 binds to collagen IV and crosslinks the 7S domain. Together, our data demonstrate that proteolytic processing is an important event that allows LOXL2-mediated crosslinking of basement membrane collagen IV. Lysyl oxidase–like-2 (LOXL2) is an enzyme secreted into the extracellular matrix that crosslinks collagens by mediating oxidative deamination of lysine residues. Our previous work demonstrated that this enzyme crosslinks the 7S domain, a structural domain that stabilizes collagen IV scaffolds in the basement membrane. Despite its relevant role in extracellular matrix biosynthesis, little is known about the structural requirements of LOXL2 that enable collagen IV crosslinking. In this study, we demonstrate that LOXL2 is processed extracellularly by serine proteases, generating a 65-kDa form lacking the first two scavenger receptor cysteine-rich domains. Site-specific mutagenesis to prevent proteolytic processing generated a full-length enzyme that is active in vitro toward a soluble substrate, but fails to crosslink insoluble collagen IV within the extracellular matrix. In contrast, the processed form of LOXL2 binds to collagen IV and crosslinks the 7S domain. Together, our data demonstrate that proteolytic processing is an important event that allows LOXL2-mediated crosslinking of basement membrane collagen IV. Lysyl oxidase–like-2 (LOXL2) is a member of the lysyl oxidase family of copper-dependent amine oxidases (1Kagan H.M. Ryvkin F. Lysyl oxidase and lysyl oxidase-like enzymes.in: Mecham R.P. The Extracellular Matrix: An Overview. Springer-Verlag Berlin Heidelberg, Berlin, Germany2011: 303-335Crossref Google Scholar). Like the rest of the family members, LOXL2 catalyzes the oxidative deamination of peptidyl lysine residues, which promotes the formation of lysyl-derived crosslinks in collagens and elastin (1Kagan H.M. Ryvkin F. 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Targeting LOXL2 for cardiac interstitial fibrosis and heart failure treatment.Nat. Commun. 2016; 7: 13710Crossref PubMed Scopus (134) Google Scholar17Ikenaga N. Peng Z.W. Vaid K.A. Liu S.B. Yoshida S. Sverdlov D.Y. Mikels-Vigdal A. Smith V. Schuppan D. Popov Y.V. Selective targeting of lysyl oxidase-like 2 (LOXL2) suppresses hepatic fibrosis progression and accelerates its reversal.Gut. 2017; 66: 1697-1708Crossref PubMed Scopus (169) Google Scholar, 18Aumiller V. Strobel B. Romeike M. Schuler M. Stierstorfer B.E. Kreuz S. Comparative analysis of lysyl oxidase (like) family members in pulmonary fibrosis.Sci. Rep. 2017; 7: 149Crossref PubMed Scopus (70) Google Scholar, 19Chien J.W. Richards T.J. Gibson K.F. Zhang Y. Lindell K.O. Shao L. Lyman S.K. Adamkewicz J.I. Smith V. Kaminski N. O'Riordan T. Serum lysyl oxidase-like 2 levels and idiopathic pulmonary fibrosis disease progression.Eur. Respir. J. 2014; 43: 1430-1438Crossref PubMed Scopus (101) Google Scholar20Barry-Hamilton V. Spangler R. Marshall D. McCauley S. Rodriguez H.M. Oyasu M. Mikels A. Vaysberg M. Ghermazien H. Wai C. Garcia C.A. Velayo A.C. Jorgensen B. Biermann D. Tsai D. et al.Allosteric inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment.Nat. Med. 2010; 16: 1009-1017Crossref PubMed Scopus (639) Google Scholar). extracellular matrix scavenger receptor cysteine-rich decanoyl chloromethylketone β-aminopropionitrile proprotein convertase lysyl-tyrosyl quinone single guide RNA. The amino acid sequence of human LOXL2 consists of 774 residues organized by an N-terminal signal peptide contiguous to four N-terminal scavenger receptor cysteine-rich (SRCR) domains followed by a highly conserved lysyl oxidase catalytic domain. SRCR domains, commonly found on secreted and cell surface–bound proteins, have been proposed to mediate protein-protein interactions, ligand binding, and more recently deacetylation and deactylimination activity over peptidyl-acetyl lysines (21Ma L. Huang C. Wang X.J. Xin D.E. Wang L.S. Zou Q.C. Zhang Y.S. Tan M.D. Wang Y.M. Zhao T.C. Chatterjee D. Altura R.A. Wang C. Xu Y.S. Yang J.H. et al.Lysyl oxidase 3 is a dual-specificity enzyme involved in STAT3 deacetylation and deacetylimination modulation.Mol. Cell. 2017; 65: 296-309Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 22Resnick D. Pearson A. Krieger M. The SRCR superfamily: A family reminiscent of the Ig superfamily.Trends Biochem. Sci. 1994; 19: 5-8Abstract Full Text PDF PubMed Scopus (309) Google Scholar). Although the predicted molecular mass derived from the amino acid sequence is about 84 kDa, LOXL2 is found as a ∼100-kDa protein in both the intracellular and the extracellular compartments, most likely because of posttranslational modifications such as N-glycosylation (23Xu L. Go E.P. Finney J. Moon H. Lantz M. Rebecchi K. Desaire H. Mure M. Post-translational modifications of recombinant human lysyl oxidase-like 2 (rhLOXL2) secreted from Drosophila S2 cells.J. Biol. Chem. 2013; 288: 5357-5363Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Interestingly, several studies in cultured cells and tissues, including our own, have identified a smaller extracellular 65-kDa form (7Añazco C. López-Jiménez A.J. Rafi M. Vega-Montoto L. Zhang M.Z. Hudson B.G. Vanacore R.M. Lysyl oxidase-like-2 cross-links collagen IV of glomerular basement membrane.J. Biol. Chem. 2016; 291: 25999-26012Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 24Akiri G. Sabo E. Dafni H. Vadasz Z. Kartvelishvily Y. Gan N. Kessler O. Cohen T. Resnick M. Neeman M. Neufeld G. Lysyl oxidase-related protein-1 promotes tumor fibrosis and tumor progression in vivo.Cancer Res. 2003; 63: 1657-1666PubMed Google Scholar, 25Vadasz Z. Kessler O. Akiri G. Gengrinovitch S. Kagan H.M. Baruch Y. Izhak O.B. Neufeld G. Abnormal deposition of collagen around hepatocytes in Wilson's disease is associated with hepatocyte specific expression of lysyl oxidase and lysyl oxidase like protein-2.J. Hepatol. 2005; 43: 499-507Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar26Fong S.F. Dietzsch E. Fong K.S. Hollosi P. Asuncion L. He Q. Parker M.I. Csiszar K. Lysyl oxidase-like 2 expression is increased in colon and esophageal tumors and associated with less differentiated colon tumors.Genes Chromosomes Cancer. 2007; 46: 644-655Crossref PubMed Scopus (94) Google Scholar). The addition of a protease inhibitor mixture in the conditioned media of MCF-7 cells prevented the formation of the 65-kDa form, suggesting that it results from proteolytic processing of the large 100-kDa form (27Hollosi P. Yakushiji J.K. Fong K.S. Csiszar K. Fong S.F. Lysyl oxidase-like 2 promotes migration in noninvasive breast cancer cells but not in normal breast epithelial cells.Int. J. Cancer. 2009; 125: 318-327Crossref PubMed Scopus (63) Google Scholar). This observation is particularly relevant as other members of the LOX family such as LOX and LOXL1 become active only after they are proteolytically processed by bone morphogenetic protein-1 (28Uzel M.I. Scott I.C. Babakhanlou-Chase H. Palamakumbura A.H. Pappano W.N. Hong H.H. Greenspan D.S. Trackman P.C. Multiple bone morphogenetic protein 1-related mammalian metalloproteinases process pro-lysyl oxidase at the correct physiological site and control lysyl oxidase activation in mouse embryo fibroblast cultures.J. Biol. Chem. 2001; 276: 22537-22543Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 29Borel A. Eichenberger D. Farjanel J. Kessler E. Gleyzal C. Hulmes D.J. Sommer P. Font B. Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1.J. Biol. Chem. 2001; 276: 48944-48949Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Although the crosslinking function of LOXL2 has been characterized in many biological systems, it is not known whether proteolytic processing regulates its enzymatic activity (30Moon H.J. Finney J. Ronnebaum T. Mure M. Human lysyl oxidase-like 2.Bioorg. Chem. 2014; 57: 231-241Crossref PubMed Scopus (89) Google Scholar). In this study, we investigated the role of LOXL2 processing on the crosslinking of the 7S domain of collagen IV, a key structural component of basement membranes. We identified the cleavage site in LOXL2 sequence by classical Edman microsequencing and site-directed mutagenesis. Furthermore, using CRISPR-Cas9 system, we developed a LOXL2-null cell line that allowed us to explore the effect of proteolytic processing on LOXL2 enzymatic activity. Our results suggest that LOXL2 proteolytic processing is a regulatory step on collagen IV and basement membranes biosynthesis. To purify sufficient 65-kDa extracellular form of LOXL2 for the identification of the site of proteolytic cleavage by Edman microsequencing, we stably expressed the LOXL2 human ORF (NM_002318) in HEK293 cells (7Añazco C. López-Jiménez A.J. Rafi M. Vega-Montoto L. Zhang M.Z. Hudson B.G. Vanacore R.M. Lysyl oxidase-like-2 cross-links collagen IV of glomerular basement membrane.J. Biol. Chem. 2016; 291: 25999-26012Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Immunoblotting characterization of HEK293 cells expressing this construct using a polyclonal antibody raised against the C-terminal region of the LOXL2 sequence showed that the intracellular fraction produced the full-length form of LOXL2 (∼100 kDa) without any sign of proteolytic cleavage (Fig. 1A). In contrast, the conditioned media of these cells showed the presence of both full-length and processed forms of LOXL2, similar to those observed in other cell types (24Akiri G. Sabo E. Dafni H. Vadasz Z. Kartvelishvily Y. Gan N. Kessler O. Cohen T. Resnick M. Neeman M. Neufeld G. Lysyl oxidase-related protein-1 promotes tumor fibrosis and tumor progression in vivo.Cancer Res. 2003; 63: 1657-1666PubMed Google Scholar, 25Vadasz Z. Kessler O. Akiri G. Gengrinovitch S. Kagan H.M. Baruch Y. Izhak O.B. Neufeld G. Abnormal deposition of collagen around hepatocytes in Wilson's disease is associated with hepatocyte specific expression of lysyl oxidase and lysyl oxidase like protein-2.J. Hepatol. 2005; 43: 499-507Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar26Fong S.F. Dietzsch E. Fong K.S. Hollosi P. Asuncion L. He Q. Parker M.I. Csiszar K. Lysyl oxidase-like 2 expression is increased in colon and esophageal tumors and associated with less differentiated colon tumors.Genes Chromosomes Cancer. 2007; 46: 644-655Crossref PubMed Scopus (94) Google Scholar), and glomeruli (7Añazco C. López-Jiménez A.J. Rafi M. Vega-Montoto L. Zhang M.Z. Hudson B.G. Vanacore R.M. Lysyl oxidase-like-2 cross-links collagen IV of glomerular basement membrane.J. Biol. Chem. 2016; 291: 25999-26012Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We purified LOXL2 forms from cell media by affinity chromatography and fractionated proteins by SDS-PAGE. The 65-kDa processed form was cut out of the gel and prepared for N-terminal Edman microsequencing. The results revealed the first 11 amino acid residues of processed form N-terminal end starting at Ala-318 of human LOXL2 sequence, indicating that proteolytic processing occurred at Lys-317. This site resides in the linker region between the SRCR2 and SRCR3 domains (Fig. 1B). Further inspection of the sequence showed that the P1 and P2 positions from the Lys-317 processing site are occupied by basic residues that are highly conserved in LOXL2 sequences of vertebrates (Fig. 1C). The presence of these basic residues in P1 and P2 suggests that a serine protease cleaves LOXL2 at Lys-317. To test this hypothesis, HEK293 cells stably expressing human LOXL2 were grown in media containing serine protease inhibitors including leupeptin, aprotinin, and PMSF (Fig. 1D). Immunoblotting analyses of the culture media from these cells showed that the formation of the 65-kDa LOXL2 processed form was significantly reduced by aprotinin and PMSF, but not leupeptin, suggesting that serine proteases may be responsible for LOXL2 cleavage. HEK293 cells were also cultured in the presence of decanoyl-RVKR-chloromethylketone (Dec-RVKR-CMK), an inhibitor that blocks the activity of members of the subtilisin-like family of proprotein convertases such as furin (31Denault J.B. D'Orleans-Juste P. Masaki T. Leduc R. Inhibition of convertase-related processing of proendothelin-1.J. Cardiovasc. Pharmacol. 1995; 26: S47-S50Crossref PubMed Scopus (23) Google Scholar). As shown in Fig. 1D, Dec-RVKR-CMK exhibited the highest inhibition of LOXL2 processing. Pepstatin, a potent inhibitor of aspartyl proteases, had no effect on LOXL2 cleavage. In summary, these data suggest that LOXL2 is proteolytically processed by a serine protease at Lys-317 in the extracellular compartment, removing the first two SRCR domains from LOXL2 sequence. To evaluate the effect of proteolytic processing on amine oxidase activity, we performed site-directed mutagenesis to generate either full-length or processed forms of LOXL2. To block proteolytic processing and generate a full-length LOXL2 form, the two basic residues preceding the cleavage site were mutated from RK to GE. The introduction of negatively charged glutamic acid at P1 was expected to block proteolytic processing by serine proteases (32Remacle A.G. Shiryaev S.A. Oh E.S. Cieplak P. Srinivasan A. Wei G. Liddington R.C. Ratnikov B.I. Parent A. Desjardins R. Day R. Smith J.W. Lebl M. Strongin A.Y. Substrate cleavage analysis of furin and related proprotein convertases. A comparative study.J. Biol. Chem. 2008; 283: 20897-20906Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 33Duckert P. Brunak S. Blom N. Prediction of proprotein convertase cleavage sites.Protein Eng. Des. Sel. 2004; 17: 107-112Crossref PubMed Scopus (406) Google Scholar34Barré O. Dufour A. Eckhard U. Kappelhoff R. Béliveau F. Leduc R. Overall C.M. Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs) using PICS with proteome-derived peptide libraries.PLoS One. 2014; 9: e105984Crossref PubMed Scopus (43) Google Scholar). Transient expression of this construct in HEK293 cells predominantly generated the full-length form of LOXL2, indicating that proteolytic processing was abrogated (Fig. 2A). Initial studies in which only Lys-317 was mutated uncovered an anticipated atypical form of LOXL2 of about 70 kDa that is not observed in cells expressing wild-type LOXL2. A careful inspection of LOXL2 sequence identified Arg-256 as a potential cleavage site of serine proteases. Thus, to completely eliminate proteolytic processing and generate the full-length 100-kDa form, Arg-257 was mutated to glycine in addition to 316–317 residues RK (Fig. 2A). LOXL2BP was used to describe this “blocked processing LOXL2 mutant” construct. As most serine proteases have a preference for Arg at P1 (33Duckert P. Brunak S. Blom N. Prediction of proprotein convertase cleavage sites.Protein Eng. Des. Sel. 2004; 17: 107-112Crossref PubMed Scopus (406) Google Scholar, 34Barré O. Dufour A. Eckhard U. Kappelhoff R. Béliveau F. Leduc R. Overall C.M. Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs) using PICS with proteome-derived peptide libraries.PLoS One. 2014; 9: e105984Crossref PubMed Scopus (43) Google Scholar), we generated a second mutant by changing Lys-317 to Arg-317 to potentially enhance proteolytic cleavage. The resulting K317R processing LOXL2 mutant was named LOXL2K317R. To evaluate the effect of mutagenesis on proteolytic processing, these two new LOXL2BP and LOXL2K317R constructs, together with the catalytically inactive LOXL2Y689F mutant and wild-type LOXL2 as controls, were expressed in HEK293 cells. Immunoblot analyses for LOXL2 detection in whole cell extracts showed that all constructs produced LOXL2 proteins of 100 kDa. However, LOXL2 processing differences became evident when analyzing culture media containing the extracellular forms of LOXL2. As expected, the LOXLBP construct produced only the 100-kDa form. In contrast to the wild-type construct, the LOXLK317R construct showed a significant increase of the 65-kDa processed LOXL2 form (Fig. 2B). To assess the effect of proteolytic processing on the amine oxidase activity, conditioned media of cells expressing LOXL2 wild type and mutants were assayed with Amplex Red assay using 1,5-diaminopentane as substrate (35Palamakumbura A.H. Trackman P.C. A fluorometric assay for detection of lysyl oxidase enzyme activity in biological samples.Anal. Biochem. 2002; 300: 245-251Crossref PubMed Scopus (186) Google Scholar). As shown in Fig. 2C, the amine oxidase activity showed no significant difference between processing mutants and wild-type LOXL2. These results show that cleavage at Lys-317 does not influence LOXL2 amine oxidase activity. To evaluate the role of LOXL2 proteolytic processing on collagen IV crosslinking, we generated a LOXL2-null PFRH9 cell line by using CRISPR-Cas9 gene editing to target exon 2 of the mouse Loxl2 gene (Fig. 3A). Immunoblot analysis of the ECM-derived fraction, where both 100-kDa and 65-kDa forms are normally deposited in the wild-type cells, showed that both LOXL2 forms are absent in the knock-out cell line (Fig. 3B). In addition, no residual amine oxidase activity inhibited by BAPN was detected in LOXL2-null cell conditioned medium (Fig. 3C). Fig. 3D shows that 7S dodecamers extracted after collagenase digestion of the ECM derived from LOXL2-null PFHR9 cells are composed of only 7S monomeric subunits, confirming our previous crosslinking studies using pharmacological inhibitors of lysyl oxidase activity (7Añazco C. López-Jiménez A.J. Rafi M. Vega-Montoto L. Zhang M.Z. Hudson B.G. Vanacore R.M. Lysyl oxidase-like-2 cross-links collagen IV of glomerular basement membrane.J. Biol. Chem. 2016; 291: 25999-26012Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Interestingly, the nonreduced 7S dodecamer of the LOXL2-null cells showed slightly faster electrophoretic mobility in comparison to the wild-type cells (Fig. 3E). These results show that LOXL2 is the sole source of lysyl oxidase activity for crosslinking the 7S dodecamer of collagen IV in PFHR9 cells. Furthermore, generation of a LOXL2-null PFHR9 cell line provides an excellent system to test the effect of LOXL2 proteolytic processing on collagen IV crosslinking. To evaluate the 7S crosslinking activity of LOXL2 processing mutants, these constructs were expressed in the LOXL2-null PFHR9 cells. As expected, monomeric 7S subunits are predominant in the catalytically defective LOXL2Y689F mutant, indicating that the enzyme failed to reestablish the crosslinking profile observed in wild-type cells (Fig. 4). In contrast, the 7S dodecamer from wild-type LOXL2 and the enhanced processing mutant LOXL2K317R exhibited the typical crosslinking profile of oligomeric forms observed in wild-type cells. Surprisingly, the full-length, processing defective mutant LOXL2BP failed to crosslink the 7S dodecamer. These results demonstrate that, despite being catalytically active, the full-length processing mutant LOXL2BP was unable to crosslink the 7S dodecamer. These results suggest that removal of the first two SRCR domains by proteolytic processing is required for effective collagen IV crosslinking in the insoluble matrix. Although full-length and processed forms of LOXL2 have been reported to localize in the ECM of different tissues (7Añazco C. López-Jiménez A.J. Rafi M. Vega-Montoto L. Zhang M.Z. Hudson B.G. Vanacore R.M. Lysyl oxidase-like-2 cross-links collagen IV of glomerular basement membrane.J. Biol. Chem. 2016; 291: 25999-26012Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 15Bignon M. Pichol-Thievend C. Hardouin J. Malbouyres M. Bréchot N. Nasciutti L. Barret A. Teillon J. Guillon E. Etienne E. Caron M. Joubert-Caron R. Monnot C. Ruggiero F. Muller L. Germain S. Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane.Blood. 2011; 118: 3979-3989Crossref PubMed Scopus (141) Google Scholar), our results showed that only the processed LOXL2 crosslinks the 7S dodecamer. Thus, we hypothesized that the processed, but not the full-length, form of LOXL2 interacts with collagen IV. To test this hypothesis, we performed a collagenase digestion to solubilize proteins interacting with the insoluble collagen IV scaffold. As we wanted to detect a potential LOXL2–7S dodecamer interaction, we first performed immunoblotting analyses of purified LOXL2 to characterize its electrophoretic mobility under reducing and nonreducing conditions. As shown in Fig. 5A, under nonreducing conditions the full-length and processed LOXL2 forms migrated with an apparent molecular mass of ∼75 and ∼45 kDa, respectively. The faster electrophoretic mobility of both LOXL2 forms is consistent with the presence of many disulfide bridges that restrain the polypeptide chain yielding a smaller apparent molecular mass in SDS-PAGE. Immunoblotting analyses of collagenase-soluble proteins from wild-type PFHR9 matrix show a 45-kDa band equivalent to that of processed LOXL2 (Fig. 5B, right panel). Surprisingly, the immunoblot also showed an unexpected high molecular mass form of LOXL2 with an electrophoretic mobility slightly slower than the 7S dodecamer shown in the SDS-PAGE (Fig. 5B). None of these two LOXL2 forms were observed in the collagenase digest of LOXL2-null PFHR9 matrix (Fig. 5B, right panel). Thus, the presence of the high molecular mass LOXL2 form suggests a stable interaction between processed LOXL2 and the 7S dodecamer that is resistant to the highly dissociative conditions of SDS-PAGE analysis. To further characterize the interaction between 7S dodecamer and processed LOXL2, we performed immunoblotting analyses of purified 7S dodecamer, urea-soluble, and collagenase-soluble PHFR9 matrix proteins under reducing conditions. As shown in Fig. 5C, some bands are similar, but there are striking differences in the number and size of LOXL2 forms. Consistent with a LOXL2–7S dodecamer interaction, the unbound 65-kDa processed LOXL2 form was not detected in purified 7S dodecamer (Fig. 5C, left panel), as it is likely removed during chromatographic purification. However, a ladder of three or more additional bands, larger than the 65-kDa processed LOXL2, was observed in purified 7S dodecamer samples and collagenase digests of PFHR9 and glomerular matrices (Fig. 5C). Of note, the bands detected in the collagenase digest of bovine glomerular matrix showed a more complex pattern, which is probably a consequence of the highly heterogeneous collagenous material isolated from this tissue. The striking differences between the electrophoretic profiles of LOXL2 forms present in urea-soluble and collagenase-soluble protein samples suggest that the enzyme interacts with collagen IV in different ways (Fig. 5C, right panel). For instance, the 100-kDa full-length LOXL2 band is detected in urea-extracted proteins, but not in collagenase digests, suggesting that it does not interact with collagen. In contrast, the 65-kDa processed LOXL2 (free form, equivalent to the 45-kDa band observed under nonreducing conditions) is present in both preparations, suggesting that this form associates with collagen IV in a noncovalent fashion. Lastly, the ladder of LOXL2 bands is only present in the collagenase digest, suggesting that processed LOXL2 forms a covalent complex with peptide fragments derived from 7S dodecamer that were not digested by collagenase under native conditions. Taken together, these results suggest that processed, but not full-length, LOXL2 is bound both covalently and noncovalently to collagen IV. To validate that LOXL2 is bound to 7S polypeptides, we subjected collagenase-soluble PFHR9 proteins to pepsin digestion under nondenaturing conditions. As expected, the 7S dodecamer was resistant to pepsin digestion (Fig. 5D, left panel). However, LOXL2 was completely degraded under the same conditions (Fig. 5D, right panel), indicating that the LOXL2 signal detected by the antibody is specific despite the presence of excessively abundant 7S oligomeric subunits. In contrast, because it is possible that processed LOXL2 may have protected the attached 7S peptide fragments from collagenase digestion under native conditions, collagenase