Cellular Fibronectin Binds to Lysyl Oxidase with High Affinity and Is Critical for Its Proteolytic Activation

Abstract

Lysyl oxidase (LOX) is a copper-containing amine oxidase known to catalyze the covalent cross-linking of fibrillar collagens and elastin at peptidyl lysine residues. In addition, its involvement in cancer, wound healing, cell motility, chemotaxis, and differentiation reflect a remarkable functional diversity of LOX. To investigate novel mechanisms of LOX regulation and function, we performed a yeast two-hybrid screen to identify LOX-interacting proteins. Three overlapping positive clones were identified as C-terminal fragments of fibronectin (FN). Glutathione S-transferase pull-downs and solid phase binding assays confirmed this interaction. LOX binds to the cellular form of FN (cFN) with a dissociation constant (Kd) of 2.5 nm. This was comparable with our measured Kd of LOX binding to tropoelastin (1.9 nm) and type I collagen (5.2 nm), but LOX demonstrated a much lower binding affinity for the plasma form of FN (pFN). Immunofluorescent microscopy revealed co-localization of FN and LOX in normal human tissues, where these proteins may interact in vivo. LOX enzymatic activity assays showed that cFN does not seem to be a substrate of LOX. However, cFN can act as a scaffold for enzymatically active 30-kDa LOX. Furthermore, in FN-null mouse embryonic fibroblasts, we observed dramatically decreased proteolytic processing of the 45-kDa LOX proenzyme to the 30-kDa active form, with a corresponding decrease in LOX enzyme activity. Our results suggest that the FN matrix may provide specific microenvironments to regulate LOX catalytic activity. Lysyl oxidase (LOX) is a copper-containing amine oxidase known to catalyze the covalent cross-linking of fibrillar collagens and elastin at peptidyl lysine residues. In addition, its involvement in cancer, wound healing, cell motility, chemotaxis, and differentiation reflect a remarkable functional diversity of LOX. To investigate novel mechanisms of LOX regulation and function, we performed a yeast two-hybrid screen to identify LOX-interacting proteins. Three overlapping positive clones were identified as C-terminal fragments of fibronectin (FN). Glutathione S-transferase pull-downs and solid phase binding assays confirmed this interaction. LOX binds to the cellular form of FN (cFN) with a dissociation constant (Kd) of 2.5 nm. This was comparable with our measured Kd of LOX binding to tropoelastin (1.9 nm) and type I collagen (5.2 nm), but LOX demonstrated a much lower binding affinity for the plasma form of FN (pFN). Immunofluorescent microscopy revealed co-localization of FN and LOX in normal human tissues, where these proteins may interact in vivo. LOX enzymatic activity assays showed that cFN does not seem to be a substrate of LOX. However, cFN can act as a scaffold for enzymatically active 30-kDa LOX. Furthermore, in FN-null mouse embryonic fibroblasts, we observed dramatically decreased proteolytic processing of the 45-kDa LOX proenzyme to the 30-kDa active form, with a corresponding decrease in LOX enzyme activity. Our results suggest that the FN matrix may provide specific microenvironments to regulate LOX catalytic activity. Lysyl oxidase (LOX), 1The abbreviations used are: LOX, lysyl oxidase; bLOX, bovine LOX; FN, fibronectin; cFN, cellular FN; pFN, plasma FN; ECM, extracellular matrix; BMP-1, bone morphogenic protein-1; DMEM, Dulbecco's modified Eagle's medium; MEF, mouse embryonic fibroblast; CCM, conditioned cell medium; GST, glutathione S-transferase; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PBST, PBS with Tween 20; BSA, bovine serum albumin; CRL, cytokine receptor-like; BD, binding domain; AD, activation domain; BAPN, β-aminopropionitrile. a copper-dependent amine oxidase, catalyzes the oxidative deamination of peptidyl lysine residues in collagen and elastin molecules to δ-aminoadipic-β-semialdehyde or allysine, which can then spontaneously condense with neighboring amino groups or other peptidyl aldehydes to form covalent cross-links in fibrillar collagens and elastin (1Csiszar K. Prog. Nucleic Acid Res. Mol. Biol. 2001; 70: 1-32Crossref PubMed Google Scholar, 2Smith-Mungo L.I. Kagan H.M. Matrix Biol. 1998; 16: 387-398Crossref PubMed Scopus (459) Google Scholar). The covalent cross-linking of these extracellular matrix (ECM) proteins by LOX is essential to the formation of insoluble collagen and elastic fibers and for normal mammalian development (3Maki J.M. Rasanen J. Tikkanen H. Sormunen R. Makikallio K. Kivirikko K.I. Soininen R. Circulation. 2002; 106: 2503-2509Crossref PubMed Scopus (410) Google Scholar, 4Hornstra I.K. Birge S. Starcher B. Bailey A.J. Mecham R.P. Shapiro S.D. J. Biol. Chem. 2002; 278: 14387-14393Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). In many human pathologies, including cardiovascular disease, fibrosis, and cancer, LOX expression and activity have been demonstrated to be altered as compared with normal conditions (1Csiszar K. Prog. Nucleic Acid Res. Mol. Biol. 2001; 70: 1-32Crossref PubMed Google Scholar, 2Smith-Mungo L.I. Kagan H.M. Matrix Biol. 1998; 16: 387-398Crossref PubMed Scopus (459) Google Scholar). The human LOX protein is synthesized as a prepro-enzyme of 417 amino acids with an N-terminal signal peptide sequence for secretion. During protein trafficking in the Golgi, a copper atom is incorporated at the copper-binding site (residues 286–296) (5Gacheru S.N. Trackman P.C. Shah M.A. O'Gara C.Y. Spacciapoli P. Greenaway F.T. Kagan H.M. J. Biol. Chem. 1990; 265: 19022-19027Abstract Full Text PDF PubMed Google Scholar, 6Krebs C.J. Krawetz S.A. Biochim. Biophys. Acta. 1993; 1202: 7-12Crossref PubMed Scopus (33) Google Scholar, 7Kosonen T. Uriu-Hare J.Y. Clegg M.S. Keen C.L. Rucker R.B. Biochem. J. 1997; 327: 283-289Crossref PubMed Scopus (38) Google Scholar), which allows the formation of a lysyl-tyrosylquinone cofactor derived from Tyr-355 and Lys-320 residues (8Wang S.X. Mure M. Medzihradszky K.F. Burlingame A.L. Brown D.E. Dooley D.M. Smith A.J. Kagan H.M. Klinman J.P. Science. 1996; 273: 1078-1084Crossref PubMed Scopus (308) Google Scholar). After secretion into the extracellular space, proteolytic cleavage between residues Gly-168 and Asp-169 removes the N-terminal propeptide, yielding an active enzyme of 30 kDa (9Uzel M.I. Shih S.D. Gross H. Kessler E. Gerstenfeld L.C. Trackman P.C. J. Bone Miner. Res. 2000; 15: 1189-1197Crossref PubMed Scopus (28) Google Scholar, 10Cronshaw A.D. Fothergill-Gilmore L.A. Hulmes D.J. Biochem. J. 1995; 306: 279-284Crossref PubMed Scopus (77) Google Scholar, 11Panchenko M.V. Stetler-Stevenson W.G. Trubetskoy O.V. Gacheru S.N. Kagan H.M. J. Biol. Chem. 1996; 271: 7113-7119Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). This processing is mainly accomplished by the procollagen C-proteinase bone morphogenic protein-1 (BMP-1), and to a lesser degree, by mammalian Tolloid-like 1 protein (12Uzel M.I. Scott I.C. Babakhanlou-Chase H. Palamakumbura A.H. Pappano W.N. Hong H.H. Greenspan D.S. Trackman P.C. J. Biol. Chem. 2001; 276: 22537-22543Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 13Kessler E. Takahara K. Biniaminov L. Brusel M. Greenspan D.S. Science. 1996; 271: 360-362Crossref PubMed Scopus (458) Google Scholar). However, it is not known where and how the proteolytic activation and amine oxidase activity of LOX are regulated in physiological and pathological conditions in the ECM. Fibronectin (FN) has been well characterized as an extracellular matrix glycoprotein that can regulate many cellular functions such as proliferation, differentiation, migration, adhesion, and apoptosis (14Pankov R. Yamada K.M. J. Cell Sci. 2002; 115: 3861-3863Crossref PubMed Scopus (1488) Google Scholar, 15Magnusson M.K. Mosher Biol. 1998; PubMed Scopus Google Scholar, J. Cell Sci. PubMed Scopus Google Scholar, Scholar, Google Scholar). FN this of functions with a of proteins including cell and FN is as a by C-terminal and it has been as plasma FN or insoluble cellular FN (cFN) (14Pankov R. Yamada K.M. J. Cell Sci. 2002; 115: 3861-3863Crossref PubMed Scopus (1488) Google Scholar, Cell Res. 1995; PubMed Scopus (172) Google Scholar, H. 1996; PubMed Scopus Google Scholar). has been that activation of FN a in an insoluble that is revealed binding H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. 1996; PubMed Scopus (38) Google Scholar, Mosher J. Biol. Chem. Full Text PDF PubMed Google Scholar, M.S. S.A. Biochem. J. 1990; PubMed Scopus Google Scholar, M. M. R. J. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In tissues, FN is and into a which has been observed to be with collagen of wound have revealed a sequence of FN by cell then type collagen by type I collagen and of FN Scholar, Google Scholar). the formation of the FN matrix has been to be for the of I and collagen T. J. K. Mosher S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Mol. Biol. 2002; PubMed Scopus Google Scholar). has been demonstrated that cFN is for collagen in wound H. S. K. Cell Res. PubMed Scopus Google Scholar, L. S.D. Sci. 2000; Google Scholar, T. K. S. T. Sci. PubMed Scopus Google Scholar). Lysyl oxidase enzyme activity has been to be a in collagen M. J. Full Text PDF PubMed Scopus Google Scholar, Res. PubMed Scopus Google Scholar). The FN matrix was to act as a scaffold to regulate the and of type I and but the mechanisms by which the FN matrix may this have not been In this we the of FN as a binding for LOX by a yeast two-hybrid screen the 30-kDa LOX protein as that LOX has a binding affinity for cFN, but not pFN, and that the cytokine of LOX to a in this that FN does not seem to be a substrate of LOX but that LOX its amine oxidase activity. Immunofluorescent microscopy revealed LOX and FN co-localization in and human In FN-null mouse embryonic we dramatically decreased proteolytic processing of LOX with a corresponding decrease in catalytic activity. was from which of FN, was from S-transferase was from was characterized and the and the LOX from human and mouse T. K. J. M. K. PubMed Scopus Google Scholar). which the was from used as an for was from for which to to the and to to the and the were from active bovine LOX was by Kagan H.M. A.L. Biochem. J. PubMed Scopus Google Scholar). pFN, cFN, bovine serum and tropoelastin proteins were from collagen type I was from were to and to sequence LOX to the into and with the The used to the LOX and with the and To the form of LOX acids a was that at the known cleavage site A.D. Fothergill-Gilmore L.A. Hulmes D.J. Biochem. J. 1995; 306: 279-284Crossref PubMed Scopus (77) Google with the site After the were and into the The and were by was to a human which clones from human with The was into yeast that with an measured by of proteins were by a and and by activation of the The positive clones were these to clones and to The were from the positive yeast and the were an The of the identified FN clones were characterized by assays in or was with or The for positive was with the conditions as the two-hybrid Cell were from and Dulbecco's modified Eagle's with fetal bovine serum and embryonic from FN-null and were by Mosher S. Mosher S.D. Sci. S. PubMed Scopus Google Scholar). were in the conditions as human as was used to the The of FN from the was accomplished by of with of and then to the human and mouse of were used for protein cell were to with PBS, and with After an in the conditioned cell was To proteins from the for we of to the of CCM, and then the was and for 30 at J. T. Biochem. 1997; PubMed Scopus Google Scholar). After the was and we to the it for and then the and was used to expression for LOX fragments an N-terminal The was used as a for the LOX corresponding to amino acids LOX the and were and into the of protein expression was by to and for an at proteins were from a a and with a into proteins were and and then the and were measured of or was to and with pFN, of cFN, or 1 of human for 1 at The was with Tween and then in 30 of and for and were by FN was by an the binding affinity of LOX to FN as compared with and solid phase binding assays were performed P. M. Matrix Scholar). of were with proteins at in PBS or at were with in PBS for at After the were with in protein was to the at nm) in PBS or and at The were then with PBST, and was with or and then with activity was the substrate with measured at nm. were performed in The dissociation were performed by Immunofluorescent human were as and then with PBS and with for at After the were with in PBS, and with in PBS for 30 at The were with in in PBS for 1 at and then in PBS with for The were with in in PBS for 1 at and then the were in PBS with for After a in PBS for the were Immunofluorescent human was performed human and The were by at for 30 and in for were then in and by in was performed by the in and then for The were in PBS for and then with normal serum at for 30 The were with for at in PBST, and then with for at After in PBS, was used to were with a LOX LOX enzyme activity was measured the A.H. Trackman P.C. Biochem. 2002; PubMed Scopus Google Scholar), which we have for in The of and The protein were to the in the or of BAPN, which was then at The was at and the was at a To LOX activity is by binding to FN, of was with of cFN or at for 1 To LOX amine oxidase activity to a solid phase FN we performed an to the solid phase binding with cFN, and with BSA, we in the at a of at the with and with PBS and then of the were at for and the was measured as To LOX activity in an of proteins from and were to well and with the as a were with as a The were at and the was measured The LOX activity was as the in the were used to decrease LOX activity assays performed from the of proteins LOX with investigate novel mechanisms of LOX regulation and function, we performed a yeast two-hybrid screen to identify LOX-interacting proteins. The expression of the lysyl oxidase is in tissues, and we that a be for proteins with was the N-terminal of LOX acids protein as it has been to interact with the active site of LOX to enzyme activity H.M. J. Biochem. PubMed Scopus Google Scholar). the LOX protein in the has been to be the active 30-kDa a to the 30-kDa form of LOX acids was used to screen a human yeast two-hybrid After of into yeast expression of the protein was confirmed by not for expression of the and showed activity by the was into the yeast and the were to screen for expression of the the was that were and the was this yeast two-hybrid of the positive clones we were overlapping FN the was the FN fragments were the C-terminal of the The FN amino acids which a a cell the type and the FN of the which is in cellular of To that the binding of FN was specific to LOX and to FN to the form of LOX of and were into yeast and were with of LOX and FN proteins with as was used as a to of the FN interaction. the yeast that LOX or and FN were to the demonstrated that the between LOX and FN was not the of binding and that the does not the of LOX with the FN clones from the in and to the of LOX and FN, of LOX LOX has been in M. Hulmes D.J. 1996; PubMed Scopus Google Scholar, M.S. J.Y. PubMed Scopus Google Scholar), we LOX into the to LOX as a protein in E. The N-terminal for of of the LOX protein the affinity binding to were to and of from the a modified of protein that of M.S. J.Y. PubMed Scopus Google Scholar). the as a but to the at the and it was not used for of LOX we other fragments that the of the LOX and where the to the amino the proteins and were to in the as for To the protein observed in yeast was used for of or were to glutathione and with FN and FN was by FN as we were to plasma and cellular of FN with but not with In addition, from human as we were to FN with but not with To the binding of LOX to FN and to an constant (Kd) for this we performed solid phase binding assays to the binding affinity of LOX to FN type I collagen and In the were with pFN, cFN, type I or as a in a of was with the proteins at After the of was measured in The protein to cFN, type I and tropoelastin with as Kd was at and binding to was The binding of to was lower that to cFN in these assays but that of the that may be a with the plasma form of To the binding of cFN to solid phase and LOX we with or cFN, in a of was with the proteins as observed that cFN to solid phase with a Kd of with the affinity in the solid phase binding cFN binding to the at a lower affinity the 30-kDa form of LOX, which the cFN demonstrated binding to and the in binding to was FN and LOX in LOX and FN in was performed human and human and by were with FN and LOX with with the for the LOX to the of the LOX In the of human fibroblasts, LOX was in a as compared with the matrix of FN in normal human demonstrated many of the of an in between FN and LOX. The of LOX, observed in fibroblasts, was with that in microenvironments of In tissues, FN and LOX the of the LOX was to the and the the and In the LOX with FN the the and the have and showed LOX and FN co-localization cell In where is for function, FN and LOX were in the of and and In the and showed LOX that with the FN the the of the FN was observed in the between the cFN a for LOX, but LOX cFN to LOX with the affinity as LOX tropoelastin and type I we LOX lysyl residues of cFN in was with of cFN, type I or and the of the oxidative was measured and A.H. Trackman P.C. Biochem. 2002; PubMed Scopus Google Scholar). a was to to activity. The LOX activity was as the in The results that cFN was not at as compared with type I or the of which was and type I the showed that is by LOX tropoelastin or type I in in LOX activity was used it was to a of substrate in the for of LOX activity. To binding to cFN the amine oxidase activity of LOX, we performed an activity with that been with an of decrease in LOX activity was as compared with and with In addition, we used a of solid phase binding and amine oxidase activity to LOX to solid phase cFN in an active we or of with cFN as the proteins to we the protein and performed an amine oxidase activity in the by the and with in The results that LOX, to cFN, enzymatically active in LOX in FN-null to investigate the of the the LOX protein and its proteolytic activation in we LOX by embryonic derived from FN-null and S. Mosher S.D. Sci. S. PubMed Scopus Google Scholar). were in with many LOX dramatically the were LOX protein was performed with to and then with for cell was from the and to the LOX of protein from these a showed dramatically decreased of the 30-kDa LOX in the of In addition, of was observed in the as compared with the and the of LOX in the the of 30-kDa LOX was decreased by in the as compared with the However, the of LOX and was in and was in the cell protein which of in cell not of the demonstrated that was decrease in in the To the in LOX proteolytic processing observed by to in enzyme the LOX activity was measured from of proteins from and was as the in the of the The results showed that LOX amine oxidase activity was decreased by in the cell as compared with the cell with The decrease in 30-kDa LOX protein and corresponding decrease in LOX activity in FN-null conditions were in The results of this that FN binds the LOX protein and provide this as a novel by which FN may regulate the proteolytic activation of LOX and to the of the collagen of FN and collagen have been Scholar, Google Scholar), and have that the of I and collagen into fibers is the of FN T. J. K. Mosher S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Mol. Biol. 2002; PubMed Scopus Google Scholar). FN has been to to collagen molecules S. M. Biochem. J. PubMed Scopus Google Scholar, A.J. J. Cell Biol. Google Scholar, E. E. J. PubMed Scopus Google Scholar), but this binding does not the of FN in the regulation of the collagen matrix LOX the covalent cross-linking of fibrillar collagen which is the enzymatic in the formation of collagen the that FN can LOX and the activation of LOX an how FN can the formation of collagen the solid phase binding we demonstrated that the binding affinity of LOX for cFN was much its affinity for the of plasma cellular FN may have been is that in a with binding In cFN is in an (14Pankov R. Yamada K.M. J. Cell Sci. 2002; 115: 3861-3863Crossref PubMed Scopus (1488) Google Scholar, J. Cell Sci. PubMed Scopus Google Scholar). The affinity of LOX for cFN may be to this a revealed The other between and cFN, which from the is is that the which is in cFN molecules (14Pankov R. Yamada K.M. J. Cell Sci. 2002; 115: 3861-3863Crossref PubMed Scopus (1488) Google Scholar, Cell Res. 1995; PubMed Scopus (172) Google Scholar), the FN in the yeast two-hybrid screen the and the The the LOX protein to be between residues and but the C-terminal may a in the interaction. In the solid phase binding we binding of cFN to but at a lower affinity which the the is to the N-terminal extracellular of the 1 cytokine Sci. S. 1990; PubMed Scopus Google Scholar, K. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). However, for the of LOX has been Our results suggest that this C-terminal may to the protein of LOX. In a we our which was amino residues be used to the with decrease in cFN binding was measured was with of not which is with the site of to the of LOX. microscopy of FN and LOX in showed a of FN matrix with overlapping LOX. this the of LOX in the extracellular matrix and with the FN at many of LOX and FN was observed in a of normal human we observed co-localization of such as in and but we co-localization at the of in the and In the and tissues, LOX was observed in a to have been of LOX with FN to has been well characterized Scholar, Google Scholar, L. Res. 1996; PubMed Google Scholar). type and collagens and elastin are not of the this of LOX that LOX may have functions or The results of in activity assays suggest that cFN is not a substrate for the LOX results not the that lysyl residues FN are by LOX specific conditions in vivo. cross-links in the FN matrix have not been in conditions of wound and FN is to by J. Chem. 1997; 16: PubMed Scopus Google Scholar, R.B. Sci. PubMed Scopus Google Scholar, Sci. PubMed Scopus (33) Google Scholar). The that LOX its enzyme activity that the active site of LOX is of how these collagen to be However, this to an active LOX enzyme at specific may the FN matrix to provide specific microenvironments to the cross-linking of To LOX in the of FN in we embryonic from FN-null and the of 30-kDa LOX in the cell was decreased by in FN-null observed a corresponding of the 45-kDa in the cell of the and an of a which does in the in This protein but it may an form of LOX or a novel LOX with the decrease in 30-kDa LOX by LOX amine oxidase activity assays demonstrated that LOX activity in cell was of was measured in The of active in and that the decrease in LOX activation was not to an of suggest that the of FN is for LOX activation in vivo. FN regulation of LOX activation the that the extracellular is a of the of LOX enzyme This regulation may be a in the in LOX expression and LOX activity. have or of LOX in to but in many of these the of between LOX and LOX activity was not A.H. P. Trackman P.C. J. Biol. Chem. 278: Full Text Full Text PDF PubMed Scopus Google Scholar, H.H. Trackman P.C. J. 2002; PubMed Scopus Google Scholar, M. L. Trackman P.C. PubMed Scopus Google Scholar, K. L.I. K.M. Kagan H.M. J. Biochem. PubMed Scopus Google Scholar, H.H. M.I. Trackman P.C. Google Scholar, Trackman P.C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, C.J. M. R. J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, the of LOX in to has been demonstrated to be H. M. M. H. M. T. S. Res. 1996; PubMed Scopus Google Scholar). However, LOX enzyme activity has been to Res. PubMed Google Scholar), which that the LOX may be in the for a of activation of LOX has been to be performed by which is for the C-terminal of type and M.V. Stetler-Stevenson W.G. Trubetskoy O.V. Gacheru S.N. Kagan H.M. J. Biol. Chem. 1996; 271: 7113-7119Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, M.I. Scott I.C. Babakhanlou-Chase H. Palamakumbura A.H. Pappano W.N. Hong H.H. Greenspan D.S. Trackman P.C. J. Biol. Chem. 2001; 276: 22537-22543Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, D.J. A.L. Matrix Biol. 1998; 16: PubMed Scopus Google Scholar). for a between FN and has been our results suggest that may be FN LOX processing by into or by the of to the cleavage site FN may act as a scaffold to LOX, BMP-1, and procollagen molecules in of collagen may the by which FN LOX processing and how the the of the extracellular matrix in The may have for pathological that have been with of of these such as cardiovascular disease, and of the of or FN in pathological conditions may be to the in LOX activity. In pathological conditions in which FN may a such as the FN form in and it be to investigate the LOX and M. for LOX and H. Kagan for Mosher for from FN-null and R. Mecham and P. Trackman for