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Imaging and targeting LOX-mediated tissue remodeling with a reactive collagen peptide

Nature Chemical Biology volume 17, pages 865–871 (2021)Cite this article

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Abstract

Collagens are fibrous proteins that are integral to the strength and stability of connective tissues. During collagen maturation, lysyl oxidases (LOX) initiate the cross-linking of fibers, but abnormal LOX activity is associated with impaired tissue function as seen in fibrotic and malignant diseases. Visualizing and targeting this dynamic process in healthy and diseased tissue is important, but so far not feasible. Here we present a probe for the simultaneous monitoring and targeting of LOX-mediated collagen cross-linking that combines a LOX-activity sensor with a collagen peptide to chemoselectively target endogenous aldehydes generated by LOX. This synergistic probe becomes covalently anchored and lights up in vivo and in situ in response to LOX at the sites where cross-linking occurs, as demonstrated by staining of normal skin and cancer sections. We anticipate that our reactive collagen-based sensor will improve understanding of collagen remodeling and provide opportunities for the diagnosis of fibrotic and malignant diseases.

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Fig. 1: Overview and design strategy.
Fig. 2: Design and characteristics of the LOX-activatable fluorescent probe and measurements of LOX activity in mouse skin lysates.
Fig. 3: In situ determination of LOX activity with collagen peptide-sensor conjugates.
Fig. 4: Collagen peptide-sensor conjugates identify sites of LOX activity in tumor tissue.

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Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information. Expression data for LOX and LOX-like isoforms is available from the Hair-GEL database (E14.5 and P5) (http://hair-gel.net/).

References

  1. Shoulders, M. D. & Raines, R. T. Collagen structure and stability. Annu. Rev. Biochem. 78, 929–958 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Eming, S. A., Martin, P. & Tomic-Canic, M. Wound repair and regeneration: mechanisms, signaling, and translation. Sci. Transl. Med. 6, 265sr266 (2014).

    Article  Google Scholar 

  3. Rockey, D. C., Bell, P. D. & Hill, J. A. Fibrosis—a common pathway to organ injury and failure. N. Engl. J. Med. 372, 1138–1149 (2015).

    Article  CAS  PubMed  Google Scholar 

  4. Kagan, H. M. & Li, W. Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell. J. Cell. Biochem. 88, 660–672 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Vallet, S. D. & Ricard-Blum, S. Lysyl oxidases: from enzyme activity to extracellular matrix cross-links. Essays Biochem. 63, 349–364 (2019).

    Article  CAS  PubMed  Google Scholar 

  6. Ramshaw, J. A. M., Shah, N. K. & Brodsky, B. Gly-X-Y tripeptide frequencies in collagen: a context for host–guest triple-helical peptides. J. Struct. Biol. 122, 86–91 (1998).

    Article  CAS  PubMed  Google Scholar 

  7. Rodriguez-Pascual, F. & Slatter, D. A. Collagen cross-linking: insights on the evolution of metazoan extracellular matrix. Sci. Rep. 6, 37374 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Piersma, B. & Bank, R. A. Collagen cross-linking mediated by lysyl hydroxylase 2: an enzymatic battlefield to combat fibrosis. Essays Biochem. 63, 377–387 (2019).

    Article  CAS  PubMed  Google Scholar 

  9. Levene, C. I. & Gross, J. Alterations in state of molecular aggregation of collagen induced in chick embryos by beta-aminopropionitrile (lathyrus factor). J. Exp. Med. 110, 771–790 (1959).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Cox, T. R. & Erler, J. T. Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer. Dis. Models Mech. 4, 165–178 (2011).

    Article  CAS  Google Scholar 

  11. Wynn, T. Cellular and molecular mechanisms of fibrosis. J. Pathol. 214, 199–210 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Montesi, S. B., Desogere, P., Fuchs, B. C. & Caravan, P. Molecular imaging of fibrosis: recent advances and future directions. J. Clin. Invest. 129, 24–33 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Baues, M. et al. Fibrosis imaging: current concepts and future directions. Adv. Drug. Deliv. Rev. 121, 9–26 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Palamakumbura, A. H. & Trackman, P. C. A fluorometric assay for detection of lysyl oxidase enzyme activity in biological samples. Anal. Biochem. 300, 245–251 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Pinnell, S. R. & Martin, G. R. The cross-linking of collagen and elastin: enzymatic conversion of lysine in peptide linkage to α-aminoadipic-delta-semialdehyde (allysine) by an extract from bone. Proc. Natl Acad. Sci. USA 61, 708–716 (1968).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Holt, A. & Palcic, M. M. A peroxidase-coupled continuous absorbance plate-reader assay for flavin monoamine oxidases, copper-containing amine oxidases and related enzymes. Nat. Protoc. 1, 2498–2505 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Trackman, P. C., Zoski, C. G. & Kagan, H. M. Development of a peroxidase-coupled fluorometric assay for lysyl oxidase. Anal. Biochem. 113, 336–342 (1981).

    Article  CAS  PubMed  Google Scholar 

  18. Rodriguez, H. M. et al. Modulation of lysyl oxidase-like 2 enzymatic activity by an allosteric antibody inhibitor. J. Biol. Chem. 285, 20964–20974 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Aslam, T. et al. Optical molecular imaging of lysyl oxidase activity—detection of active fibrogenesis in human lung tissue. Chem. Sci. 6, 4946–4953 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Chen, G., Yee, D. J., Gubernator, N. G. & Sames, D. Design of optical switches as metabolic indicators: new fluorogenic probes for monoamine oxidases (MAO A and B). J. Am. Chem. Soc. 127, 4544–4545 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Albers, A. E., Rawls, K. A. & Chang, C. J. Activity-based fluorescent reporters for monoamine oxidases in living cells. Chem. Commun. 44, 4647–4649 (2007).

  22. Wu, X., Shi, W., Li, X. & Ma, H. A. Strategy for specific fluorescence imaging of monoamine oxidase a in living cells. Angew. Chem. Int. Ed. 56, 15319–15323 (2017).

    Article  CAS  Google Scholar 

  23. Sun, W.-C., Gee, K. R. & Haugland, R. P. Synthesis of novel fluorinated coumarins: excellent UV-light excitable fluorescent dyes. Bioorg. Med. Chem. Lett. 8, 3107–3110 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Cohen, J. D., Thompson, S. & Ting, A. Y. Structure-guided engineering of a pacific blue fluorophore ligase for specific protein imaging in living cells. Biochemistry 50, 8221–8225 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Lee, M. M., Gao, Z. & Peterson, B. R. Synthesis of a fluorescent analogue of paclitaxel that selectively binds microtubules and sensitively detects efflux by P-glycoprotein. Angew. Chem. Int. Ed. 56, 6927–6931 (2017).

    Article  CAS  Google Scholar 

  26. Chang, D., Kim, K. T., Lindberg, E. & Winssinger, N. Accelerating turnover frequency in nucleic acid templated reactions. Bioconj. Chem. 29, 158–163 (2018).

    Article  CAS  Google Scholar 

  27. Klein, G. & Reymond, J. L. An enantioselective fluorimetric assay for alcohol dehydrogenases using albumin-catalyzed beta-elimination of umbelliferone. Bioorg. Med. Chem. Lett. 8, 1113–1116 (1998).

    Article  CAS  PubMed  Google Scholar 

  28. Csiszar, K. Lysyl oxidases: a novel multifunctional amine oxidase family. Prog. Nucleic Acid Res. Mol. Biol. 70, 1–32 (2001).

    Article  CAS  PubMed  Google Scholar 

  29. Brown-Augsburger, P., Tisdale, C., Broekelmann, T., Sloan, C. & Mecham, R. P. Identification of an elastin cross-linking domain that joins three peptide chains: possible role in nucleated assembly. J. Biol. Chem. 270, 17778–17783 (1995).

    Article  CAS  PubMed  Google Scholar 

  30. Sanada, H. et al. Changes in collagen cross-linking and lysyl oxidase by estrogen. Biochim. Biophys. Acta, Gen. Subj. 541, 408–413 (1978).

    Article  CAS  Google Scholar 

  31. Szauter, K. M., Cao, T., Boyd, C. D. & Csiszar, K. Lysyl oxidase in development, aging and pathologies of the skin. Pathol. Biol. (Paris) 53, 448–456 (2005).

    Article  CAS  Google Scholar 

  32. Chattopadhyay, S. & Raines, R. T. Collagen-based biomaterials for wound healing. Biopolymers 101, 821–833 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Bennink, L. L. et al. Visualizing collagen proteolysis by peptide hybridization: from 3D cell culture to in vivo imaging. Biomaterials 183, 67–76 (2018).

    Article  CAS  PubMed  Google Scholar 

  34. Dones, J. M. et al. Optimization of interstrand interactions enables burn detection with a collagen-mimetic peptide. Org. Biomol. Chem. 17, 9906–9912 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Li, Y. et al. Targeting collagen strands by photo-triggered triple-helix hybridization. Proc. Natl Acad. Sci. USA 109, 14767–14772 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Takita, K. K., Fujii, K. K., Kadonosono, T., Masuda, R. & Koide, T. Cyclic peptides for efficient detection of collagen. Chem. Bio. Chem. 19, 1613–1617 (2018).

    Article  CAS  PubMed  Google Scholar 

  37. Kalia, J. & Raines, R. T. Hydrolytic stability of hydrazones and oximes. Angew. Chem. Int. Ed. 47, 7523–7526 (2008).

    Article  CAS  Google Scholar 

  38. Kölmel, D. K. & Kool, E. T. Oximes and hydrazones in bioconjugation: mechanism and catalysis. Chem. Rev. 117, 10358–10376 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Waghorn, P. A. et al. Molecular magnetic resonance imaging of lung fibrogenesis with an oxyamine-based probe. Angew. Chem. Int. Ed. 56, 9825–9828 (2017).

    Article  CAS  Google Scholar 

  40. Hentzen, N. B., Smeenk, L. E. J., Witek, J., Riniker, S. & Wennemers, H. Cross-linked collagen triple helices by oxime ligation. J. Am. Chem. Soc. 139, 12815–12820 (2017).

    Article  CAS  PubMed  Google Scholar 

  41. Merkel, J. R., DiPaolo, B. R., Hallock, G. G. & Rice, D. C. Type I and type III collagen content of healing wounds in fetal and adult rats. Proc. Soc. Exp. Biol. Med. 187, 493–497 (1988).

    Article  CAS  PubMed  Google Scholar 

  42. Volk, S. W., Wang, Y., Mauldin, E. A., Liechty, K. W. & Adams, S. L. Diminished type III collagen promotes myofibroblast differentiation and increases scar deposition in cutaneous wound healing. Cells Tissues Organs 194, 25–37 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Xue, M. & Jackson, C. J. Extracellular matrix reorganization during wound healing and its impact on abnormal scarring. Adv. Wound Care 4, 119–136 (2015).

    Article  Google Scholar 

  44. Wietecha, M. S. et al. Activin-mediated alterations of the fibroblast transcriptome and matrisome control the biomechanical properties of skin wounds. Nat. Commun. 11, 2604 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Rucklidge, G. J., Milne, G., McGaw, B. A., Milne, E. & Robins, S. P. Turnover rates of different collagen types measured by isotope ratio mass spectrometry. Biochim. Biophys. Acta 1156, 57–61 (1992).

    Article  CAS  PubMed  Google Scholar 

  46. Campagnola, P. J. et al. Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. Biophys. J. 82, 493–508 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Zoumi, A., Yeh, A. & Tromberg, B. J. Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc. Natl Acad. Sci. USA 99, 11014–11019 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Perryman, L. & Erler, J. T. Lysyl oxidase in cancer research. Future Oncol. 10, 1709–1717 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Cangkrama, M. et al. A paracrine activin A–mDia2 axis promotes squamous carcinogenesis via fibroblast reprogramming. EMBO Mol. Med. 12, e11466 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Pensalfini, M. et al. The mechanical fingerprint of murine excisional wounds. Acta Biomater. 65, 226–236 (2018).

    Article  PubMed  Google Scholar 

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Acknowledgements

We thank P. Bruckner, Freiburg, Germany, for critical comments on the manuscript, M. Wietecha, ETH Zurich, for discussions and assistance compiling LOX expression data, M. Cangkrama, ETH Zurich, for the skin tumor samples and J. Kusch for microscopy assistance. SHG imaging was carried out at the Scientific Center for Optical and Electron Microscopy (ScopeM) at the ETH Zurich. This work was funded by the ETH Zurich and the European Union’s Seventh Framework Program with an ETH Postdoctoral Fellowship (to M.R.A.), the Fonds National de la Recherche Luxembourg with an AFR Ph.D. Fellowship (to N.B.H.), the Swiss National Science Foundation (SNF grant nos. 31003B-189364 (to S.W.) and 2000020_178805 (to H.W.)), University Medicine Zurich (Flagship project SKINTEGRITY to S.W. and H.W.) and by the ETH Zurich OpenETH Project SKINTEGRITY.CH (to S.W. and H.W.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Laboratory of Organic Chemistry, ETH Zurich, Zurich, Switzerland

    Matthew R. Aronoff, Nina B. Hentzen & Helma Wennemers

  2. Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland

    Paul Hiebert & Sabine Werner

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Contributions

M.R.A. and H.W. conceived the project. M.R.A. performed the chemical syntheses together with N.B.H., and carried out the spectroscopic analyses and the ex vivo and in situ assays after P.H. collected the tissue samples. P.H. performed the in vivo experiments and histological/immunofluorescence analyses. S.W. designed the in vivo experiments together with P.H. M.R.A. and H.W. drafted the paper. All authors contributed to data analysis and writing of the paper.

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Correspondence to Helma Wennemers.

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Competing interests

ETH Zurich has applied for a patent (EP19213787.5) related to technology described in this publication with M.R.A and H.W. listed as inventors.

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Peer review information Nature Chemical Biology thanks Fernando Rodriguez-Pascual and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Information

Supplementary Figs. 1–35, Table 1 and Note: Synthetic protocols and characterization.

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Aronoff, M.R., Hiebert, P., Hentzen, N.B. et al. Imaging and targeting LOX-mediated tissue remodeling with a reactive collagen peptide. Nat Chem Biol 17, 865–871 (2021). https://doi.org/10.1038/s41589-021-00830-6

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