Expanding limits of artificial enzymes: unprecedented catalysis by an oxidase nanozyme in activating a structural protein for covalent crosslinking and conferring remarkable proteolytic resistance

Abstract

Nature has endowed us with some complex enzymes capable of utilizing proteins as their substrates to generate functional proteins through post-translational modification. However, nanozymes' interplay with proteins as substrates is scarce, with their chemistry predominantly established using only small molecule substrates, featuring a significant gap in this area. Due to the huge prospects of nanozymes in biotechnological and therapeutic interventions, studies establishing the unexplored roles of nanozymes in the biological environments and their interplay beyond small molecule substrates warrant immediate attention. In this study, we unveil the unprecedented role of a Mn-based oxidase nanozyme (MnN) in activating a structural protein, collagen, and covalently crosslinking its tyrosine residues with only a trace amount of tannic acid (TA) without compromising its triple-helical structural integrity. While therapeutic applications demand materials prepared from collagen, the current chemical and physical crosslinking of collagen often presents significant challenges such as toxicity, denaturation, or high costs. MnN lucidly accomplishes crosslinking interplay at its 101 facets using oxygen as a co-substrate under mild conditions. This process takes advantage of MnN being active at mild acidic pH where collagen preferentially exists as a soluble triple helix (monomeric form), exposing functionalities and enhancing the crosslinking degree. Importantly, this reaction also confers 100% resistance to collagenase attack on the collagen tendon-derived biological material. The catalyzed TA-tyrosine linkage in the telopeptide region of collagen probably impedes the initial recognition step of collagenase, providing robust protection against its degradative action. Our study not only expands the repertoire of nanozymes' substrates beyond the existing library of small molecules but also establishes a significant step toward designing a gold standard for collagen crosslinking. With biomedical applications demanding biomaterials derived from protein scaffolds with preserved structural integrity, our investigation bridges the gap between nanozymes' chemistry and crosslinking proteins, opening exciting prospects for biomaterial development.

Fig. 1. (a) The triple helical structure of collagen. (b) Hydrogen bonding in the triple helix. (c) Schematics showing the assembled collagen molecules in fibrils. (d) Collagen crosslinking methods and the use of crosslinked collagen biomaterials in biomedical applications.

Fig. 2. (a) Powder XRD pattern of MnN. (b and c) The low and high-resolution SEM images of MnN showing octahedral nanoparticles, respectively. (d) TEM image of MnN (inset shows a representative red colored octahedron). (e) HRTEM image of MnN showing lattice fringes. Inset shows the SAED pattern exhibiting clear spots due to the crystalline nature of MnN. (f) The magnified portion of the area marked as a red box in panel (e), showing (101) and (211) planes. (g) Survey XPS spectrum of MnN. (h and i) Core-level Mn 2p and O 1s XPS spectra of MnN.

Fig. 3. (a) Scheme showing the oxidase-like activity of MnN converting TMB to the oxidized blue-colored charge-transfer adduct that absorbs at 652 nm. (b) Time-dependent monitoring of the formation of oxidized TMB at 652 nm in the presence and absence of MnN. (c) Bar graph showing the comparison of the rate of oxidase-like activity of MnN with other control reactions. (d) Rates of oxidase-like activity at different pH. (e) Effect of increase in the concentration of MnN on its oxidase-like activity. (f) Effect of increase in the concentration of TMB on the oxidase-like activity of MnN, showing Michaelis–Menten kinetics. (g) Lineweaver–Burk plot corresponding to the plot shown in panel (f).

Fig. 4. (a) Conversion of TyrMe to its dimer as a result of oxidase-like activity of MnN. (b) Fluorescence spectra showing a time-dependent increase in the intensity of fluorescence due to the continuous formation of TyrMe dimer during the reaction. (c) The line graph corresponding to the fluorescence spectra presented in panel (b). (d) Coupling of TA with TA itself as a result of oxidase-like activity of MnN. (e) Fluorescence spectra showing time-dependent increase in the intensity of fluorescence due to the continuous formation of the TA–TA species during the reaction. Inset shows a cuvette containing the product that exhibits bluish fluorescence when excited at 365 nm. (f) MnN-catalyzed crosslinking of RTT collagen in the presence of TA. (g) SDS PAGE analysis of the crosslinked collagen and other controls, LAD = ladder. (h) Representative photographs of control RTTs and experimental RTT obtained after MnN-catalyzed crosslinking. (i) The possible modes of covalent crosslinking of collagen in addition to the oxidative coupling of phenolics. Inset shows a dried crosslinked RTT that was found to be stable after 12 months of storage. SEM image of (j) RTT, (k) RTT + TA, (l) RTT + MnN and (m) RTT + TA + MnN, after treatment at pH 5.5 for 24 h.

Fig. 5. (a) Scheme showing treatment of the crosslinked RTT with collagenase. (b) Photographs of the RTTs obtained after collagenase treatment. Note that RTTs were destroyed in control reactions. However, the MnN-catalyzed crosslinked RTT is intact. (c) Comparison of % degradation of RTTs by collagenases as analyzed by hydroxyproline estimation assay. (d) The crystal structure of MnN showing the arrangement of ions at the exposed 101 planes of the octahedral nanoparticle. The structure is made using a cif file of Mn₃O₄ (ID: mp-18759) provided in the materials project database. (e) Probing oxidase mechanism by studying the effect of using reactive oxygen species scavenging enzymes such as SOD and catalase, and the addition of H₂O₂ on the oxidase-like activity of MnN. (f) The proposed mechanism by which the molecular oxygen is converted to water as a result of the transfer of electrons from electron donors such as TMB, TA or tyrosyl residues of collagen.