Abstract:
Matrix metalloproteinases (MMPs) belong to a family of over 20 structurally related zinc-dependent proteases. They play a crucial role in every stage of wound healing by regulating the balance between tissue synthesis and destruction. However, excessive proteolytic activity, especially of MMP9, results in non-healing chronic wounds by degrading newly formed extracellular matrix and other required proteins. Thus, controlled activity of MMPs is essential for systematic wound healing. The development of new and efficient biomaterials in wound care is an area of intense research. Amyloid-like cross-β-structures have been explored for the development of novel biomaterials. The conversion of soluble protein into amyloid fibrils is accompanied by an increased resistance to proteolytic degradation, and this, along with other desirable features, recommends protein nanofibrils (PNFs) as a new scaffold for wound healing therapy. MMPs share a homologous catalytic domain, required for all MMP functions. In this thesis, the catalytic domain along with the pro-domain and fibronectin domain of human MMP9 (proMMP9cat) was expressed in E. coli. The protein was solubilised from inclusion bodies, refolded, and purified to homogeneity, with improved yields (1 mg/mL) compared to other methods followed in this thesis. The secondary structure of the refolded proMMP9cat was confirmed by circular dichroism. Following activation of the refolded proMMP9cat, the catalytic ability was confirmed using a colorimetric assay. The specific activity (50 U/μL) was comparable with commercially obtained MMP9 (48 U/μL). Whey protein isolate (WPI) was used as a commercially viable source of PNFs. To improve consistency of prepared biomaterials, the peptide composition of amyloid fibrils formed from WPI with and without limited proteolysis was systematically studied. The kinetics of fibril formation was assessed using a β-sheet binding dye, thioflavin T, and whey protein nanofibrils (WPNFs) were visually confirmed by transmission electron microscopy. Analysis of hydrolysates using matrix-assisted laser desorption/ionisation coupled with Fourier transform ion cyclotron resonance (MALDI-FTICR) revealed critical sequence motifs required for WPNFs formation. The peptide composition of mature WPNFs identified by liquid chromatography-mass spectrometry coupled with quadrupole time-of-flight (LC-MS/MS Q-TOF) generated fragments largely consistent with those in literature. Previously identified minimal sequences containing phenylalanine residues in β-Lactoglobulin (β-Lac), the major protein involved in WPNF formation, were surveyed against the generated WPNF fragments. Those peptide fragments common between the WPNF formed from untreated and protease treated WPI solution, which contained the minimal sequences, were examined in the context of the β-Lac crystal structure to understand the role of quaternary structure. This revealed the absence of a specific peptide fragment in WPNFs formed after trypsin and proteinase K treatment. This observation, in the context of the quaternary structure of β-Lac showed the influence of protein structure in generating peptides available for fibrillation. This observation will enable the preparation of a more consistent biomaterial. The proteolytic resistance of WPNF was investigated against purified MMP9cat, and commercial MMP1, 2, and 9 to confirm its stability, along with other relevant proteases. The resistance of WPNF against MMPs was also compared to other PNFs formed from soy and apo-haemoglobin. Varied resistance of PNFs to MMPs was observed. While partial stability was achieved overall against MMPs, minimal resistance to MMP1 (40 %) was observed compared to MMP2 (> 50 %) and MMP9 (> 70 %). Furthermore, the ability of PNFs to inhibit MMP catalytic activity (> 50 % for MMP1, > 70 % for MMP2 and 9) was confirmed using a chromogenic substrate. The selectivity for MMPs observed here could potentially be due to the differences between their catalytic subsites (S1) pocket. These preliminary results demonstrate the potential of WPNFs to modify MMP activity, overexpression of which is indicated in chronic wounds. The findings of this thesis encourage further development to utilise PNFs to develop functional, cost-effective biomaterial that can regulate protease activity.