Allosteric Regulation of Fibronectin/α5β1 Interaction by Fibronectin-Binding MSCRAMMs

Citation: Liang, X. W., Garcia, B. L., Visai, L., Prabhakaran, S., Meenan, N. A. G., Potts, J. R., . . . Hook, M. (2016). Allosteric Regulation of Fibronectin/alpha(5)beta(1) Interaction by Fibronectin-Binding MSCRAMMs. Plos One, 11(7), 17. doi:10.1371/journal.pone.0159118Adherence ofmicrobes to host tissues is a hallmark of infectious disease and is often mediated by a class of adhesins termed MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Numerous pathogens express MSCRAMMs that specifically bind the heterodimeric human glycoprotein fibronectin (Fn). In addition to roles in adhesion, Fn-binding MSCRAMMs exploit physiological Fn functions. For example, several pathogens can invade host cells by a mechanism whereby MSCRAMM-bound Fn bridges interaction with alpha(5)beta(1) integrin. Here, we investigate two Fn-binding MSCRAMMs, FnBPA (Staphylococcus aureus) and BBK32 (Borrelia burgdorferi) to probe structure-activity relationships of MSCRAMM-induced Fn/alpha(5)beta(1) integrin activation. Circular dichroism, fluorescence resonance energy transfer, and dynamic light scattering techniques uncover a conformational rearrangement of Fn involving domains distant from the MSCRAMM binding site. Surface plasmon resonance experiments demonstrate a significant enhancement of Fn/alpha(5)beta(1) integrin affinity in the presence of FnBPA or BBK32. Detailed kinetic analysis of these interactions reveal that this change in affinity can be attributed solely to an increase in the initial Fn/alpha(5)beta(1) on-rate and that this rate-enhancement is dependent on high-affinity Fn-binding by MSCRAMMs. These data implicate MSCRAMM-induced perturbation of specific intramolecular contacts within the Fn heterodimer resulting in activation by exposing previously cryptic alpha(5)beta(1) interaction motifs. By correlating structural changes in Fn to a direct measurement of increased Fn/alpha(5)beta(1) affinity, this work significantly advances our understanding of the structural basis for the modulation of integrin function by Fn-binding MSCRAMMs

Thin films of vitronectin transferred by MAPLE

We report on matrix-assisted pulsed laser evaporation (MAPLE) transfer of intact and functional protein molecules from a cryogenic aliquot obtained by freezing a protein-saline buffer solution. Vitronectin (Vn), an extracellular matrix protein with distinctive active domains for cell attachment and signalization, was expelled from frozen targets by KrF*excimer laser irradiation, and then immobilized on substrates. Particulates surrounded by a dense matrix were observed by optical, profilometry and AFM studies. The composition preservation of MAPLE-deposited protein films versus drop-cast films was demonstrated by FTIR and immunostaining studies. The stability and integrity of Vn after transfer was shown by their interaction with human osteoprogenitor cells in which actin filaments stretched across the entire cell area and clear focal points with surface were formed. The absence of detectable degradation of protein structure after MAPLE immobilization could provide benefits to surface functionalization for biomedical applications. © 2011 Springer-Verlag

Polymer-Based Honeycomb Films on Bioactive Glass : Toward a Biphasic Material for Bone Tissue Engineering Applications

The development of innovative materials for bone tissue engineering to promote bone regeneration while avoiding fibrous tissue infiltration is of paramount importance. Here, we combined the known osteopromotive properties of bioactive glasses (BaGs) with the biodegradability, biocompatibility, and ease to shape/handle of poly-l-co-d,l-lactic acid (PLDLA) into a single biphasic material. The aim of this work was to unravel the role of the surface chemistry and topography of BaG surfaces on the stability of a PLDLA honeycomb membrane, in dry and wet conditions. The PLDLA honeycomb membrane was deposited using the breath figure method (BFM) on the surface of untreated BaG discs (S53P4 and 13-93B20), silanized with 3-aminopropyltriethoxysilane (APTES) or conditioned (immersed for 24 h in TRIS buffer solution). The PLDLA membranes deposited onto the BaG discs, regardless of their composition or surface treatments, exhibited a honeycomb-like structure with pore diameter ranging from 1 to 5 μm. The presence of positively charged amine groups (APTES grafting) or the precipitation of a CaP layer (conditioned) significantly improved the membrane resistance to shear as well as its stability upon immersion in the TRIS buffer solution. The obtained results demonstrated that the careful control of the substrate surface chemistry enabled the deposition of a stable honeycomb membrane at their surface. This constitutes a first step toward the development of new biphasic materials enabling osteostimulation (BaG) while preventing migration of fibrous tissue inside the bone defect (honeycomb polymer membrane).publishedVersionPeer reviewe

Labeling of fibronectin by fluorescent and paramagnetic nanoprobes for exploring the extracellular matrix: bioconjugate synthesis optimization and biochemical characterizatio

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