Inclusion of an RGD Motif Alters Invasin Integrin-Binding Affinity and Specificity

Abstract

Invasin is a key adhesin displayed on the outer membrane of Yersinia enterocolitica and Y. pseudotuberculosis that mediates the initial stages of infection. Invasin specifically targets microfold (M) cells in the small intestine by binding β1 integrins and is sufficient to trigger eukaryotic uptake of invasin-coated particles, including Yersinia, Escherichia coli, and latex beads. As a result, invasin has generated interest to mediate oral delivery of vaccines and other biologics. Integrin binding affinity has been shown to correlate with particle uptake; thus we hypothesized that invasin variants with higher affinity would confer enhanced internalization. We first performed alanine scanning of surface-exposed tyrosine residues to identify those contributing to integrin binding. We identified two residues, which, when substituted with alanine, reduced binding to soluble α5β1 integrin. Next, we constructed four targeted mutagenesis libraries spanning these and other residues known to contribute to binding, followed by enrichment of variants able to mediate Caco-2 cellular invasion and to bind soluble α5β1 integrin. We identified three amino acid substitutions that increased α5β1 integrin binding affinity as measured by flow cytometry and ELISA assays, two of which created a novel RGD motif surrounding the D911 residue critical for binding. This variant confers enhanced internalization into CHO cells but not Caco-2 cells when expressed on the E. coli surface. Further analysis showed that inclusion of an RGD expands invasin-integrin specificity, thereby impacting cellular selectivity. This work provides a molecular explanation for the lack of an RGD motif in invasin that is present in many other adhesins.

Release of astroglial vimentin by extracellular vesicles: Modulation of binding and internalization of C3 transferase in astrocytes and neurons

“…Indeed, incubation of spinal cord synaptosomes for 1 hr at 4 C with 8 and 40 ng/μl of recombinant protein followed by stringent washing resulted in a concentration-dependent binding of vimentin ( Figure 6b). Amongst them are collagen (Lee, Sodek, & McCulloch, 1996), fibronectin (Bowditch et al, 1991), Yersinia pseudotuberculosis invasin (Khan, Wang, & Maynard, 2016), or Epstein-Barr virus (Chesnokova, Nishimura, & Hutt-Fletcher, 2009). Incubation with C3bot alone resulted in a detectable binding but was sig- (Mak & Brüggemann, 2016).…”

“…Since transmembrane integrins are main cell adhesion and signal transduction molecules for extracellular cues, a multitude of ligands has been described under both physiological and pathophysiological conditions for the extracellular N-terminal domains of integrin. Amongst them are collagen (Lee, Sodek, & McCulloch, 1996), fibronectin (Bowditch et al, 1991), Yersinia pseudotuberculosis invasin (Khan, Wang, & Maynard, 2016), or Epstein-Barr virus (Chesnokova, Nishimura, & Hutt-Fletcher, 2009). Therefore, interaction with clostridial C3bot harboring the integrin binding motif RGD seems also feasible for astrocytes, as demonstrated by reduced binding of the RGD-mutated C3bot-G89I.…”

Integrin‐Targeting Fluorescent Proteins: Exploration of RGD Insertion Sites

“…The relatively low affinity and integrin selectivity of linear RGD sequences can be increased by rigidification strategies, such as the use of cyclic peptides . The RGD sequence has also been inserted into protein loops for binding and uptake applications …”

Cell Adhesion on RGD-Displaying Knottins with Varying Numbers of Tryptophan Amino Acids to Tune the Affinity for Assembly on Cucurbit[8]uril Surfaces

“… 20 A combination of factors account for this observation, among which the increase in the effective concentration of the supramolecular components under spread cells and the weak rupture force between linear RGD and integrin played major roles. 20 , 21 When using RGD with a high affinity for integrin receptors, the interaction between cells and our supramolecular surfaces would be more prone to sense differences in the binding affinity of the host–guest complex. Compared to linear RGD, improved integrin binding, functionality, and specificity have been achieved when using synthetic cyclic RGD sequences 22 and recombinant proteins with RGD grafted within one of their loops, for example, in VEGF, 23 fluorescent proteins, 24 and miniprotein scaffolds.…”