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. 2010 Oct 1;285(40):31046-54.
doi: 10.1074/jbc.M110.151357. Epub 2010 Jul 30.

An engineered alpha1 integrin-binding collagenous sequence

Neungseon Seo  1 , Brooke H Russell, Jose J Rivera, Xiaowen Liang, Xuejun Xu, Vahid Afshar-Kharghan, Magnus Höök
Affiliations

An engineered alpha1 integrin-binding collagenous sequence

Neungseon Seo et al. J Biol Chem. .

Abstract

Collagen is an extracellular matrix structural component that can regulate cellular processes through its interaction with the integrins, α1β1, α2β1, α10β1, and α11β1. Collagen-like proteins have been identified in a number of bacterial species. Here, we used Scl2 from Streptococcus pyogenes serotype M28 strain MGAS6274 as a backbone for the introduction of discrete integrin-binding sequences. The introduced sequences GLPGER, GFPGER, or GFPGEN did not affect triple helix stability of the Scl (Streptococcal collagen-like) protein. Using ELISA and surface plasmon resonance, we determined that Scl2(GLPGER) and Scl2(GFPGER) bound to recombinant human α1 and α2 I-domains in a metal ion-dependent manner and without a requirement for hydroxyproline. We predicted a novel and selective integrin-binding sequence, GFPGEN, through the use of computer modeling and demonstrated that Scl2(GFPGEN) shows specificity toward the α1 I-domain and does not bind the α2 I-domain. Using C2C12 cells, we determined that intact integrins interact with the modified Scl2 proteins with the same selectivity as recombinant I-domains. These modified Scl2 proteins also acted as cell attachment substrates for fibroblast, endothelial, and smooth muscle cells. However, the modified Scl2 proteins were unable to aggregate platelets. These results indicate that Scl2 is a suitable backbone for the introduction of mammalian integrin-binding sequences, and these sequences may be manipulated to individually target α1β1 and α2β1.

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Figures

FIGURE 1.
FIGURE 1.
SDS-PAGE analysis reveals the presence of trimers by collagen-like proteins containing integrin-binding sequences. Coomassie-stained SDS-PAGE analysis of purified recombinant Scl2GLPGER, Scl2GFPGER, and Scl2GFPGEN. Proteins were kept at 4 °C (− Heat) or heat-treated at 95 °C for 5 min in the presence of 0.1% SDS (+ Heat) prior to electrophoresis. Trimers demonstrate decreased electrophoretic migration in comparison with monomers as determined by molecular mass standards (M).
FIGURE 2.
FIGURE 2.
CD analysis demonstrates triple helical structures within Scl2, Scl2GLPGER, Scl2GFPGER, and Scl2GFPGEN. A, CD wavelength scans revealed the presence of a peak at 220 nm indicative of a triple helical structure. B, CD thermal transition monitored at 220 nm with a temperature slope of 10 °C/h revealed a transition at ∼37 °C.
FIGURE 3.
FIGURE 3.
Integrin α1 and α2 I-domains bind to Scl2GLPGER and Scl2GFPGER. Recombinant human α1 (A) and α2 (B) I-domains (5 μm) were incubated with immobilized Scl2, Scl2GLPGER, Scl2GFPGER, and collagen type I (Collagen) (1 μg/well) with 1 mm Mg2+ (black bars) or 1 mm EDTA (white bars). Integrin binding was detected with Scl2GLPGER, Scl2GFPGER, and collagen type I in the absence of EDTA indicating a metal ion-dependent binding mechanism. C and D, Biacore analysis indicated that both I-domains interacted with Scl2GFPGER and Scl2GLPGER. In an SPR-based binding assay, 0.8 μm of α1 (C) or 25 μm of α2 (D) I-domains in binding buffer (10 mm HEPES, pH 7.3, 150 mm NaCl, 5 mm β-mercaptoethanol, 1 mm MgCl2, 0.005% Tween 20) was injected over the captured Scl2GFPGER and Scl2GLPGER surface. The captured Scl2 surface was used as a control (which did not have detectable binding by either I-domain; data not shown), and SPR response was obtained by subtraction of that from a reference surface. For comparison, each response curve was rescaled to the response equivalent to that generated by every 1000 resonance units (RU) of ligand captured.
FIGURE 4.
FIGURE 4.
Scl2GFPGEN binds the recombinant integrin α1 I-domain. Recombinant human α1 (A) and α2 (B) I-domains were incubated with immobilized Scl2, Scl2GFPGER, Scl2GFPGEN, and collagen type I (Collagen I) (1 μg/well) with 1 mm Mg2+. α1 Binding was detected with Scl2GFPGER, Scl2GFPGEN, and collagen type I and α2 binding was detected with Scl2GFPGER and collagen type I only. C–E, Biacore analysis revealed that unlike Scl2GFPGER, Scl2GFPGEN is only recognized by the α1 I-domain, and this recognition is Mg2+-dependent and can be inhibited by collagen type I. In an SPR-based binding assay, 0.8 μm of α1 (C) or 25 μm of α2 (D) I-domains in the binding buffer were injected over the captured Scl2GFPGER and Scl2GFPGEN surfaces. E, 2 μm of α1 in the binding buffer, binding buffer without MgCl2, or in the presence of 0.5 μm collagen type I was injected over the Scl2GFPGEN surface. All SPR responses were obtained by subtraction of that from a reference surface.
FIGURE 5.
FIGURE 5.
Collagen-like proteins act as substrates for cell adherence and spreading. Increasing concentrations of Scl2 (gray square), Scl2GLPGER (pink triangle), Scl2GFPGER (green triangle), Scl2GFPGEN (purple inverted triangle), and collagen type I (yellow diamond) were coated on microtiter wells (0–10 μg/well) in duplicate. C2C12-α1 (A), C2C12-α2 (B), and C2C12 (C) cells were added to wells for 1 h and washed to remove unbound cells. Bound cells were stained with crystal violet and quantified by optical density measurement at 590 nm (A590). D, glass chamber slides were coated with Scl2, collagen type I (Collagen I), Scl2GLPGER, Scl2GFPGER, or Scl2GFPGEN (1 μg/well). C2C12-α1 (top row) or C2C12-α2 (bottom row) cells were added to wells, and unbound cells were washed after 1 h of incubation. Bound cells were stained with fluorescently tagged phalloidin (green) and DAPI (blue). Bound cells demonstrated a spread morphology as apposed to a rounded morphology. Details are as described under “Experimental Procedures.” The representative experiments and images are shown.
FIGURE 6.
FIGURE 6.
Human fibroblast, endothelial, and smooth muscle cells adhere to collagen-like protein substrates. Recombinant proteins Scl2, Scl2GFPGER, and Scl2GFPGEN and collagen type I (Collagen I) were coated on microtiter wells (1 μg/well) in triplicate. Human fibroblast cells (HT10180), human fibroblast cells (MRC5), HUVECs, and mouse vascular smooth muscle cells (SMC), were allowed to adhere to substrates for 1 h, and unbound cells were washed away. Bound cells were stained with crystal violet and quantified by measurement at 590 nm. Details are as described under “Experimental Procedures.” A representative experiment is shown.
FIGURE 7.
FIGURE 7.
Collagen-like proteins do not aggregate platelets. Collagen type I (Collagen I), Scl2GLPGER, Scl2GFPGER, and Scl2GFPGEN were tested as agonists for platelet aggregation. Briefly, platelet-rich plasma was obtained and stimulated by collagen-like proteins (2 μm) or collagen type I (0.2 μm) in an aggregometer under stirring conditions for 10 min. Platelet aggregation (y axis) was measured by optical turbidometry over time (x axis). A representative experiment is shown.
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