An Engineered α1 Integrin-binding Collagenous Sequence

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

doi:10.1074/jbc.m110.151357

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.

Highlights

Surface proteins with collagen-like domains recently have been found on a number of prokaryotic organisms, including
We demonstrate that the introduction of integrin-binding sequences does not disrupt triple helix formation or alter thermal stability of the modified Scl2 protein and that, in general, the presence of hydroxyproline is not required for integrin binding or cell adherence to substrates composed of the modified Scl2 proteins
Two previously identified integrin-binding sequences, GLOGER and GFOGER [24, 25], were introduced in Scl2 by site-directed mutatgenesis, except that hydroxyproline was replaced by proline

Summary

To whom correspondence should be addressed

Streptococcus pyogenes, Streptococcus equi, and Bacillus anthracis (4 – 6) These collagen-like domains contain conserved GXY repeats but lack the hydroxyproline found in mammalian collagens. The collagen-like domain of Scl from S. pyogenes serotype M41 is recognized by the collagen-binding ␣2␤1 integrin in an interaction that can lead to bacterial host cell invasion [8, 11]. The structure of the ␣1 I-domain indicated a similar overall structure, but the ligand-binding trench in ␣1 is predicted to be longer and more flexible than in ␣2 [28] These observations suggest that ␣1 and ␣2 integrinspecific sequences may exist in collagen and may contribute to the observed differences in ␣1 and ␣2 integrin binding to various collagens [23, 25, 29]. We describe a novel recombinant system for the study of collagen-integrin interactions with potential bioengineering applications

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION