A Streptococcal Collagen-like Protein Interacts with the α2β1 Integrin and Induces Intracellular Signaling

Abstract

The streptococcal collagen-like proteins Scl1 and Scl2 are prokaryotic members of a large protein family with domains containing the repeating amino acid sequence (Gly-Xaa-Yaa) n that form a collagen-like triple-helical structure. Here, we test the hypothesis that Scl variant might interact with mammalian collagen-binding integrins. We show that the recombinant Scl protein p176 promotes adhesion and spreading of human lung fibroblast cells through an ␣ 2 ␤ 1 integrin-mediated interaction as shown in cell adhesion inhibition assays using anti-␣ 2 ␤ 1 and anti-␤ 1 integrins monoclonal antibodies. Accordingly, C2C12 cells stably expressing ␣ 2 ␤ 1 integrin as the only collagen-binding integrin show productive cell adhesion activities on p176 that can be blocked by an anti-␣ 2 ␤ 1 integrin antibody. In addition, p176 promotes tyrosine phosphorylation of p125 FAK of C2C12 cells expressing ␣ 2 ␤ 1 integrin, whereas parental C2C12 cells do not. Furthermore, adhesion of human lung fibroblast cells to p176 induces phosphorylation of p125 FAK , p130 CAS , and p68 Paxillin proteins. In a domain swapping experiment, we show that integrin binds to the collagenous domain of the Scl protein. Moreover, the recombinant inserted domain of the ␣ 2 integrin interacts with p176 with a relatively high affinity (K D ‫؍‬ 17 nM). Attempts to identify the integrin sites in p176 suggest that more than one site may be involved. These studies, for the first time, suggest that the collagen-like proteins of prokaryotes retained not only structural but also functional characteristics of their eukaryotic counterparts.The collagens are a family of extracellular matrix (ECM) 1 proteins that provide structural support to all multicellular animals (1). The repeating sequence Gly-Xaa-Yaa (GXY), where X is often proline and Y is often hydroxyproline, is a unique feature of the collagen polypeptides (2-4). Long tracks of repeated GXY sequences fold into left-handed polyproline type II-like chains, and three such chains cooperatively twist around a central axis to form a right-handed rope-like superhelix (2, 5-7), considered a defining feature of collagens. The collagens also contain C-and N-terminal noncollagenous domains, which often are proteolyzed during secretion from the cells (7-9). Mature collagen molecules are deposited in the ECM in the form of fibers, networks, and beaded filaments (8). One group of mammalian proteins that fulfill rudimentary host defense functions such as the complement factor C1q (9) and several mammalian lectins (10) also have collagenous domains, but they are not conventional collagens. These proteins form characteristic lollipop-like structures, where the collagenous domains form the stalks, and globular heads correspond to the noncollagenous regions. Collagen-like molecules also have been found in lower eukaryotes such as mussels, worms, and sponges (11), and collagen-like sequences have been identified from analyses of the genomes of prokaryotes (12-15).The streptococcal collagen-like proteins Scl1 and Scl2 (also know...

Identification of the First Prokaryotic Collagen Sequence Motif That Mediates Binding to Human Collagen Receptors, Integrins α2β1 and α11β1

“…Mapping of ␣ 2 ␤ 1 Integrin Binding Region in rScl1-Our previous study reported binding between the CL region of the recombinant P176 construct, derived from Scl1.41 protein, and the ␣ 2 ␤ 1 integrin on human fibroblasts (20); however, the integrin-binding motif on P176 was not defined. To map the ␣ 2 ␤ 1 integrin binding site of P176 we inserted fragments of the P176-CL region into the integrin binding-negative protein, P163 (Table 1).…”

“…Rotary Shadowing and Electron Microscopy-rScl proteins and rScl-r␣ 2 I complexes were rotary shadowed and analyzed by EM as described previously (17,20). Briefly, recombinant proteins were dialyzed against 0.1 M sodium bicarbonate containing 1 mM magnesium chloride.…”

“…Cell Attachment Assays-The ability of cells to use rScl proteins for attachment was tested using a previously described assay (20). BSA or type I collagen (10 g/well) or rScl proteins (100 nM) in Tris-buffered saline (TBS) were coated overnight onto 24-well tissue culture plates at 4°C.…”

“…1 tion of the O residues in mammalian collagens (19). A recombinant Scl1-derived protein binds to the ␣ 2 ␤ 1 integrin on human cells and this interaction resulted in the phosphorylation of integrin pathway-associated cell signaling proteins (20). Thus, Scl proteins are both structural and functional mimics of human collagen.…”

Bioengineered collagens

“…28 The few collagen-like structures from pathogenic species that have been well studied suggest that the collagen motif is typically associated with the outer membrane of the bacteria, and may interact with the host to assist invasion or to help a pathogen evade the host immune system. 28 This binding can be, for example, to integrin receptors 29 or to other extracellular matrix molecules, mediating pathogen internalization by human cells. 30 The identification of this new group of collagens provides potential for development of new, recombinant biomedical materials.…”