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

Abstract

Many pathogenic bacteria interact with human integrins to enter host cells and to augment host colonization. Group A Streptococcus (GAS) employs molecular mimicry by direct interactions between the cell surface streptococcal collagen-like protein-1 (Scl1) and the human collagen receptor, integrin ␣ 2 ␤ 1 . The collagen-like (CL) region of the Scl1 protein mediates integrin-binding, although, the integrin binding motif was not defined. Here, we used molecular cloning and site-directed mutagenesis to identify the GLPGER sequence as the ␣ 2 ␤ 1 and the ␣ 11 ␤ 1 binding motif. Electron microscopy experiments mapped binding sites of the recombinant ␣ 2 -integrin-inserted domain to the GLPGER motif of the recombinant Scl (rScl) protein. rScl proteins and a synthetic peptide harboring the GLPGER motif mediated the attachment of C2C12-␣ 2 ؉ myoblasts expressing the ␣ 2 ␤ 1 integrin as the sole collagen receptor. The C2C12-␣ 11 ؉ myoblasts expressing the ␣ 11 ␤ 1 integrin also attached to GLPGER-harboring rScl proteins. Furthermore, the C2C12-␣ 11 ؉ cells attached to rScl1 more efficiently than C2C12-␣ 2 ؉ cells, suggesting that the ␣ 11 ␤ 1 integrin may have a higher binding affinity for the GLPGER sequence. Human endothelial cells and dermal fibroblasts adhered to rScl proteins, indicating that multiple cell types may recognize and bind the Scl proteins via their collagen receptors. This work is a stepping stone toward defining the utilization of collagen receptors by microbial collagen-like proteins that are expressed by pathogenic bacteria.The integrins are a family of ␣␤ heterodimeric cell surface receptors found in metazoans that bind proteins of the extracellular matrix (ECM) and function in diverse cellular processes, including cell adhesion, spreading, and migration. There are currently 18 ␣ and 8 ␤ known subunits that combine to form 24 distinct heterodimers with various ligand binding specificities (1). One group of integrins binds several collagen types with varying affinities and includes the integrins ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , ␣ 10 ␤ 1 , and ␣ 11 ␤ 1 (2). The ␣ subunit of these integrins contains an inserted (I) 2 domain, and the ␣I domain confers binding specificity to the integrin heterodimer. Over the past decade much emphasis has been directed toward defining the parameters of collagen recognition by these integrins, as well as the specific amino acid sequences in collagens that are required for binding.The GFOGER (where O is hydroxyproline) sequence has been identified in some collagens as a major binding motif for the ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins (3, 4). However, not all collagen types harbor the GFOGER motif. Using synthetic collagen peptides, additional sequences with the base sequence GXOGER were proposed to bind the ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins with lower affinity as compared with the GFOGER sequence (5, 6). The hydroxylated proline residue of GXOGER is required for binding by the ␣ 1 ␤ 1 integrin but not by the ␣ 2 ␤ 1 integrin (7). Furthermore, the E residue of the GER triplet is essential for integ...

Disease Manifestations and Pathogenic Mechanisms of Group A Streptococcus

“…Focal adhesion kinase (FAK); integrin-linked kinase (ILK); Rac; CDC42; and the Src kinases Src, Yes, and Fyn have also been shown to be involved in ␣5␤1-mediated GAS internalization (144,156). Similarly, the collagen-like protein Scl1 can also mediate epithelial cell adherence and internalization by directly binding to integrins ␣2␤1 and ␣11␤1 through their collagen-like domains (159,160). Once internalized, some GAS strains are susceptible to autophagy following escape from endosomes into the cytoplasm (161,162).…”

Noncollagenous region of the streptococcal collagen‐like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains

“…6,[16][17][18][19][20] The collagen-like domain of Scl1, but not Scl2 can interact with the a2b1 integrin collagen receptor, promoting adherence to mammalian cells, internalization and re-emergence into the extracellular environment. [21][22][23][24] The Scl1 and Scl2 proteins can bind to thrombin-activatable fibrinolysis inhibitor, which may counteract the host response. 25 Both Scl1 and Scl2 related proteins have been expressed in E. coli, and their conformation, stability, and folding have been studied.…”

The Crystal Structure of the Streptococcal Collagen-like Protein 2 Globular Domain from Invasive M3-type Group A Streptococcus Shows Significant Similarity to Immunomodulatory HIV Protein gp41

“…Thus, some Scl1 variants may aid host colonization by binding to cellular fibronectin and laminin, which are major components of human extracellular matrix, and integrins ␣ 2 ␤ 1 and␣ 11 ␤ 1 , which are present on the host cell surface (24,31). Scl1 has also been shown to bind the plasma lipoproteins and complement regulators of the immune system (18,27,28).…”