Interaction between Collagen and the α2 I-domain of Integrin α2β1

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A panel of variants with alanine substitutions in the small loop of anthrax toxin protective antigen domain 4 was created to determine individual amino acid residues critical for interactions with the cellular receptor and with a neutralizing monoclonal antibody, 14B7. Substituted protective antigen proteins were analyzed by cellular cytotoxicity assays, and their interactions with antibody were measured by plasmon surface resonance and analytical ultracentrifugation. Residue Asp 683 was the most critical for cell binding and toxicity, causing an ϳ1000-fold reduction in toxicity, but was not a large factor for interactions with 14B7. Substitutions in residues Tyr 681 , Asn 682 , and Pro 686 also reduced toxicity significantly, by 10 -100-fold. Of these, only Asn 682 and Pro 686 were also critical for interactions with 14B7. However, residues Lys 684 , Leu 685 , Leu 687 , and Tyr 688 were critical for 14B7 binding without greatly affecting toxicity. The K684A and L685A variants exhibited wild type levels of toxicity in cell culture assays; the L687A and Y688A variants were reduced only 1.5-and 5-fold, respectively.Bacillus anthracis secretes two toxins: edema toxin and lethal toxin. Each is composed of a common binding component, protective antigen (PA), 1 together with an enzymatic component, edema factor (EF), in the case of edema toxin and lethal factor (LF) in the case of lethal toxin (1-3). The current model for toxin entry into the cell illustrates the centrality of PA for toxin action. PA binds to cellular receptors, recently identified as splice variants of either tumor endothelial marker 8 (TEM8) (4 -6) or the closely related capillary morphogenesis protein 2 (CMG2) (7). Furin cleaves PA, releasing a 20-kDa fragment and leaving behind a 63-kDa portion (PA 63 ) capable of forming a heptamer, which has a newly exposed surface that binds . Heptamer complexes enter the endocytic pathway by receptor-mediated endocytosis (13), and upon acidification of the vesicle, the PA 63 heptamer undergoes a conformational change to form a pore through which EF and LF translocate into the cytoplasm (10, 11, 14 -16). Once in the cytoplasm, EF and LF exert their toxic effects.The PA protein can be divided into four domains based on its crystal structure, and functions can be attributed to the different domains based on mutational and biochemical analyses (16). Domain 1 (residues 1-258) contains the furin cleavage site as well as the hydrophobic portion of PA, which is exposed upon furin cleavage to allow EF and LF to bind (16,17). Several lines of evidence indicate that domain 2 (residues 259 -487) is involved in oligomerization and contains the loop that inserts into the membrane to form the channel through which the LF and EF enter the cytosol (16, 18 -20). Various amino acids in domain 3 (residues 488 -595) are necessary for oligomerization, and this has been the only function attributed to domain 3 to date (21,22). Domain 4 (residues 596 -735) is essential for binding to cellular receptor as indicated by several lines of...

Section: Discussionmentioning

confidence: 99%

Alanine-scanning Mutations in Domain 4 of Anthrax Toxin Protective Antigen Reveal Residues Important for Binding to the Cellular Receptor and to a Neutralizing Monoclonal Antibody

Rosovitz

Schuck

Varughese

et al. 2003

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136

“…Thus, antibodies that block ␣ 2 ␤ 1 interaction with collagen recognize epitopes within the I domain (6 -8). Three groups have shown that recombinant ␣2-I domain fusion proteins bind specifically to collagen in a divalent cation-dependent manner (9 -11), and a fourth group has presented conflicting data suggesting that collagen binding is independent of divalent cation (12).…”

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confidence: 99%

“…In the crystal structure (14), the side chains of residues Ser-153, Ser-155, and Asp-254 directly coordinate a Mg 2ϩ ion, and Asp-151 and Thr-221 provide water-mediated bonds. One study reported that mutation of residues Asp-151, Thr-221, or Asp-254 abolishes adhesion of the intact integrin ␣ 2 ␤ 1 to collagen (12), whereas of these three residues only Thr-221 is absolutely critical for the binding of the recombinant glutathione S-transferase-␣2-I fusion protein to collagen.…”

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confidence: 99%

Mapping the Collagen-binding Site in the I Domain of the Glycoprotein Ia/IIa (Integrin α2β1)

Smith¹,

Estavillo²,

Emsley³

et al. 2000

Self Cite

The I domain present within the ␣2 chain of the integrin ␣ 2 ␤ 1 (GPIa/IIa) contains the principal collagenbinding site. Based on the crystal structure of the ␣2-I domain, a hypothetical model was proposed in which collagen binds to a groove on the upper surface of the I domain (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). We have introduced point mutations into 13 residues on the upper surface of the domain. Recombinant mutant proteins were assayed for binding to monoclonal antibodies 6F1 and 12F1, to collagen under static conditions, and for the ability to retain adhesive activity under flow conditions. The mutations to residues surrounding the metal ion-dependent adhesion site that caused the greatest loss of collagen binding under both static and flow conditions are N154S in the ␤A-␣1 turn, N190D in the ␤B-␤C turn, D219R in the ␣3-␣4 turn, and E256V and H258V in the ␤D-␣5 turn. Mutation in one of the residues that coordinate the metal binding, S155A, completely lost the adhesive activity under flow but bound normally under static conditions, whereas the mutation Y285F had the converse effect. We conclude that the upper surface of the domain, including the metal ion-dependent adhesion site motif, defines the collagen recognition site. The glycoprotein (GP)1 Ia/IIa (integrin ␣ 2 ␤ 1 ) is a major platelet adhesion receptor for collagen. Integrins are a family of heterodimeric cell adhesion molecules that mediate cell-cell and cell-matrix adhesion (1). The integrin ␣ 2 ␤ 1 is expressed on several different cell types. Although it is a collagen receptor in platelet and fibroblastic cells, it functions both as a collagen and laminin receptor on endothelial and epithelial cells (2, 3). It also acts as the receptor for the human pathogen echovirus-1 (4).␣ 2 ␤ 1 is composed of a 150-kDa ␣2 and a 130-kDa ␤1 subunit (5). Within the ␣2 subunit, the 200-amino acid I domain shares homology with the A domains of von Willebrand factor, complement proteins, cartilage matrix protein, and certain other integrins. There is increasing evidence that I (A) domains play an important role in cell-adhesion protein and cell-matrix interaction.Within the ␣ 2 ␤ 1 integrin, the I domain (amino acids 140 -349) provides the principal binding site for collagen. Thus, antibodies that block ␣ 2 ␤ 1 interaction with collagen recognize epitopes within the I domain (6 -8). Three groups have shown that recombinant ␣2-I domain fusion proteins bind specifically to collagen in a divalent cation-dependent manner (9 -11), and a fourth group has presented conflicting data suggesting that collagen binding is independent of divalent cation (12).Like other I domains, the ␣2-I domain contains a cationbinding site called the metal ion-dependent adhesion site (MI-DAS) motif (13). In the crystal structure (14), the side chains of residues Ser-153, Ser-155, and Asp-254 directly coordinate a Mg 2ϩ ion, and Asp-151 and Thr-221 provide water-mediated bonds. One study reported that mutation of residues A...

“…Both ␣ 2 ␤ 1 and VWF bind to collagen through their I-domains, in VWF known as A-domains (13)(14)(15)(16)(17)(18)(19). A-domains form independent globular modules of some 200 amino acid residues.…”

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confidence: 99%

A Consensus Tetrapeptide Selected by Phage Display Adopts the Conformation of a Dominant Discontinuous Epitope of a Monoclonal Anti-VWF Antibody That Inhibits the von Willebrand Factor-Collagen Interaction

Vanhoorelbeke

Depraetere

Romijn

et al. 2003

Monoclonal antibody (mAb) 82D6A3 is an anti-vonIn conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.Platelet adhesion to subendothelial structures, more specifically to the thrombogenic compound collagen, is one of the first steps in a sequence of reactions that can lead to arterial thrombosis. Platelets interact with collagen both in a direct manner via their collagen receptors (e.g. ␣ 2 ␤ 1 (1, 2) and glycoproteins IV (3) and VI (4, 5)) and indirectly with VWF, 1 forming the bridge between collagen and its platelet receptor glycoprotein Ib/IX/V (6). Binding via both ␣ 2 ␤ 1 and VWF is necessary to sustain platelet adhesion under high shear forces (7-9); VWF-mediated interaction results in rolling of the platelets over the collagen surface (10), upon which the collagen receptors can interact with the damaged vessel wall, leading to firm adhesion. This is the result of platelet activation by the signal-transducing glycoprotein VI (11), leading to a gain-in-affinity of ␣ 2 ␤ 1 (12) and activation of ␣ IIb ␤ 3 with platelet aggregation as a consequence.Both ␣ 2 ␤ 1 and VWF bind to collagen through their I-domains, in VWF known as A-domains (13-19). A-domains form independent globular modules of some 200 amino acid residues. In VWF three such domains have been identified. The A1-domain contains the binding site for glycoprotein Ib (20,21), sulfatides (22), heparin (23), and collagen VI (24, 25), which constitutes the main reactive collagen in the extracellular matrix of endothelial cells. The A2-domain has no clear binding function but is sensitive to protease ADAMTS13-mediated enzymatic degradation (26,27), whereas the A3-domain (residues 920 -1111) contains the main binding site for fibrillar collagens such as type I and III (19,28). Recombinant A3-domain also binds to collagen (28), whereas deletion of A3 results in a VWF that binds 40 times less to collagen (19). By using synthetic triple helical collagen-related peptides, the VWF-binding site has been localized to residues 541-558 of the ␣1CB4(III) fragment of collagen type III (29). Recently, we identified the collagen binding site by cocrystallization of the A3-domain with an inhibitory anti-A3 antibody, RU5, which was confirmed by showing that especially an H1023A mutant abolished binding of VWF to collagen (30). This study was further extended by the analysis of a series of 27 VWF-A3 mutants, which defined the collagen binding site of the VWF-A3-domain to the "front" face of the domain (31), an observation confirmed by Nishida et al. (32).We raised a monoclonal antibody (mAb), 82D6A3, against human VWF that prevents the binding of VWF to collagen (24) and that is antithrombotic in a baboon arterial thrombosis model (33). Since a previous effort to determine the epitope of 82D6A3 using phage display, was not successful (34, 35), we repeated this study us...