Identification of Two Amino Acids within the EIIIA (ED-A) Segment of Fibronectin Constituting the Epitope for Two Function-blocking Monoclonal Antibodies

Abstract

Alternative splicing of the fibronectin gene transcript gives rise to a group of adhesive glycoproteins showing restricted spatial and temporal expression during embryonic development, tumor growth, and tissue repair. Alternative splicing occurs in three segments termed EIIIB, EIIIA, and V. The EIIIA (or ED-A) segment of fibronectin is expressed prominently but transiently in healing wounds coincident with fibroblast expression of an activation marker, smooth muscle cell ␣-actin. A monoclonal antibody (IST-9) to the EIIIA segment blocks transforming growth factor-␤-mediated smooth muscle cell ␣-actin expression by fibroblasts in culture. A second monoclonal antibody (DH1) blocks chondrocyte condensation in chicken embryos. We find that IST-9 and DH1 react with human, rat, and chicken but not with mouse or frog EIIIA, suggesting that His 44 may be important for antibody binding. A series of deletion mutants of rat EIIIA, constructed as glutathione S-transferase fusion proteins, do not react with either IST-9, DH1, or a third monoclonal antibody (3E2 ) is sufficient to restore fully IST-9 binding and much of the activity of DH1 and 3E2. Our findings demonstrate that the function-blocking antibodies, IST-9 and DH1, bind to the Ile 43 and His 44 residues in a conformationally dependent fashion, implicating the loop region encompassing both residues as critical for mediating EIIIA function.The fibronectins (FNs) 1 comprise a group of extracellular matrix proteins that mediate cell adhesion, migration, proliferation, and differentiation (1). FNs play significant roles in embryonic development and are prominent components of the provisional matrix following tissue injury in adults (2, 3). The fundamental importance of the FNs is substantiated by the observation that homozygous mutations in either the FN gene or in the ␣ 5 integrin, a FN-specific receptor, are lethal (4, 5). The FNs are disulfide-linked, dimeric glycoproteins with structural domains that bind cells, collagen, proteoglycans, and fibrin. Each FN consists of homologous repeats, either type I, II, or III. Individual type III repeats within FN exhibit high sequence similarity between species (greater than 90% identity (6)). Despite variations in protein sequence identity between different type III repeats within FN (20 -40% (6)), these repeats show a high degree of structural homology (7-11). X-ray crystallographic studies demonstrate that each type III repeat consists of two ␤ sheets, made up of four strands (G, F, C, CЈ) and three strands (A, B, E) respectively, folded into a ␤ sandwich (7). This structural arrangement is also conserved in other proteins, including growth hormone (12), tenascin (13), neuroglian (14), tissue factor (15), and chitinases (16).Diversity in the FNs occurs by alternative splicing in two type III repeats termed EIIIA (or ED-A) and EIIIB (or ED-B) and one non-homologous repeat called V (or IIICS) (1). The EIIIA and EIIIB segments are either entirely included or excluded, whereas the V region may be included, excluded, or partial...

The EIIIA Segment of Fibronectin Is a Ligand for Integrins α9β1 and α4β1Providing a Novel Mechanism for Regulating Cell Adhesion by Alternative Splicing

“…One integrin, ␣ 9 ␤ 1 , binds to a peptide sequence within the B-C loop of tenascin-C (34). This sequence (AEIDGIEL) is similar to the EDGIHEL sequence that we identified in the EIIIA segment (26). The ␣ 9 subunit binds unrelated sequences in other ligands including the vascular cell adhesion molecule-1 (VCAM-1) (35), osteopontin (36), the propolypeptide of von Willebrand factor (pp-vWF) (37), tissue transglutaminase (tTG) (37), blood coagulation factor XIII (FXIII) (37), and L1-CAM (38).…”

“…Protein-coated wells were washed with PBS and blocked with 1% BSA in PBS at 37°C for 1 h. Cells were harvested and resuspended in Hanks' buffered salt solution (HBSS) (10 6 Thrombin Cleavage of the GST-tagged EIIIA Segment-GST-tagged wild type and deletion mutants of the EIIIA segment were purified as previously described (26). Proteins were re-attached to 200 l of glutathione-agarose (50% slurry) in microfuge tubes at 4°C for 1 h with gentle agitation, followed by three washes with PBS.…”

“…Samples were then analyzed by Western blotting (26). Proteins were resolved in precast Tris glycine polyacrylamide gels (4 -20%) (Invitrogen) in duplicate.…”

“…We recently reported detailed epitope maps for functionblocking monoclonal antibodies that bind to the C-CЈ loop of the EIIIA segment (26). The FN type III (FN-III) repeats, of which the EIIIA segment is one, exhibit high structural homology (27)(28)(29)(30)(31) despite only 20 -40% identity in amino acid sequence (32).…”

“…The canonical FN type III repeat is a conserved ␤-sandwich conformation consisting of two ␤ sheets comprising four strands (G, F, C, CЈ) and three strands (A, B, and E) (27). Epitope mapping of the EIIIA segment reveals that functionblocking mAbs interact with the loop between the C and CЈ ␤-strands and the adjacent Ile 43 and His 44 residues are critical to the epitope (26). Given that these monoclonal antibodies blocked EIIIA function we reasoned that the peptide comprising the C-CЈ loop region (EDGIHEL) could encode a sequence that bound cell surface receptors, possibly integrins.…”