Analysis of an [Alpha]-Actinin-Like Protein That May Cross-Link Intermediate Filaments and Microfilaments
Date
1997-05
Authors
Bolanos, Sandra H.
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Abstract
We previously described a novel intermediate filament-associated protein (IFAP) designated the G.3.5 antigen (after the monoclonal antibody that recognizes it). This IFAP has been found in a variety of tissue types including human and rat astrocytes (Malhotra et al., 1984b), rat skeletal and cardiac myocytes (Price et al., 1993), fibroblasts (Malhotra et al,, 1984a), rat hepatocytes (Jeffcoat et al., 1995), and chicken (Garcia, personal communication) and fish retinal tissues (Zamora et al., 1994). The G.3.5 antigen has been purified from rat skeletal and cardiac muscle (Price et al., 1993) and from bovine skeletal muscle (Jeffcoat et al., 1995). The G.3.5 antigen is an IFAP that colocalizes with desmin in skeletal and cardiac myocytes. Because of sequence similarities to the actin-binding site of a-actinin, the G.3.5 antigen is thought to be an a-actinin-like protein which may cross link actin to intermediate filaments (Price et al., 1993). In this study, we have compared the G.3.5 antigen isolated from bovine skeletal muscle with a smooth muscle isofonn of a-actinin using biochemical analysis and binding assays. We find that cytoplasmic a actinin and the G.3.5 antigen migrate similarly but not identically in a nonreducing environment When both antigens are subjected to partial proteolysis by the V8 protease, similar fragments are generated. Overlay-immunoblotting assays indicate that cytoplasmic a-actinin and the G.3.5 antigen bind filamentous actin and desmin simultaneously. On the other hand, analysis of the G.3.5 antigen by SDS-PAGE (12% separating/4% stacking) results in a 100 kDa 'doublet' band, which is further resolved into a 114 kDa single band and a 107 kDa doublet when separated by 7%/4% SDS-PAGE; cytoplasmic a-actinin produces only a single band (107 kDa by 7%/4% SDS-PAGE). Although the G.3.5 mAB and anti-cytoplasmic a-actinin mAB cross-react with each other's antigens, anti-sarcomeric a-actinin mAB does not recognize cytoplasmic a-actinin but does react with the G.3.5 antigen, therefore indicating differences in their immunorecognition. These findings support the hypothesis that the G.3.5 antigen is a novel isofonn of a-actinin with binding sites for both actin and desmin and serves to cross-link the two cytoskeletal fibers.
Description
Keywords
proteins, crosslinking, antigens, cytoplasmic filaments
Citation
Bolanos, S.H. (1997). Analysis of an [alpha]-actinin-like protein that may cross-link intermediate filaments and microfilaments (Unpublished thesis). Southwest Texas State University, San Marcos, Texas.