Specific immunological detection of the (V+C)(-) fibronectin isoform.

The population of fibronectins in adult mammalian cartilage includes high levels of a cartilage-specific (V+C)(-) isoform which lacks the V, III-15, and I-10 segments and thus contains a novel junction between protein segments III-14 and I-11. We report production of a monoclonal antibody specific for (V+C)(-) fibronectin without cross-recognition of V(+)C(+) and V(-)C(+) isoforms found in plasma and other tissues. Presentation of epitope to this antibody requires the III-14/I-11 junction, but the epitope itself extends beyond 14 amino acids immediately surrounding the junction site and involves a conformational change in III-14 and/or the N-terminal portion of I-11.

Absence of the I-10 protein segment mediates restricted dimerization of the cartilage-specific fibronectin isoform.

The cartilage-specific (V + C)(-) fibronectin isoform does not efficiently heterodimerize with other V-region splice variants of fibronectin. To understand better the structural elements that determine this restricted dimerization profile, a series of truncated fibronectin expression constructs with various internal deletions in the V, III-15, or I-10 segments were constructed and co-transfected into COS-7 cells with either the V(+)C(+) or the (V + C)(-) isoform. SDS-PAGE and immunoblot analyses of the resulting conditioned media suggest that the I-10 segment must either be present in both monomeric subunits of fibronectin or absent from both subunits for efficient dimerization to occur.