EXPRESSION OF THE ALTERNATIVELY SPLICED EIIB SEGMENT OF FIBRONECTIN

Previous descriptions of the expression and distribution of the altern atively spliced EIIIB segment of fibronectin (FN) relied upon an antib ody which, on subsequent testing, was shown not to recognize this segm ent directly. This raises concerns regarding the reliability of all su ch previous descriptions. In order to prepare reagents directly reacti ve with this segment, we raised polyclonal antibodies to two different bacterial fusion proteins containing intact EIIIB segments, and to a synthetic 36 amino acid peptide from the center of the EIIIB segment. Antibodies raised to each of these three immunogens recognized fusion proteins containing the EIIIB segment, but failed to recognize full le ngth EIIIB(+) FNs produced by mammalian cells, suggesting that oligosa ccharide linked to Asn(139) within the EIIIB segment, or potentially t o other residues in FN, might interfere with antibody recognition of t his segment. Consistent with this hypothesis, N-deglycosylation of rec ombinant full and partial length EIIIB(+) FNs permitted their specific recognition by the anti-fusion protein (but not anti-peptide) antibod ies. Using anti-fusion protein antibodies coupled with deglycosylation procedures, we provide a series of new results relevant to the functi ons of the EIIIB segment: 1) Endogenously synthesized EIIIB(+) FN is i ncorporated into the extracellular matrix of cultured fibroblasts, whe re it appears by immunofluorescence microscopy and radioimmunoprecipit ation analyses to have a distribution very similar to both EIIIA(+) fo rms and the total pool of FNs. 2) No reproducible changes can be shown to occur in the extent of synthesis or matrix incorporation of EIIIB( +) FNs upon cellular transformation. 3) Low levels of EIIIB(+) FN are normally present in blood plasma and consequently also in purified pre parations of plasma FN. 4) EIIIB(+) FN is present in blood platelets, where it constitutes a minor fraction of total platelet FN, yet is gre ater than 4-fold enriched relative to plasma FN. 5) EIIIB(+) FN is syn thesized by first passage cultured endothelial cells, suggesting that the endothelium could constitute a source for this FN isoform in the c irculating blood. The antibodies and methods used in this study consti tute the first direct assays of EIIIB(+) FN protein expression and are applicable to a variety of species.