Expression of the Alternatively Spliced EIIIB Segment of Fibronectin

Abstract

Previous descriptions of the expression and distribution of the alternatively spliced EIIIB segment of fibronectin (FN) relied upon an antibody which, on subsequent testing, was shown not to recognize this segment directly. This raises concerns regarding the reliability of all such previous descriptions. In order to prepare reagents directly reactive 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 length EIIIB⁺ FNs produced by mammalian cells, suggesting that oligosaccharide linked to Asn¹³⁵⁹ within the EIIIB segment, or potentially to other residues in FN, might interfere with antibody recognition of this segment. Consistent with this hypothesis, N-deglycosylation of recombinant full and partial length EIIIB⁺ FNs permitted their specific recognition by the anti-fusion protein (but not anti-peptide) antibodies. Using anti-fusion protein antibodies coupled with deglycosylation procedures, we provide a series of new results relevant to the functions of the EIIIB segment: 1) Endogenously synthesized EIIIB⁺ FN is incorporated into the extracellular matrix of cultured fibroblasts, where it appears by immunofluorescence microscopy and radioimmunoprecipitation analyses to have a distribution very similar to both EIIIA⁺ forms 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 preparations of plasma FN. 4) EIIIB⁺ FN is present in blood platelets, where it constitutes a minor fraction of total platelet FN, yet is greater than 4-fold enriched relative to plasma FN. 5) EIIIB⁺ FN is synthesized by first passage cultured endothelial cells, suggesting that the endothelium could constitute a source for this FN isoform in the circulating blood. The antibodies and methods used in this study constitute the first direct assays of EIIIB⁺ FN protein expression and are applicable to a variety of species.

Key Words:

• fibronectin
• alternative splicing
• epitopes
• extracellular matrix