Abstract

We have identified the biological activity of three polypeptides released by limited proteolysis of human plasma fibronectin by leukocyte elastase. A Mr = 140,000 peptide contains cell-spreading activity; a Mr = 60,000 peptide mediates binding to denatured collagen (gelatin), and a Mr = 29,000 peptide contains glutaminyl residues responsible for the transglutaminase (blood coagulation factor XIIIa)-catalyzed incorporation of amines. More extensive proteolysis yielded numerous peptides, including a Mr = 40,000 peptide derived from the Mr = 60,000 peptide which retains gelatin-binding activity. Quantification of the gelatin-binding peptides is consistent with two binding sites per dimeric fibronectin molecule of Mr = 440,000. Both Mr = 60,000 and 40,000 gelatin-binding peptides were enriched with half-cystine residues, containing 28 and 25, respectively, but devoid of cysteine. This, coupled with the electrophoretic behavior of both peptides, was consistent with the presence of intramolecular disulfide bonds in the gelatin-binding domain. Intact fibronectin contains 1 free cysteine residue/monomer, as recently described. This cysteine reacts with 5,5'-dithiobis(2-nitrobenzoic acid) very slowly under nondenaturing conditions but rapidly when fibronectin is denatured. The free cysteine is located in the Mr = 140,000 peptide. While the Mr = 40,000 and 60,000 gelatin-binding peptides bind to gelatin with an affinity about 30-fold and 5-fold less than intact fibronectin (based on a monomeric fibronectin Mr = 220,000), neither gelatin-binding peptide supports spreading of fibronectin-deficient test cells on gelatin or tissue culture plastic substrates. The purified Mr = 140,000 peptide supported cell spreading on plastic, retaining about one-half of the spreading activity of intact fibronectin on a weight basis. These data confirm recent results, suggesting multiple, protease- resistant domains with discrete biological functions within fibronectin. Our results, together with established data, suggest a model for the location of the transglutaminase-reactive glutaminyl residues, gelatin binding, and cell-adhesive domains in fibronectin. The release of univalent, biologically active fibronectin fragments by elastase, a major physiologically released inflammatory protease of human leukocytes, suggests a new potential mechanism for alteration of cell connective tissue interactions at sites of inflammation in vivo.

Highlights

  • We have identified the biological activity of three sized by a variety of cultured cells

  • The common feature apparently underlying the diverse biological activities of fibronectin is the capacity to bind to extracellular macromolecules, including collagen (6, 7), heparin (8,9),hyaluronic acid (8),fibrin,and sulfated proteoglycans (11).The interaction of fibronectin with extracellular macromolecules in turn modifies cell behavior, possibly by bonds in thegelatin-binding domain

  • Identification and Characterization of HLE-released Gelatin-binding Peptides-Using the strategy of Hahn and Yamada (17), fibronectin was bound to gelatin-Sepharose, digested with HLE, and nonbinding and gelatin-binding peptides displayed by SDS-polyacrylamide gel electrophoresis

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Summary

Introduction

We have identified the biological activity of three sized by a variety of cultured cells. This, coupled with the electrophoretic behavior of both peptides, was consistent with the presence of intramolecular disulfide nectin share common structural organization and immunological identity, but therelationship between soluble, circulating plasma fibronectin (cold-insolubleglobulin)and sparingly soluble cellular fibronectin (cell surface or LETS protein) is unclear (1).The biological activities of fibronectin’ are extensive, including promotion of cell adhesion, mediation of cellcell and cell-extracellular matrix interactions, partial restoration of normal cell behavior to aberrant transformed cells deficient in fibronectin, opsonic activity for the reticuloendothelial system, and substrate for plasma transglutaminase Intact fibronectin direct binding of fibronectin to cell membrane receptors (12, contains 1free cysteine residue/monomer, as recently 13).In cultured cells,membrane binding of fibronectin appears described. This cysteine reacts with 5,5’-dithiobis(2-ni- to be concentrated at specialized sites (adhesion plaques), trobenzoic acid) very slowly under nondenaturingcon- providing a mechanism forlinking the intracellular contractile ditions but rapidly when fibronectin is denatured.

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