Characterization of the Binding Domains in the Fragments Cleaved by Cathepsin G from Human Plasma Fibronectin

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Abstract
Purified plasma fibronectin was digested by the tissue proteinase cathepsin G isolated from human leukocytes. Using an enzyme‐substrate ratio of 1 : 200 (w/w), fibronectin was degraded to relatively stable peptides of Mr= 38000, 33500, 30000, 28500, 26000, 25000, and 15500 within 4‐8 h. These fragments contained distinct binding domains of the molecule as determined by exposing the peptides to Sepharose‐linked gelatin, arginine, spermidine, spermine and heparin. The bound material was analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The gelatin‐binding activity was located according to the kinetic analysis of the digestion to peptides of Mr= 64000, 40000, and 30000 appearing in that order. Previously uncharacterized domains, having affinity for amino‐containing compounds, were detected by allowing them to bind to Sepharoselinked arginine, spermidine or spermine. The final fragments binding to these molecules had Mr= 25000 and 15 500. The gelatin‐binding domain of fibronectin also bound to these ligands. Heparin‐Sepharose bound several of the 5‐h‐digestion peptides, one of which, with Mr= 28 500, also bound to Staphylococcus aureus and is, on the basis of other studies, located at the NH2 terminus of fibronectin [Mosher and Proctor (1980) Science (Wash. DC) 209, 927‐929]. This peptide was rich in intramolecular disulfide bonds as judged by comparison of the electrophoretic migration under nonreducing and reducing conditions. The results show that in fibronectin there are two distinct heparin‐binding domains. One is located near the COOH terminus of the molecule as described earlier [Ruoslahti et al. (1981) J. Biol. Chem. 256, 7277‐7281] and the other is, according to the present results, located NH2‐terminally between the S. aureus and gelatin‐binding regions. The peptides were eluted from the ligands with 100‐200 mM salt concentrations except for the gelatin‐binding fragment which resisted all concentrations tested (up to 3 M NaCI). In these conditions the peptides were bound generally more weakly than intact plasma fibronectin. In isoelectric focusing the gelatin, arginine and polyamine‐binding peptides had PI = 5.6‐6.2. The heparin‐binding peptides had a PI > 9 and were analyzed using two‐dimensional nonequilibrium pH‐gradient reverse electrophoresis. The binding properties of the fragments suggest an asymmetric cleavage of the subunit polypeptides of fibronectin so that the 64 000‐Mr, gelatin and heparin‐binding fragment is generated from one of the chains whereas the 40000‐Mr, gelatin‐binding and the 30000‐Mr, S. aureus and heparin‐binding fragments are derived from the other one.