Early and Late Cathepsin D-Derived Fragments of Fibronectin Containing theC-terminal Interchain Disulfide Cross-Link

Abstract

Fibronectin [human plasma] consists of 2 very similar subunits connected by S-S bonds close to their C-terminal ends. After short digestion with cathepsin D a fragment containing an intact subunit linked to a C-terminal piece of the other chain was isolated. Both possible combinations were realized. The remnant chain, removed by reduction, had a relative molecular mass (Mr) of 65,000 or 75,000, respectively, whether it originated from the shorter or the longer fibronectin subunit. After prolonged cathepsin D digestion another S-S-linked fragment of Mr 90,000 containing peptide chains of Mr 65,000 and Mr 36,000 was found. The same fragment also formed during prolonged digestion of a previously described cathepsin D-derived heparin-binding piece of Mr 140,000 which consisted of S-S-linked chains of Mr 65,000 and Mr 75,000. The cut-off material emerged as a mixture of 2 similar heparin-binding peptides of Mr 35,000 and Mr 37,000. The residual S-S-linked peptide of Mr 90,000 was retained by heparin-Sepharose, too, indicating that the non-degraded chain of Mr 65,000 also contained a heparin-binding site. Plasminolysis of the Mr 140,000 fragment first liberated the 2 individual chains and subsequently cleaved the longer one into 2 domains of nearly equal size. A rather basic one (pI [isoelectric point] 8.4-8.8) with low cystine content bound to heparin-Sepharose, while the other cystine-rich and more acidic domain (pI 5.0-5.8) was not retained. The shorter peptide chain, also showing affinity to heparin, was attacked by plasmin rather slowly. It, however, also appeared to be composed to 2 differently charged regions as it had an isoelectric point in the neutral range (pI 6.4-6.8) in spite of its heparin-binding properties. In the longer chain the heparin-binding domain was located in a distal position to the C-terminal disulfide link. The same is assumed for the shorter chain by reason of homology.