Possible involvement of aminotelopeptide in self-assembly and thermal stability of collagen I as revealed by its removal with proteases

The functions of aminotelopeptide and N-terminal cross-linking of collagen I were examined. Acetic acid-soluble collagen I (ASC) was purified from neo natal bovine skin and treated with three kinds of proteases, The amino acid sequencing analysis of the N terminus showed that ASC contained a full-len gth aminotelopeptide. Pepsin and papain cleaved the aminotelopeptide of the alpha 1 chain at the same site and the aminotelopeptide of the alpha 2 cha in at different sites. Proctase-treated ASC lost the whole aminotelopeptide , and the N-terminal sequence began from the tenth residue inside the tripl e helical region. The rates of fibril formation of pepsin-treated ASC and p roctase-treated ASC were the same and were slower than that of ASC. The den aturation temperatures, monitored by CD ellipticity at 221 nm, of ASC, peps in-treated, or papain-treated collagens were the same at 41.8 degrees C, Pr octase-treated ASC showed a lower denaturation temperature of 39.9 degrees C. We also observed the morphology of the collagen fibrils under an electro n microscope. The ASC fibrils were straight and thin, whereas the fibrils o f pepsin-treated ASC were slightly twisted, and the fibrils from papain- an d proctase-treated ASC were highly twisted and thick. When the collagen gel strength was examined by a modified method of viscosity-measurement, ASC w as the strongest, followed by pepsin-treated ASC, and papain- and proctase- treated ASCs were the weakest. These results suggest that the aminotelopept ide plays important roles in fibril formation and thermal stability. In add ition, the functions of intermolecular cross-linking in aminotelopeptides m ay contribute to the formation of fibrils in the correct staggered pattern and to strengthening the collagen gel.