The functions of aminotelopeptide and N-terminal cross-linking of collagen I were examined. Acetic acidsoluble collagen I (ASC) was purified from neonatal bovine skin and treated with three kinds of proteases. The amino acid sequencing analysis of the N terminus showed that ASC contained a full-length aminotelopeptide. Pepsin and papain cleaved the aminotelopeptide of the ␣1 chain at the same site and the aminotelopeptide of the ␣2 chain at different sites. Proctase-treated ASC lost the whole aminotelopeptide, and the N-terminal sequence began from the tenth residue inside the triple helical region. The rates of fibril formation of pepsintreated ASC and proctase-treated ASC were the same and were slower than that of ASC. The denaturation temperatures, monitored by CD ellipticity at 221 nm, of ASC, pepsin-treated, or papain-treated collagens were the same at 41.8°C. Proctase-treated ASC showed a lower denaturation temperature of 39.9°C. We also observed the morphology of the collagen fibrils under an electron microscope. The ASC fibrils were straight and thin, whereas the fibrils of pepsin-treated ASC were slightly twisted, and the fibrils from papain-and proctase-treated ASC were highly twisted and thick. When the collagen gel strength was examined by a modified method of viscosity-measurement, ASC was the strongest, followed by pepsin-treated ASC, and papain-and proctase-treated ASCs were the weakest. These results suggest that the aminotelopeptide plays important roles in fibril formation and thermal stability. In addition, the functions of intermolecular cross-linking in aminotelopeptides may contribute to the formation of fibrils in the correct staggered pattern and to strengthening the collagen gel.The collagens of types I, II, III, and V/XI are grouped as fibrillar collagens, and collagens I, II, III and V are able to form fibrils in exactly a 67-nm staggered manner in vitro (1). The existence of collagen amino-and carboxytelopeptides in the collagen molecules accelerates the assembly of collagen fibrils (2) and are necessary for contraction of the collagen gel lattice by dermal cells (3). Collagens solubilized in acetic acid solution began to assemble fibrils on warming and neutralization of pH.The speed of fibril formation, the fibril thickness, and the fibril length were easily affected by pH, concentration of NaCl, and temperature (4, 5). The fibril diameter of type I collagen is reduced by the addition of type V collagen (6) or the existence of age-related cross-linking in the triple helical region (7). The fibril formation of acid-soluble collagen (ASC) 1 is faster than that of pepsin-treated ASC, and pepsin-treated ASC fibril is released from the fibrils to the solution again on incubation at low temperature (8). In fact, the pepsin-treated ASC have short telopeptides, because the molecular mass of pepsin-treated ASC is smaller than that of intact ASC by SDS-PAGE analysis (2, 3, 9). The telopeptide may affect the lag time and speed of fibril formation, but it is not known which portion of the telope...

Alkaline treatment is a good method for extracting collagen with high recovery even from an aged animal specimen. However, the properties of collagen treated under alkaline conditions have not been well established yet. By the treatment with a solution of 3% sodium hydroxide and 1.9% monomethylamine, the isoelectric point of type I collagen was lowered from 9.3 to 4.8 because of the conversions of Asn and Gln to Asp and Glu. With the acidification of the pI, the denaturation temperature of the collagen was decreased from 42 to 35 degrees C after 20 d treatment, but the collagen-specific triple helical conformation was maintained. Human keratinocytes and fibroblasts adhered to the alkali-treated collagen via the collagen receptor integrin alpha2beta1. This indicates that the alkali-treated collagen maintained its property as a biological adherent molecule. Unlike acid-soluble collagen, alkali-treated collagen lost the ability to form fibrils at neutral pH under physiological conditions. This ability was lost even after 4 h of alkaline treatment, when the denaturation temperature of the collagen did not change. On the other hand, the alkali-treated collagen formed a fibrous precipitate with a uniform diameter of 50-70 nm under acidic conditions at 30 degrees C.

In order to elucidate how chronic inflammation affects the organization of the extracellular matrix in the skin, a prolonged allergic contact dermatitis was induced in a mouse by repeated application to the ear of 2,4-dinitrofluorobenzene every 3 d for 66 d. Subsequently, the spatiotemporal changes of fibronectin, tenascin-C, fibulin-1, and fibulin-2 in the skin were examined. In the acute phase of inflammation (day 3-day 12), the amount of fibronectin and tenascin-C increased markedly and were degraded, whereas the amount of fibulin-2 changed slightly. Abundant deposition of tenascin-C was observed in the connective tissue. Fibulin-1 and fibulin-2 distributed as fine fibrils. In contrast, the amounts of fibronectin and tenascin-C decreased and their degradation was suppressed in the chronic phase (day 15-day 66), but the amount of fibulin-2 increased. Tenascin-C was observed mainly at and underneath the epidermal basement membrane. In the subepidermal region, many fibulin-2-positive microfibrils were distributed. The amount and distribution of fibulin-1 did not change markedly in either phase. MMP-like enzymes of 62 kDa, probably activated MMP-2, were upregulated in the chronic phase, whereas components of 92, 85, or 67 kDa were highly induced in the acute phase. These results suggest that chronic inflammation in allergic contact dermatitis is associated with temporal changes in the expression, deposition, and degradation of inducible extracellular matrix components.

No abstract

No abstract