Analysis of matrix protein components of the dermis-like structure formed in a long-term culture of human fibroblasts: type VI collagen is a major component.
Formation of a dermis-like structure by a long-term culture of fibroblasts in the presence of ascorbic acid is a potential model for tissue organization or wound healing, and has its practical use as a skin graft. In the present study, solubilization of the dermis-like structure without pepsin treatment was attempted for analysis of pepsin-labile matrix components that might be involved in the formation of the dermis-like structure, as well as quantification of mutated type I collagen that could be susceptible to pepsin. The whole dermis-like structure was dissolved in a Tris buffer containing SDS and urea at 80 degreesC. Analysis of the extract by SDS-PAGE revealed several protein bands that were not found in the pepsin-treated extract. Among them, the polypeptide band migrating at 140k under reducing condition showed a similar intensity of protein staining to the alpha2(I) chain band. The N-terminal amino acid sequences of cyanogen bromide peptides derived from the 140k polypeptide band as well as the amino acid composition of the band suggested that the band essentially consisted of alpha1(VI) and alpha2(VI) chains. The results demonstrated that the type VI collagen was a major component, being a comparable in amount to type I collagen, in the dermis-like structure.
Hazeki
N
N
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153.
Yamato
M
M
Imamura
Y
Y
Sasaki
T
T
Nakazato
K
K
Yamamoto
K
K
Konomi
H
H
Hayashi
T
T
D000293:Adolescent; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002478:Cells, Cultured; D003094:Collagen / Q000032:analysis*; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology; D006801:Humans; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000737:chemistry; D012867:Skin / Q000737:chemistry* / Q000166:cytology
D000596:Amino Acids; D016326:Extracellular Matrix Proteins; D003094:Collagen; D010434:Pepsin A