부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.bbrc.2005.08.018 Byline: Hsiu-Chuan Li (a), Chuan-Chuan Huang (a), Sung-Fang Chen (b), Min-Yuan Chou (a) Keywords: FACIT; Collagen; Type XXI collagen; Minicollagen; Prolyl 4-hydroxylase; Drosophila melanogaster S2 cells Abstract: We established stably transfected insect cell lines containing cDNAs encoding the [alpha] and [beta] subunits of human prolyl 4-hydroxylase in both Trichoplusia ni and Drosophila melanogaster S2 cells. The expression level and enzymatic activity of recombinant prolyl 4-hydroxylase produced in the Drosophila expression system were significantly higher than those produced in the T. ni system. We further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three [alpha] chains to form trimeric type XXI minicollagen, which comprises the intact C-terminal non-collagenous (NC1) and collagenous domain (COL1), in the Drosophila system. When minicollagen XXI was stably expressed in Drosophila S2 cells alone, negligible amounts of interchain disulfide-bonded trimers were detected in the culture media. However, minicollagen XXI was secreted as disulfide-bonded homotrimers by coexpression with prolyl 4-hydroxylase in the stably transfected Drosophila S2 cells. Minicollagen XXI coexpressed with prolyl 4-hydroxylase contained sufficient amounts of hydroxyproline to form thermal stable pepsin-resistant triple helices consisting of both interchain and non-interchain disulfide-bonded trimers. These results demonstrate that a sufficient amount of active prolyl 4-hydroxylase is required for the assembly of type XXI collagen triple helices in Drosophila cells and the trimeric assembly is governed by the C-terminal collagenous domain. Author Affiliation: (a) Department of Applied Gene Technology, Biomedical Engineering Center, Industrial Technology Research Institute, Taiwan, ROC (b) Department of Proteomics, Biomedical Engineering Center, Industrial Technology Research Institute, Taiwan, ROC Article History: Received 12 July 2005 Article Note: (footnote) [star] Abbreviations: P4H, human prolyl 4-hydroxylase; P4H[alpha], human prolyl 4-hydroxylase [alpha] subunit; P4H[beta], human prolyl 4-hydroxylase [beta] subunit; FACIT, fibril-associated collagen with interrupted triple-helices; [alpha]1(XXI) collagen, [alpha]1-chain of type XXI collagen; kDa, kilodalton(s); mC21, type XXI minicollagen(s); MES, morpholineethanesulfonic acid; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight.