Assembly of homotrimeric type XXI minicollagen by coexpression of prolyl 4-hydroxylase in stably transfected Drosophila melanogaster S2 cells

Hsiu Chuan Li, Chuan Chuan Huang, Sung Fang Chen, Min Yuan Chou

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

We established stably transfected insect cell lines containing cDNAs encoding the α and β 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 α 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.

Original languageEnglish
Pages (from-to)375-385
Number of pages11
JournalBiochemical and Biophysical Research Communications
Volume336
Issue number2
DOIs
Publication statusPublished - 2005 Oct 21

Keywords

  • Collagen
  • Drosophila melanogaster S2 cells
  • FACIT
  • Minicollagen
  • Prolyl 4-hydroxylase
  • Type XXI collagen

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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