Abstract
The processing of type I procollagen is essential for fibril formation; however, the steps involved remain controversial. We constructed a live cell imaging system by inserting fluorescent proteins into type I pre-procollagen α1. Based on live imaging and immunostaining, the C-propeptide is intracellularly cleaved at the perinuclear region, including the endoplasmic reticulum, and subsequently accumulates at the upside of the cell. The N-propeptide is also intracellularly cleaved, but is transported with the repeating structure domain of collagen into the extracellular region. This system makes it possible to detect relative increases and decreases in collagen secretion in a high-throughput manner by assaying fluorescence in the culture medium, and revealed that the rate-limiting step for collagen secretion occurs after the synthesis of procollagen. In the present study, we identified a defect in procollagen processing in activated hepatic stellate cells, which secrete aberrant collagen fibrils. The results obtained demonstrated the intracellular processing of type I procollagen, and revealed a link between dysfunctional processing and diseases such as hepatic fibrosis.
Highlights
IntroductionCollagen is a primary component of the ECM and accounts for ~30% of the total protein in the body (Hall, 1964; Lehninger, 1975), making it the most essential protein
Collagen α-chains twist to form a triple helical structure, and are widely distributed in tissues and organs such as skin, the basement membrane, cartilage, tendons, muscle, and blood vessel walls. 28 types of collagens have been identified to date, and most are categorized into two classes: fibrillar and non-fibrillar collagens
These results suggested that the tagged collagen passed through the ER quality control system (Copito, 1997; Araki & Nagata, 2011) and was transferred into the cis- and trans-Golgi, which was supported by data showing that the expression of the ER stress marker CHOP was not up-regulated in NIH3T3 cells stably expressing Gr-CC (Fig S4)
Summary
Collagen is a primary component of the ECM and accounts for ~30% of the total protein in the body (Hall, 1964; Lehninger, 1975), making it the most essential protein. Most of the previous studies reported that N-pp and C-pp are enzymatically cleaved after secretion by procollagen N- and C-propeptidases, respectively, to form collagen (Prockop & Kivirikko, 1995; Brodsky & Ramshaw, 1997; Lamande & Bateman, 1999). The processing step of type I procollagen is essential in vertebrates, but has not yet been elucidated
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