Lowering the culture temperature corrects collagen abnormalities caused by HSP47 gene knockout

Kazunori K Fujii,Shunji Hattori,Shinya Ito,Kazuhiro Nagata,Takaki Koide,Takayuki Sakai,Yuki Taga

doi:10.1038/s41598-019-53962-0

Kazunori K Fujii, Shunji Hattori + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-53962-0

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Abstract

Heat shock protein 47 (HSP47) is an endoplasmic reticulum (ER)-resident molecular chaperone that specifically recognizes triple helical portions of procollagens. The chaperone function of HSP47 is indispensable in mammals, and hsp47-null mice show an embryonic lethal phenotype accompanied by severe abnormalities in collagen-based tissue structures. Two leading hypotheses are currently accepted for the molecular function of HSP47 as a procollagen-specific chaperone. One is facilitation of procollagen folding by stabilizing thermally unstable triple helical folding intermediates, and the other is inhibition of procollagen aggregation or lateral association in the ER. The aim of this study was to elucidate the functional essence of this unique chaperone using fibroblasts established from hsp47−/− mouse embryos. When the cells were cultured at 37 °C, various defects in procollagen biosynthesis were observed, such as accumulation in the ER, over-modifications including prolyl hydroxylation, lysyl hydroxylation, and further glycosylation, and unusual secretion of type I collagen homotrimer. All defects were corrected by culturing the cells at a lower temperature of 33 °C. These results indicated that lowering the culture temperature compensated for the loss of HSP47. This study elucidated that HSP47 stabilizes the elongating triple helix of procollagens, which is otherwise unstable at the body temperature of mammals.

Highlights

Heat shock protein 47 (HSP47) is an endoplasmic reticulum (ER)-resident molecular chaperone that recognizes triple helical portions of procollagens
The amount of HSP47 expressed in hsp47+/+ and −/− MEF clones cultured at 37 °C and 33 °C was compared by western blotting (Fig. 1a)
To investigate whether the band shifts found in hsp47−/− MEF clones were caused by over-modifications in the triple helical region of collagens, samples were digested with pepsin at 4 °C prior to separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Summary

Introduction

Heat shock protein 47 (HSP47) is an endoplasmic reticulum (ER)-resident molecular chaperone that recognizes triple helical portions of procollagens. Approximately one third of Xaa and Yaa positions are occupied by proline (Pro) and 4-hydroxyproline (4-Hyp) residues, respectively Such high content of imino acid residues and post-translational prolyl hydroxylations greatly contribute to stabilizing the triple helical structure[1]. Three pro-α-chains are first trimerized at the C-propeptide domain, followed by triple helix formation in the C to N direction[2] During this process, pro-α-chains receive significant modifications including hydroxylations of Pro and lysine (Lys) residues forming 3-hydroxyproline (3-Hyp) (at Xaa positions), 4-Hyp, and hydroxylysine (Hyl) residues (both at Yaa positions). HSP47 dissociates from procollagens in a pH-dependent manner at the cis-Golgi or ER-Golgi intermediate compartment (ERGIC), which is transported back to the ER by the ER-retrieval signal at its C-terminus[14,15,16,17]

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