Multiple functions of the first EGF domain in matrilin-3: Secretion and endoplasmic reticulum stress.
Mutations in matrilin-3 are associated with common skeletal diseases, such as hand osteoarthritis (HOA), as well as rare chondrodysplasias, such as multiple epiphyseal dysplasia (MED) and spondyloepimetaphyseal dysplasia (SEMD). In the present study, we constructed the mutations R116W [at the von Willebrand factor, type A (vWFA) domain], T298M [at the first epidermal growth factor (EGF) domain] and C299S (at the first EGF domain), according to the mouse sequence, which are associated with human MED, HOA and SEMD, respectively, by overlap extension PCR and inserted them into an expression vector (pcDNA3.1/v5-His). We transfected these contructs into the COS-1 or MCT cells, and the results revealed that the HOA-related matrilin-3 mutation (T298M) leads to a high expression level of growth arrest DNA damage-inducible gene 153 (GADD153, also known as CHOP; an endoplasmic reticulum stress marker), as shown by western blot analysis and does not significantly affect protein secretion, as shown by immunofluorescence staining; however, osteochondroplasia, i.e., MED-related (R116W) and SEMD-related (C299S) mutations lead to both high levels of GADD153 expression and protein trafficking into the cytoplasm and form multiple vacuoles in cells, which in turn leads to insufficient protein secretion.
Department of Anesthesiology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China.
Yi-Chun
YC
Wang
Department of Critical Care Medicine, The Affiliated Cancer Hospital of Xiangya Medical School, Central South University, Changsha, Hunan 410013, P.R. China.
Jing-Shi
JS
Liu
Department of Critical Care Medicine, The Affiliated Cancer Hospital of Xiangya Medical School, Central South University, Changsha, Hunan 410013, P.R. China.
Jun-Yi
JY
Chen
Central Laboratory, The Affiliated Cancer Hospital of Xiangya Medical School, Central South University, Changsha, Hunan 410013, P.R. China.
Sheng-Qi
SQ
Wu
Department of Surgery, SUNY Upstate Medical University College of Medicine, Syracuse, NY 13210, USA.
Gui-Rong
GR
Wang
Department of Critical Care Medicine, The Affiliated Cancer Hospital of Xiangya Medical School, Central South University, Changsha, Hunan 410013, P.R. China.
Jing
J
Nie
Department of Pathology, Qinghai People's Provincial Hospital, Xining, Qinghai 810007, P.R. China.
Shu-Kun
SK
Zhang
Department of Anesthesiology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China.
Qu-Lian
QL
Guo
Department of Pathology, Qinghai People's Provincial Hospital, Xining, Qinghai 810007, P.R. China.
Jun-Ming
JM
Luo
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D059865:Endoplasmic Reticulum Stress / Q000235:genetics*; D006168:Guinea Pigs; D006801:Humans; D007719:Knee Joint / Q000378:metabolism; D064235:Matrilin Proteins / Q000235:genetics* / Q000378:metabolism; D008810:Mice, Inbred C57BL; D046529:Microscopy, Electron, Transmission; D050505:Mutant Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010003:Osteoarthritis / Q000235:genetics; D010009:Osteochondrodysplasias / Q000235:genetics; D021381:Protein Transport; D051742:Transcription Factor CHOP / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D064235:Matrilin Proteins; D050505:Mutant Proteins; D051742:Transcription Factor CHOP