title: Investigation of the influence of Arg555Trp and Thr538Pro TGFBI mutations on C-terminal cleavage and cell endoplasmic reticulum stress.
abstract: OBJECTIVE
  To gain insight into the mechanisms underlying the transforming growth factor-beta induced (TGFBI)-related corneal dystrophies and the influence of the Arg555Trp and Thr538Pro, TGFBI mutations on C-terminal cleavage and cell endoplasmic reticulum (ER) stress were investigated.
  
  
  METHODS
  The Arg555Trp and Thr538Pro mutations known to be associated with corneal dystrophy granular type I and lattice corneal dystrophy, respectively, were introduced with the two-sequential PCR site-directed mutagenesis technique. Wild-type and mutant TGFBI DNAs were cloned into the pcDNA3.1(-)/myc-his expression vector and overexpressed in HeLa and human corneal epithelial cells (HCE) with transient transfection. Transfection efficiency was measured by the expression of green fluorescent protein. Expression of the fusion proteins was measured with western blot analysis with anti-c-myc-tag and anti-TGFBI antibodies. For cell ER stress studies, the expression levels of GRP78/BiP in HeLa cells were analyzed with western blot analysis using an anti-GRP78 monoclonal antibody at 12, 24, and 48 h after either the wild-type or mutant plasmid was transfected.
  
  
  RESULTS
  Arg555Trp and Thr538Pro mutant TGFBIp were detected with the anti-c-myc and anti-TGFBI antibodies, while wild-type TGFBIp was detected only with the anti-TGFBI antibody, indicating that the Arg555Trp and Thr538Pro mutations prevent the C-terminal cleavage of TGFBIp. Moreover, no significant differences were seen in the expression levels of GRP78/BiP between the mutant and wild-type TGFBIp groups, suggesting that mutations in TGFBIp are unlikely to disrupt protein folding or induce cell ER stress.
  
  
  CONCLUSIONS
  This is the first time that the influence of TGFBI mutants on C-terminal cleavage and cell ER stress has been illustrated. Corneal dystrophy-related mutations are more likely to disrupt the interaction of TGFBI with critical binding proteins than affect the whole protein structure.
authors.affiliation: Department of Ophthalmology, Ophthalmic Genetic Research Centre, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
authors.forename: Miaomiao
authors.initials: M
authors.lastname: Zhu
authors.forename: Ping
authors.initials: P
authors.lastname: Yu
authors.forename: Bo
authors.initials: B
authors.lastname: Jiang
authors.forename: Yangshun
authors.initials: Y
authors.lastname: Gu
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D003315:Cornea / Q000166:cytology / Q000378:metabolism*; D003317:Corneal Dystrophies, Hereditary / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000235:genetics*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D059748:Proteolysis; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D016212:Transforming Growth Factor beta; D049452:Green Fluorescent Proteins