title: In vitro amyloid aggregate forming ability of TGFBI mutants that cause corneal dystrophies.
abstract: OBJECTIVE
  We investigated the in vitro amyloid aggregation ability of TGFBI (transforming growth factor beta-induced) mutants causing corneal dystrophies (CDs).
  
  
  METHODS
  Peripheral blood samples were collected from 42 unrelated Chinese CD patients and 185 healthy subjects for mutation screening in all TGFBI coding exons and flanking introns. The expression vector pCMV6_TGFBI containing wild-type, Arg-124, or Arg-555 mutations was transfected to HEK293 cells. Cell-free media was incubated with amyloid-beta (Aβ) (1-40) peptides with or without a chemical osmolyte, trimethylamine N-oxide (TMAO), for different time intervals. After ultracentrifugation, protein aggregates were analyzed by denatured gel electrophoresis. The effect of TMAO on chemical and morphological properties of Aβ aggregation was examined.
  
  
  RESULTS
  TGFBI sequencing analysis showed c.Arg124Cys in all 6 lattice CD patients, c.Arg555Glu in all 11 granular CD type 1 patients, and c.Arg124His in 22 of 25 granular CD type 2 patients. Double heterozygosity (c.307-308delCT and c.Arg124His) was detected in one GCD2 patient. After transfection, cell-free media containing Arg-124 TGFBIp led to Aβ aggregation within 12 hours, whereas wild-type and Arg-555 mutant displayed aggregation after 24 hours. Western blot and Congo red binding assays showed that TMAO dose-dependently suppressed Arg-124-induced Aβ aggregation. Transmission electron microscopy showed that TMAO reduced the fibrillar aggregates caused by Aβ and c.124R > H mutated TGFBIp.
  
  
  CONCLUSIONS
  TGFBI sequence heterogeneity was observed in Chinese CD patients. TMAO reduced amyloid aggregation caused by Arg-124 mutants, which suggests a potential chemical-based treatment for CDs.
authors: Department of Ophthalmology and Visual Science, The Chinese University of Hong Kong, Hong Kong. gary_yam@cuhk.edu.hk
authors: Gary Hin-Fai
authors: GH
authors: Yam
authors: Kaijie
authors: K
authors: Wang
authors: Vishal
authors: V
authors: Jhanji
authors: Kwong-Wai
authors: KW
authors: Choy
authors: Larry
authors: L
authors: Baum
authors: Chi-Pui
authors: CP
authors: Pang
mesh_terms: D000328:Adult; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D044466:Asian People; D002478:Cells, Cultured; D003317:Corneal Dystrophies, Hereditary / Q000235:genetics / Q000378:metabolism*; D058645:Denaturing Gradient Gel Electrophoresis; D006801:Humans; D008744:Methylamines / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017422:Sequence Analysis, DNA; D016212:Transforming Growth Factor beta / Q000235:genetics*
chemical_list: D016229:Amyloid beta-Peptides; D008744:Methylamines; D016877:Oxidants; D010446:Peptide Fragments; D016212:Transforming Growth Factor beta; C005855:trimethyloxamine