title: Fibronectin from alpha 1,3-galactosyltransferase knockout pigs is a xenoantigen.
abstract: BACKGROUND
  Antibody-mediated rejection continues to be an obstacle for xenotransplantation despite development of α1,3-galactosyltransferase knockout (GTKO) pigs. Fibronectin (Fn) from GTKO pigs was identified as a xenoantigen in baboons. N-glycolylneuraminic acid (Neu5Gc), similar to galactose α1,3-galactose, is an antigenic carbohydrate found in pigs. We evaluated human antibody reactivity and performed initial antigenic epitope characterization of Fn from GTKO pigs.
  
  
  METHODS
  GTKO pig aortic endothelial cells (AEC) were isolated and assessed for antibody-mediated complement-dependent cytotoxicity (CDC). Human and GTKO pig Fn were purified and analyzed using immunoblots. GTKO pig and human AEC absorbed human sera were assessed for CDC and anti-GTKO pig Fn antibodies. GTKO pig proteins were assessed for Neu5Gc. Immunoaffinity-purified human IgG anti-GTKO pig (hIgG-GTKOp) Fn using a GTKO pig Fn column were evaluated for cross-reactivity with other proteins.
  
  
  RESULTS
  GTKO pig AEC had greater human antibody binding, complement deposition and CDC compared with allogeneic human AEC. Human sera absorbed with GTKO pig AEC resulted in diminished anti-GTKO pig Fn antibody. Neu5Gc was identified on GTKO pig Fn and other proteins. The hIgG-GTKOp Fn cross-reacted with multiple GTKO pig proteins and was enriched with anti-Neu5Gc antibody.
  
  
  CONCLUSIONS
  Removal of antigenic epitopes from GTKO pig AEC would improve xenograft compatibility. GTKO pig Fn has antigenic epitopes, one identified as Neu5Gc, which may be responsible for pathology and cross-reactivity of hIgG-GTKOp Fn. Genetic knockout of Neu5Gc appears necessary to address significance and identification of non-Neu5Gc GTKO pig Fn antigenic epitopes.
authors.affiliation: Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Ray K
authors.initials: RK
authors.lastname: Chihara
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Lutz
authors.forename: Leela L
authors.initials: LL
authors.lastname: Paris
authors.forename: Zheng-Yu
authors.initials: ZY
authors.lastname: Wang
authors.forename: Richard A
authors.initials: RA
authors.lastname: Sidner
authors.forename: Alex T
authors.initials: AT
authors.lastname: Heyrman
authors.forename: Susan M
authors.initials: SM
authors.lastname: Downey
authors.forename: Christopher
authors.initials: C
authors.lastname: Burlak
authors.forename: A Joseph
authors.initials: AJ
authors.lastname: Tector
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000906:Antibodies / Q000276:immunology; D015478:Antigens, Heterophile / Q000276:immunology*; D001011:Aorta / Q000166:cytology / Q000276:immunology; D002478:Cells, Cultured; D003429:Cross Reactions / Q000276:immunology; D004730:Endothelium, Vascular / Q000166:cytology / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D005353:Fibronectins / Q000276:immunology*; D005700:Galactosyltransferases / Q000172:deficiency* / Q000235:genetics*; D055786:Gene Knockout Techniques; D006801:Humans; D023421:Models, Animal; D013552:Swine / Q000235:genetics / Q000276:immunology*; D014183:Transplantation, Heterologous
chemical_list: D000906:Antibodies; D015478:Antigens, Heterophile; D000939:Epitopes; D005353:Fibronectins; D005700:Galactosyltransferases
keywords: Antibody-mediated rejection; Fibronectin; N-glycolylneuraminic acid; Neu5Gc; Xenoantigen; Xenotransplantation