title: A novel epitope of CD59 expressed by primitive human hematopoietic progenitors.
abstract: OBJECTIVE
  The aim of this study was to determine the identity of the cell surface molecule on primitive hematopoietic cells recognized by monoclonal antibody HCC-1.
  
  
  METHODS
  Screening of a cDNA expression library prepared from human bone marrow stromal cells with HCC-1 yielded a single cDNA, which when expressed in FDCP-1 cells, resulted in the specific acquisition of HCC-1 binding. The cDNA demonstrated complete identity with CD59, a phosphoinositol glycan-linked membrane protein that protects cells against autologous complement attack. The ubiquitous expression of CD59 is in marked contrast to the restricted reactivity of HCC-1. Studies were performed to examine the basis for the novel specificity of HCC-1 for CD59. The epitope on CD59 identified by HCC-1 was mapped using a series of rat/human CD59 chimeric proteins. Immunoprecipitation analyses were performed to determine whether CD59 associates with other membrane proteins.
  
  
  RESULTS
  Mutagenesis of Asn18 did not alter the binding of HCC-1 to CD59, suggesting that N-linked carbohydrates are not responsible for the binding specificity of HCC-1. The epitope for HCC-1 was shown to differ from that identified by previously described CD59 antibodies, encompassing residues A31, L33, R55, and L59. An 80 kDa protein co-immunoprecipitated with CD59 in the HCC-1(-) cell line HL-60 but not in HCC-1(+) K562 cells.
  
  
  CONCLUSIONS
  Collectively, these data support the hypothesis that the unique specificity of HCC-1 for CD59 is due in part to recognition of a novel epitope, which is masked as a result of association with an as yet unidentified 80 kDa protein.
authors.affiliation: Stem Cell Laboratory, The Peter MacCallum Cancer Institute, East Melbourne, Victoria, Australia. p.simmons@pmci.unimelb.edu.au
authors.forename: P J
authors.initials: PJ
authors.lastname: Simmons
authors.forename: A C
authors.initials: AC
authors.lastname: Zannettino
authors.forename: D
authors.initials: D
authors.lastname: Harrison-Findik
authors.forename: B
authors.initials: B
authors.lastname: Swart
authors.forename: S
authors.initials: S
authors.lastname: Tomlinson
authors.forename: B
authors.initials: B
authors.lastname: Hill
authors.forename: J A
authors.initials: JA
authors.lastname: Javni
mesh_terms: D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology; D001798:Blood Proteins / Q000235:genetics; D001854:Bone Marrow Cells / Q000166:cytology / Q000276:immunology; D018957:CD59 Antigens / Q000235:genetics*; D019742:Chemokines, CC / Q000235:genetics; D000939:Epitopes / Q000032:analysis*; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D018448:Models, Immunological; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics; D015183:Restriction Mapping; D017154:Stromal Cells / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D001798:Blood Proteins; C097720:CCL14 protein, human; D018957:CD59 Antigens; D019742:Chemokines, CC; D000939:Epitopes; D011994:Recombinant Proteins