A novel epitope of CD59 expressed by primitive human hematopoietic progenitors.
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.
Simmons
P J
PJ
Stem Cell Laboratory, The Peter MacCallum Cancer Institute, East Melbourne, Victoria, Australia. p.simmons@pmci.unimelb.edu.au
Zannettino
A C
AC
Harrison-Findik
D
D
Swart
B
B
Tomlinson
S
S
Hill
B
B
Javni
J A
JA
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
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D001798:Blood Proteins; C097720:CCL14 protein, human; D018957:CD59 Antigens; D019742:Chemokines, CC; D000939:Epitopes; D011994:Recombinant Proteins