Full-length recombinant choline transporter-like protein 2 containing arginine 154 reconstitutes the epitope recognized by HNA-3a antibodies.
BACKGROUND
Recent reports have shown that the HNA-3a leukocyte antigen, a target for antibodies that cause severe transfusion-related acute lung injury, correlates with an arginine 154 (rather than glutamine) polymorphism in choline transporter-like protein 2 (CTL2) but did not show directly that R154 determines HNA-3a. CTL2 peptides containing R154 are recognized by only half of HNA-3a antibodies studied to date. Constructs that react with all HNA-3a antibodies are needed to fully define the HNA-3a epitope.


METHODS
HEK293 cells were transfected with cDNA encoding full-length CTL2 linked to green fluorescent protein (GFP). Transfectants were selected for GFP expression and tested with antibodies specific for HNA-3a and -3b.


RESULTS
Each of 20 HNA-3a antibodies reacted preferentially with HEK293 cells expressing the R154 CTL2 construct. An HNA-3b antibody reacted only with CTL2 (Q154).


CONCLUSIONS
These findings provide direct evidence that R154 in the context of full-length CTL2 is both necessary and sufficient to create the HNA-3a epitope but suggest that posttranslational modifications of the protein, for example, S-S bonds or addition of glycans, are necessary for recognition of HNA-3a by many antibodies. This could complicate development of an assay for large-scale screening of blood donors to detect anti-HNA-3a.
Blood Research Institute, BloodCenter of Wisconsin, 8727 Watertown Plank Road, Milwaukee, WI 53226-3548, USA.
Adam J
AJ
Kanack
Julie A
JA
Peterson
Mia J
MJ
Sullivan
Daniel W
DW
Bougie
Brian R
BR
Curtis
Richard H
RH
Aster
D001120:Arginine; D001782:Blood Donors; D000939:Epitopes; D057809:HEK293 Cells; D006801:Humans; D007518:Isoantibodies / Q000276:immunology*; D007519:Isoantigens / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000276:immunology*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000276:immunology
D000939:Epitopes; C518134:HNA-3a antigen, human; D007518:Isoantibodies; D007519:Isoantigens; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C484524:SLC44A2 protein, human; D001120:Arginine