title: HNA-3a-specific antibodies recognize choline transporter-like protein-2 peptides containing arginine, but not glutamine at Position 154.
abstract: BACKGROUND
  Antibodies specific for the neutrophil antigen HNA-3a cause severe, sometimes fatal transfusion-related acute lung disease (TRALI) when transfused, but it has not been possible to screen blood donors for anti-HNA-3a because using neutrophils as targets was impractical and molecular properties of the antigen were unknown. Recently it was shown that HNA-3a is carried on choline transporter-like protein-2 (CTL2) and that the HNA-3a/b phenotype is closely correlated with an R154Q amino acid polymorphism in CTL2. However, it has not been shown by direct experiment that R154 is essential for the HNA-3a epitope.
  
  
  METHODS
  Preliminary attempts to express recombinant full-length CTL2 (R154) recognized by anti-HNA-3a were unsuccessful. We therefore tested HNA-3a-specific antibodies from donors implicated in TRALI reactions for reactivity against chemically synthesized linear and cyclic CTL2 peptides containing R154 or Q154.
  
  
  RESULTS
  Nine of 20 HNA-3a antibodies recognized the R154, but not the Q154 version of a cyclic 36-residue CTL2 peptide (D131-K166). However, 11 others failed to distinguish between the two versions of this peptide.
  
  
  CONCLUSIONS
  The findings provide direct evidence that R154 in the context of CTL2 D131-K166 is necessary to create the HNA-3a epitope but, in the context of cyclic CTL2 peptide D131-K166, is sufficient to detect only about one-half of the HNA-3a-specific antibodies implicated in TRALI. It is likely that fragments of CTL2 longer than can be made on a large scale with an automated synthesizer will be needed to produce a target capable of detecting all examples of anti-HNA-3a in donated blood.
authors.affiliation: Platelet & Neutrophil Immunology Lab, BloodCenter of Wisconsin, Milwaukee, Wisconsin 53201-2178, USA. brian.curtis@bcw.edu
authors.forename: Brian R
authors.initials: BR
authors.lastname: Curtis
authors.forename: Mia J
authors.initials: MJ
authors.lastname: Sullivan
authors.forename: M Trudy
authors.initials: MT
authors.lastname: Holyst
authors.forename: Aniko
authors.initials: A
authors.lastname: Szabo
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Bougie
authors.forename: Richard H
authors.initials: RH
authors.lastname: Aster
mesh_terms: D001120:Arginine / Q000737:chemistry*; D001803:Blood Transfusion; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes; D005973:Glutamine / Q000737:chemistry*; D006801:Humans; D007518:Isoantibodies / Q000737:chemistry / Q000276:immunology*; D007519:Isoantigens / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D026901:Membrane Transport Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*
chemical_list: D000939:Epitopes; C518134:HNA-3a antigen, human; D007518:Isoantibodies; D007519:Isoantigens; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; C484524:SLC44A2 protein, human; D005973:Glutamine; D001120:Arginine