Molecular basis of the neutrophil glycoprotein NB1 (CD177) involved in thepathogenesis of immune neutropenias and transfusion reactions

The human granulocyte alloantigen NB1, recently clustered as CD177, is hete rogenously expressed on neutrophils of 88-97% of healthy individuals. Since its molecular nature has remained unknown, we isolated NB1 glycoprotein fr om granulocyte lysate by immunoaffinity chromatography. MALDI-TOF mass spec trometry identified a 50,556 Da glycoprotein which was reduced to 43,069 Da after removal of N-linked carbohydrates. Following N-terminal amino acid s equencing and NB1-specific primer construction, rapid amplification of cDNA ends PCR yielded a 1,614-bp cDNA for NB1. COS-7 cells transfected with the cDNA expressed immunoreactive NB1 glycoprotein. A 1,311-bp sequence was id entified to be the entire coding region. The 5' and 3' untranslated regions consist of 27 bp and 276 bp, respectively. The open reading frame codes fo r 437 amino acids of which the first 21 form the signal peptide. The remain ing 416 residues form a N-terminal extracellular protein with two cysteine- rich domains, three N-linked glycosylation sites and short transmembrane an d cytoplasmic segments including a glycosyl-phosphatidylinositol attachment (w) site. Database searches revealed homology to Ly-6 (uPAR) domain, sugge sting that NBI belongs to urokinase plasminogen activator receptor/CD59/Ly- 6 snake toxin superfamily.