Molecular basis of the K:6,-7 [Js(a+b−)] phenotype in the Kell blood group system

Endothelin-1 (ET-1), a 21-residue vasoactive peptide, is produced in vascular endothelial cells from the 38-residue inactive intermediate big endothelin-1 via a specific cleavage at Trp-21-Val-22. The protease that catalyzes the conversion, endothelin-converting enzyme (ECE), constitutes a potential regulatory site for the production of the active peptide. We report the identification of ECE-1, a novel membrane-bound neutral metalloprotease that is expressed abundantly in endothelial cells in vivo and is structurally related to neutral endopeptidase 24.11 and Kell blood group protein. When transfected into cultured cells that normally secrete only big ET-1, the ECE-1 cDNA conferred the ability to secrete mature ET-1. In transfected cells, ECE-1 processes endogenously synthesized big ET-1 as well as exogenously supplied big ET-1, which interacts with ECE-1 on the cell surface. ECE-1 may provide a target for pharmacological intervention to alter ET-1 production.

The Kell blood group is a major antigenic system in human erythrocytes. Kell antigens reside on a 93-kDa membrane glycoprotein that is surface-exposed and associated with the underlying cytoskeleton. We isolated tryptic peptides and, based on the amino acid sequence of one of the peptides and by using the PCR, prepared a specific oligonucleotide to screen a lambda gt10 human bone-marrow cDNA library. Four clones were isolated, one containing cDNA with an open reading frame for an 83-kDa protein. All known Kell amino acid sequences were present in the deduced sequence; moreover, rabbit antibody to a 30-amino acid peptide, prepared from this sequence, reacted on an immunoblot with authentic Kell protein. The Kell cDNA sequence predicts a 732-amino acid protein. Hydropathy analysis indicates a single membrane-spanning region, suggesting that Kell protein is oriented with 47 of its N-terminal amino acids in the cell cytoplasm, and a 665-amino acid segment, which contains six possible N-glycosylation sites, is located extracellularly. Computer-based search showed that Kell has structural and sequence homology to a family of zinc metalloglycoproteins with neutral endopeptidase activity.