Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: Cleavage of proteins with aspartate vs. glutamate at position 298

An endothelial nitric oxide synthase (eNOS) polymorphism in exon 7 (894 G/T ) resulting in glutamate or aspartate, respectively, at position 298 on the protein is correlated with severity of cardiopulmonary diseases. Because g lutamate and aspartate are considered to be conservative replacements, the polymorphism was thought to be a marker for a functional locus elsewhere in the gene, We now show in transfected cells, primary human endothelial cell s, and human hearts, that eNOS with aspartate, but not glutamate, at positi on 298 is cleaved, resulting in the generation of 100-kDa and 35-kDa produc ts, Recombinant or native eNOS was examined by immunoblotting either in lys ates (COS7) or after partial purification over 2',5'-ADP-Sepharose and calm odulin-Sepharose, Immunoblotting after SDS/PAGE with a carboxyl-terminal an tibody showed a single major protein band in the predicted position for eNO S at 135 kDa. An additional band at approximately 100 kDa was present only in the recombinant 298Asp eNOS and in the eNOS synthesized by primary cells and heart tissue with a G/T genotype, Using an eNOS amino-terminal-specifi c antibody, an immunoreactive band at approximately 35 kDa, corresponding t o the residual N-terminal cleavage fragment, was observed in those cells wi th a T genotype, Thus, eNOS with aspartate but not glutamate at position 29 8 is cleaved, resulting in the generation of N-terminal 35-kDa and C-termin al 100-kDa fragments. Thus, the eNOS gene with polymorphisms at nucleotide 894 generates protein products with differing susceptibility to cleavage, s uggesting that, in contrast to prior predictions, this polymorphism has a f unctional effect on the eNOS protein.