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

摘要

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 cardio- pulmonary diseases. Because glutamate and aspartate are consid- ered to be conservative replacements, the polymorphism was thought to be a marker for a functional locus elsewhere in the gene. We now show in trans fected cells, primary human endothe- lial cells, and human hearts, that eNOS with aspartate, but not glutamate, at position 298 is cleaved, resulting in the generation of 100-kDa and 35-kDa products. Recombinant or native eNOS was examined by immunoblotting either in lysates (COS7) or after partial purification over 2',5'-ADP-Sepharose and calmodu- lin-Sepharose. Immunoblotting after SDS/PAGE with a carboxyl- terminal antibody showed a single major protein band in the predicted position for eNOS 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- specific antibody, an immunoreactive band at approximately 35 kDa, corresponding to the residual N-terminal cleavage fragment, was observed in those cells with a T genotype. Thus, eNOS with aspartate but not glutamate at position 298 is cleaved, resulting in the generation of N-terminal 35-kDa and C-terminal 100-kDa frag- ments. Thus, the eNOS gene with polymorphisms at nucleotide 894 generates protein products with differing susceptibility to cleav- age. suggesting that. in contrast to prior predictions. this poly- morphism has a functional effect on the eNOS protein.