Acidic hydrolysis as a mechanism for the cleavage of the Glu(298)-->Asp variant of human endothelial nitric-oxide synthase.
The 894G-->T polymorphism within exon 7 of the human endothelial nitric-oxide synthase (eNOS) gene codes for glutamate or aspartate, respectively, at residue 298 and has been associated with several diseases of cardiovascular origin. A recent report indicates that Asp(298)-eNOS (E298D) is cleaved intracellularly to 100- and 35-kDa fragments, suggesting a mechanism for reduced endothelial function. Here we have documented the precise cleavage site of the E298D variant as a unique aspartyl-prolyl (Asp(298)--Pro(299)) bond not seen in wild-type eNOS (Glu(298)). We show that E298D-eNOS, as isolated from cells and in vitro, is susceptible to acidic hydrolysis, and the 100-kDa fragment can be generated ex vivo by increasing temperature at low pH. Importantly, cleavage of E298D was eliminated using a sample buffer system designed to limit acidic hydrolysis of Asp--Pro bonds. These results argue against intracellular processing of E298D-eNOS and suggest that previously described fragmentation of E298D could be a product of sample preparation. We also found that eNOS turnover, NO production, and the susceptibility to cellular stress were not different in cells expressing WT versus E298D-eNOS. Finally, enzyme activities were identical for the respective recombinant enzymes. Thus, intracellular cleavage mechanisms are unlikely to account for associations between the exon 7 polymorphism and cardiovascular diseases.
Fairchild
T A
TA
Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
Fulton
D
D
Fontana
J T
JT
Gratton
J P
JP
McCabe
T J
TJ
Sessa
W C
WC
D019943:Amino Acid Substitution; D001224:Aspartic Acid; D018698:Glutamic Acid; D006801:Humans; D006868:Hydrolysis; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D001224:Aspartic Acid; D018698:Glutamic Acid; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III