TY - JOUR
T1 - Acidic Hydrolysis as a Mechanism for the Cleavage of the Glu298 → Asp Variant of Human Endothelial Nitric-oxide Synthase
AU - Fairchild, Todd A.
AU - Fulton, David
AU - Fontana, Jason T.
AU - Gratton, Jean Philippe
AU - McCabe, Timothy J.
AU - Sessa, William C.
PY - 2001/7/13
Y1 - 2001/7/13
N2 - 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 Asp298 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-Pro299) 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.
AB - 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 Asp298 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-Pro299) 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.
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U2 - 10.1074/jbc.M103647200
DO - 10.1074/jbc.M103647200
M3 - Article
C2 - 11331296
AN - SCOPUS:0035854834
SN - 0021-9258
VL - 276
SP - 26674
EP - 26679
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 28
ER -