Acidic Hydrolysis as a Mechanism for the Cleavage of the Glu298 → Asp Variant of Human Endothelial Nitric-oxide Synthase

Todd A. Fairchild, David J Fulton, Jason T. Fontana, Jean Philippe Gratton, Timothy J. McCabe, William C. Sessa

Research output: Contribution to journalArticle

137 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)26674-26679
Number of pages6
JournalJournal of Biological Chemistry
Volume276
Issue number28
DOIs
StatePublished - Jul 13 2001
Externally publishedYes

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Nitric Oxide Synthase Type III
Viperidae
Hydrolysis
Polymorphism
aspartyl-proline
Exons
Cardiovascular Diseases
Enzyme activity
Enzymes
Aspartic Acid
Glutamic Acid
Buffers
Genes
Cells
Temperature
Processing

ASJC Scopus subject areas

  • Biochemistry

Cite this

Acidic Hydrolysis as a Mechanism for the Cleavage of the Glu298 → Asp Variant of Human Endothelial Nitric-oxide Synthase. / Fairchild, Todd A.; Fulton, David J; Fontana, Jason T.; Gratton, Jean Philippe; McCabe, Timothy J.; Sessa, William C.

In: Journal of Biological Chemistry, Vol. 276, No. 28, 13.07.2001, p. 26674-26679.

Research output: Contribution to journalArticle

Fairchild, Todd A. ; Fulton, David J ; Fontana, Jason T. ; Gratton, Jean Philippe ; McCabe, Timothy J. ; Sessa, William C. / Acidic Hydrolysis as a Mechanism for the Cleavage of the Glu298 → Asp Variant of Human Endothelial Nitric-oxide Synthase. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 28. pp. 26674-26679.
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