Expression and characterization of recombinant human eosinophil peroxidase. Impact of the R286H substitution on the biosynthesis and activity of the enzyme.
Hereditary eosinophil peroxidase deficiency is a genetic abnormality characterized by a decrease or absence of peroxidase activity and a reduction of the granule matrix volume. Recently, we identified two mutations associated with eosinophil peroxidase deficiency in a subject and his siblings, i.e. a base insertion causing the appearance of a premature stop codon and a base transition causing the replacement of an Arg at codon 286 with a His (R286H). In this article we report the stable expression of both the recombinant wild-type and the R286H eosinophil peroxidase precursor in the K-562 cell line, and the effects of the R286H substitution on the structure and function of the eosinophil peroxidase precursor. Heme group incorporation into both the recombinant wild-type and the recombinant R286H eosinophil peroxidase precursor was comparable, as was the stability of both proteins. Instead, the recombinant R286H eosinophil peroxidase precursor exhibited marked alterations of the catalytic properties and an increased sensitivity to four peroxidase inhibitors with respect to both the recombinant wild-type eosinophil peroxidase precursor and the native enzyme. In addition, the recombinant wild-type, but not the R286H, eosinophil peroxidase precursor was immunoprecipitated by two anti-(eosinophil peroxidase) mAbs. Altogether, our results suggest a protein misfolding of the R286H eosinophil peroxidase precursor which might account for its altered catalytic properties and the absence of expression of some epitopes.
Romano
M
M
International Center for Genetic Engineering an Biotechnology, Trieste, Italy. mromano@icgeb.trieste.it
Baralle
F E
FE
Patriarca
P
P
D019943:Amino Acid Substitution; D001560:Benzidines / Q000378:metabolism; D002460:Cell Line; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D047209:Eosinophil Peroxidase; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D010544:Peroxidases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D010655:Phenylenediamines / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D001560:Benzidines; D004792:Enzyme Precursors; D010655:Phenylenediamines; D011994:Recombinant Proteins; C021758:3,3',5,5'-tetramethylbenzidine; C034193:1,2-diaminobenzene; D006861:Hydrogen Peroxide; D047209:Eosinophil Peroxidase; D010544:Peroxidases