Biochemical and Immunologic Analysis of Hereditary Myeloperoxidase Deficiency

William M. Nauseef; Richard K. Root; Harry L. Malech

doi:10.1172/JCI110880

Myeloperoxidase (MPO), a heme enzyme present in the azurophilic granules of human polymorphonuclear neutrophils (PMN), is important in the oxygen-dependent microbicidal activity of PMN. MPO deficiency, defined as the lack of PMN peroxidative activity, is a common genetic defect of human PMN. The purpose of our study was to characterize the structural basis for this loss of enzymatic activity, using protein biochemical and immunochemical techniques to examine PMN from three subjects with partial MPO deficiency and from five subjects with complete MPO deficiency.

We purified MPO from normal PMN and defined its electrophoretic mobility after two-dimensional electrophoretic separation, using nondenaturing acidic polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecyl sulfate (SDS) denaturation and SDS-PAGE separation of MPO subunit peptides. In agreement with previous studies, we found that normal MPO had subunits of 59,000 and 13,500 mol wt when subjected to SDS-PAGE under reducing conditions.

Granule protein extracts of normal PMN, partially MPO-deficient PMN, and completely MPO-deficient PMN were analyzed with two-dimensional PAGE. Partially MPO-deficient PMN granules contained electrophoretically normal MPO in less than normal amounts, whereas completely MPO-deficient PMN granules contain no protein with the electrophoretic mobility of normal MPO.

Using rabbit antiserum against purified MPO, we used immunoautoradiographic analysis to examine whole PMN for peptides immunochemically related to MPO. PMN from normal, partially MPO-deficient, and completely MPO-deficient subjects were solubilized in SDS and component peptides separated by SDS-PAGE. The peptides were electroblotted onto nitrocellulose paper that was exposed sequentially to rabbit anti-MPO and ¹²⁵I-protein A before autoradiography. Radiolabeled bands were identical when partially purified MPO or normal PMN were compared except that whole PMN contained a small amount of an immunologically cross-reactive membrane associated material of 75,000-90,000 mol wt. Using a modification of this immunoautoradiographic analysis, we quantitated the relative amounts of MPO peptides in PMN. PMN from MPO-deficient subjects contain 41.0-52.3% the amount of MPO peptides present in normal PMN. Similar analysis showed that completely MPO-deficient PMN lacked any peptides corresponding to MPO peptides.

We conclude that partial MPO deficiency is characterized by the presence of electrophoretically and immunologically normal MPO in amounts approximately one-half that seen in PMN from normal subjects. Completely MPO-deficient PMN lack any normal MPO peptides. No MPO-deficient subject studied had an immunologically cross-reacting variant of MPO. Since this deficiency is associated with the absence of more than one peptide, it is possible that the underlying genetic defect may involve: (a) failure to synthesize a single precursor peptide; (b) abnormal regulation of the synthesis of two separate peptides; or (c) an aberration in postsynthetic processing or packaging into azurophilic granules.

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