MYELOPEROXIDASE - ITS STRUCTURE AND EXPRESSION DURING MYELOID DIFFERENTIATION

  • 1 January 1985
    • journal article
    • research article
    • Vol. 65  (2) , 484-491
Abstract
Myeloperoxidase (MPO) is a major protein present in myeloid cells and is used by these cells to help kill microbes. The human promyelocytic HL-60 line can be induced to differentiate to granulocytes or macrophagelike cells. Poly (A) containing RNA was isolated from HL-60 granulocytes, HL-60 macrophages, HL-60 blasts, and normal human granulocytes. The mRNA was translated in a reticulocyte lysate system in the presence of 35S-methionine. The MPO was precipitated from the lysate with rabbit IgG antiserum to human MPO. The resulting precipitate from HL-60 blasts gave a major band of radioactivity of .apprx. 77,000 daltons and another band at .apprx. 46,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The MPO identity of the labeled bands was confirmed by cold competition. The relative mRNA activity expressed as a percentage of radioactivity incorporated into MPO (77,000-dalton band) as compared with total trichloracetic acid (TCA) precipitable radioactivity was 0.2%. Negligible mRNA activity for MPO was present in HL-60 granulocytes, HL-60 macrophages, and normal human granulocytes. Pulse-chase experiments showed that MPO was an .apprx. 75,000-dalton major band and 77,000-dalton minor band of radioactivity after HL-60 blasts were labeled for 1/2 h with 35S-methionine and the cell lysate immunoprecipitated and subjected to SDS-PAGE. The chase experiments (1-24 h) showed that the 77,000- and 75,000-dalton bands of radioactivity were replaced with 2 major bands (55,000 and 15,000 daltons) and 1 minor band (.apprx. 39,000 daltons) of radioactivity. Six-h 35S-methionine labeling experiments showed that the relative rate of MPO synthesis compared with total TCA precipitable radioactivity was 0.5% in HL-60 blasts and almost negligible in HL-60 macrophages and granulocytes, normal human granulocytes, and B-lymphocytes. The KG-1 myeloblasts and KG-1a early myeloblasts synthesized a small amount of the 75,000-dalton MPO protein. Although HL-60 cells no longer synthesized MPO after differentiation, HL-60 granulocytes and HL-60 macrophages continued to contain MPO as measured by enzyme activity. Native MPO apparently is a 135,000-dalton protein containing 2 heavy and 2 light chains of 55,000 and 15,000 daltons, respectively. An .apprx. 77,000- to 75,000-dalton polypeptide is the direct precursor of the 55,000- and 15,000-dalton polypeptides; synthesis of the 77,000- to 75,000-dalton polypeptide precursor of MPO begins at the myeloblast (KG-1, KG-1a) stage of development; promyelocytes (HL-60) synthesize MPO, and MPO synthesis ceases as the cells differentiate to granulocytes or macrophages; functional MPO mRNA becomes negligible with myeloid maturation.