Targeting myeloperoxidase to azurophilic granules in HL‐60 cells

Myeloperoxidase (MPO) is a cationic protein and one of the major constituents of azuro- philic granules in neutrophils. Here, we examined whether intracellular transport of MPO and sergly- cin, a chondroitin sulfate (CS)-bearing proteogly- can, is correlated. First, we examined binding of MPO to CS-Sepharose and measured an ionic in- teraction, which was disrupted by 200-400 mM NaCl. Next, HL-60 promyelocytes were activated with a phorbol ester, which induced an almost com- plete rerouting of serglycin from the granular to the secretory pathway, concomitant with a similar effect on MPO transport and secretion. We then used the membrane-permeable cross-linker dithio- bis(succininmidylpropionate; DSP) after labeling HL-60 cells with ( 35 S)methionine and ( 35 S)cysteine for 19 h. Immunoprecipitation of MPO revealed its cross-linking to high molecular material having the appearance of a proteoglycan in sodium dodecyl sulfate-polyacrylamide gels. This assumption was confirmed by labeling HL-60 cells with ( 35 S)sulfate for 10 min followed by DSP cross-linking and im- munoprecipitation. From three granular enzymes immunoprecipitated, only the cationic MPO was cross-linked to ( 35 S)sulfate-labeled serglycin in ap- preciable quantities, whereas cathepsin D or ! -N- acetylhexosaminidase was not. Thus, intracellular transport of MPO appears to be linked to that of serglycin. Extracts from high buoyant density or- ganelles from human placenta containing MPO ac- tivity were subjected to CS-affinity chromatogra- phy. Proteins binding to CS were identified by mass spectrometry as MPO, lactoferrin, cathepsin G, and azurocidin/cationic antimicrobial protein of molecular weight 37 kDa, suggesting that serglycin may be a general transport vehicle for the cationic granular proteins of neutrophils. J. Leukoc. Biol. 74: 542-550; 2003.