Targeting myeloperoxidase to azurophilic granules in HL-60 cells.
Myeloperoxidase (MPO) is a cationic protein and one of the major constituents of azurophilic granules in neutrophils. Here, we examined whether intracellular transport of MPO and serglycin, a chondroitin sulfate (CS)-bearing proteoglycan, is correlated. First, we examined binding of MPO to CS-Sepharose and measured an ionic interaction, which was disrupted by 200-400 mM NaCl. Next, HL-60 promyelocytes were activated with a phorbol ester, which induced an almost complete 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 dithiobis(succininmidylpropionate; DSP) after labeling HL-60 cells with [35S]methionine and [35S]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 [35S]sulfate for 10 min followed by DSP cross-linking and immunoprecipitation. From three granular enzymes immunoprecipitated, only the cationic MPO was cross-linked to [35S]sulfate-labeled serglycin in appreciable quantities, whereas cathepsin D or beta-N-acetylhexosaminidase was not. Thus, intracellular transport of MPO appears to be linked to that of serglycin. Extracts from high buoyant density organelles from human placenta containing MPO activity were subjected to CS-affinity chromatography. 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.
Lemansky
Peter
P
Institut für Physiologische Chemie, Philipps-Universität Marburg, Germany. lemansky@home.staff.uni-marburg.de
Gerecitano-Schmidek
Mireille
M
Das
Rajesh C
RC
Schmidt
Bernhard
B
Hasilik
Andrej
A
D002809:Chondroitin Sulfates / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000201:enzymology*; D004792:Enzyme Precursors / Q000032:analysis; D018922:HL-60 Cells; D006801:Humans; D009113:Muramidase / Q000032:analysis; D009195:Peroxidase / Q000378:metabolism*; D010920:Placenta / Q000737:chemistry; D021381:Protein Transport; D011509:Proteoglycans / Q000096:biosynthesis / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D033921:Vesicular Transport Proteins
D004792:Enzyme Precursors; D011509:Proteoglycans; D033921:Vesicular Transport Proteins; C075430:serglycin; D002809:Chondroitin Sulfates; D009195:Peroxidase; D009113:Muramidase; D013755:Tetradecanoylphorbol Acetate