Transport der Myeloperoxidase in die azurophilen Granula von HL-60 Zellen

Myeloperoxidase, a cationic heme-containing enzyme, is one of the major constituents of azurophilic granules in neutrophils. In this study we examined one possible transport pathway into the azurophilic granules of HL-60 cells. The hypothesis of this work is based on the observation, that positively charged lysozyme can bind to the chondroitin sulfate(CS)-chains of serglycin (Kolset et al., 1996) and thus be transported to lysosomes (Lemansky and Hasilik, 2001). Affinity chromatography of mature MPO and proMPO with CS-Sepharose showed, that the binding-strength of both, mature MPO and proMPO, to CS-Sepharose is identical. This result indicates, that the entire MPO interacts with the carbohydrate side-chains of serglycin. We therefore assume that the propeptide is not exclusively important for the transport of MPO, as postulated by Andersson et al. (1998) and Bülow et al. (2002). Simulating the environment of azurophilic granules by lowering the pH-value to 3,5 had no effect on the binding capacity of CS-Sepharose for MPO. A pH-dependent dissociation-mechanism of MPO and serglycin is therefore not to be expected which is in accord with earlier experimental results ( Hasilik et al.; 1984). Within HL-60 cells only 50% of MPO and serglycin is sorted into the azurophilic granules. Under the influence of TPA additional major portions of the remainder (39% of MPO and 44% of serglycin) were secreted. This effect on targeting suggests that MPO and serglycin may form complexes on their way to granules. The formation of these complexes could actually be shown after labeling HL-60 cells with [35S]sulfate and cross-linking with DSP by coimmunoprecipitation. Next we showed that many different polypeptides, secreted in the presence of TPA, can bind to a CS-Sepharose-column. The results indicate that serglycin can bind other cationic proteins besides MPO, and potentially transport them to azurophilic granules. Procathepsin D was examined as a negative control for serglycin mediated transport. The binding of this nonbasic lysosomal enzyme to CS-Sepharose was significantlyweaker than that of MPO. This indicates that cationic proteins would displace procathepsin D from serglycin within HL-60 cells.