Biosynthesis, transport and processing of myeloperoxidase in the human leukaemic promyelocytic cell line HL-60 and normal marrow cells

Abstract

The processing and intracellular transport of myeloperoxidase were studied in the human promyelocytic leukaemia cell line HL-60 and in normal marrow cells labelled with [35S]methionine or [14C]leucine. Myeloperoxidase was precipitated with antimyeloperoxidase serum; the immunoprecipitates were subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and radiolabelled myeloperoxidase visualized by fluorography. During a 1 h pulse, myeloperoxidase was labelled in a chain of apparent Mr 90 000. With a subsequent chase, the Mr 90 000 polypeptide disappeared and was replaced by chains of Mr 62 000 and 12 400 corresponding roughly to the size of neutrophil myeloperoxidase subunits. The identification of the radioactive polypeptides as different forms of myeloperoxidase was established also by the similarity in patterns generated by partial proteolysis with V8 proteinase from Staphylococcus aureus. Processing of myeloperoxidase in HL-60 was slow; mature polypeptides were significantly increased only after 6 h. Another myeloperoxidase chain of apparent Mr 82 000 was an intermediate precursor or degradation form. Pulse-chase experiments in combination with sucrose-density-gradient separations of homogenates showed that the Mr 90 000 precursor was located in light density organelles only and not in granule fractions, whereas the Mr 82 000 precursor was located only in intermediate density organelles, suggesting that the latter is a product of the former. Processed mature myeloperoxidase was concentrated in the granule fraction, but some occurred in lower density organelles, which may indicate processing during intracellular transport. Only the Mr 90 000 polypeptide was secreted into the culture medium; this was also the only form found in the cytosol fraction.

The Role of the Propeptide for Processing and Sorting of Human Myeloperoxidase

“…Calreticulin, a calcium-binding protein that resides in the ER, has been suggested to function as a molecular chaperone and facilitate the critical folding of apopro-MPO to allow insertion of heme followed by conversion to pro-MPO (14). The stepwise processing of pro-MPO has been investigated in myeloid cells (10,(15)(16)(17)(18)(19)(20), and the results obtained are consistent with those later deduced from cDNA sequence data. Thus, during subsequent processing of pro-MPO the amino-terminal propeptide, a small peptide between the light and heavy chains, and a single serine residue at the carboxyl-terminal are removed (21).…”

“…Thus, during subsequent processing of pro-MPO the amino-terminal propeptide, a small peptide between the light and heavy chains, and a single serine residue at the carboxyl-terminal are removed (21). Intermedi-ate processing forms have been observed with molecular masses of 81 and 74 kDa (10,18,19,22) of which the smaller can be converted directly into mature MPO after cleavage between the heavy and the light subunit (19,22). This finding suggests that the amino-terminal propeptide, which does not seem to be part of the 74-kDa form, is removed during an intermediate step before final processing.…”

Assignment of the myeloperoxidase geneMPO to human chromosome 17 using somatic cell hybrids and flow-sorted chromosomes

“…Studies on the biosynthesis of MPO have shown that these large and small subunits are generated from a single polypeptide chain precursor (Yamada, 1982;Olsson et al, 1984;Koeffler et al, 1985;Akin and Kinkade, 1986). Our present study provided evidence that MPO is synthesized from a gene located on chromosome 17.…”

Pattern of inheritance in hereditary myeloperoxidase deficiency associated with the R569W missense mutation

“…Mature MPO can be chemically cleaved into hemimyeloperoxidase, a single heavy-light protomer with the same specific activity as the holoenzyme [31,36], although it is not known if such forms are produced in vivo. Despite significant work on the biosynthesis of MPO [37][38][39][40][41][42][43][44][45][46][47] there is limited insight into the assembly and transport of mature MPO. Taylor et al [38] demonstrated that proMPO undergoes proteolytic processing before dimerization and that assembly is a late event in MPO biosynthesis.…”