Abstract
Myeloperoxidase (MPO), stored in azurophil granules of neutrophils, is critical for an optimal oxygen-dependent microbicidal activity of these cells. Pro-MPO goes through a stepwise proteolytic trimming with elimination of an amino-terminal propeptide to yield one heavy and one light polypeptide chain. The propeptide of MPO may have a role in retention and folding of the nascent protein into its tertiary structure or in targeting of pro-MPO for processing and storage in granules. A propeptide-deleted pro-MPO mutant (MPODeltapro) was constructed to determine if deletion of the propeptide interferes with processing and targeting after transfection to the myeloid 32D cell line. Transfection of full-length cDNA for human MPO results in normal processing and targeting of MPO to cytoplasmic dense organelles. Although the efficiency of incorporation was lower for MPODeltapro, both pro-MPO and MPODeltapro showed heme incorporation indicating that the propeptide is not critical for this process. Deletion of the propeptide results in synthesis of a protein that lacks processing into mature two-chain forms but rather is degraded intracellularly or secreted. The finding of continued degradation of MPODeltapro in the presence of lysosomotrophic agents or brefeldin A rules out that the observed degradation takes place after transfer to granules. Intracellular pro-MPO has high mannose oligosaccharide side chains, whereas stored mature MPO was found to have both high mannose and complex oligosaccharide side chains as judged by only partial sensitivity to endoglycosidase H. The propeptide may normally interfere with the generation of certain complex oligosaccharide chain(s) supported by the finding of high mannose side chains in secreted pro-MPO and lack of them in MPODeltapro that contained complex oligosaccharide side chains only. In conclusion, elimination of the propeptide of pro-MPO blocks the maturation process and abolishes accumulation of the final product in granules suggesting a critical role of the propeptide for late processing of pro-MPO and targeting for storage in granules.
Highlights
Neutrophil granulocytes are specialized for a role in host defense
A pro-region segment is necessary for targeting to granules of neutrophil defensins [3], but carboxyl-terminal prodomains or asparagine-linked carbohydrates of hematopoietic serine proteases are not required in targeting storage in granules [4, 5]
We describe the consequences of these manipulations for posttranslational processing, intracellular sorting, and constitutive secretion after transfection of the cDNA for MPO and MPO lacking the propeptide (MPO⌬pro) into the murine myeloid 32D clone 3 cell line
Summary
Neutrophil granulocytes are specialized for a role in host defense. A regulated pathway targets enzymes and antibiotic proteins to a storage compartment in these cells consisting of cytoplasmic azurophil, specific, and gelatinase granules formed sequentially, whereas a constitutive pathway exports proteins to the cell surface [1]. Myeloperoxidase Processing 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. One can envision a role for the amino-terminal propeptide of MPO in retention and folding of the nascent protein into its tertiary structure or in targeting pro-MPO to pregranule structures for further processing and storage in granules. We describe the consequences of these manipulations for posttranslational processing, intracellular sorting, and constitutive secretion after transfection of the cDNA for MPO and MPO⌬pro into the murine myeloid 32D clone 3 cell line
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