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The role of the propeptide for processing and sorting of human myeloperoxidase
E Andersson1, L Hellman, U Gullberg
1Department of Hematology, Research Department 2, E-blocket, University Hospital, S-221 85 Lund, Sweden.
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.
Insights
The myeloperoxidase (MPO) propeptide is essential for its maturation and storage in neutrophil granules. Deleting this propeptide prevents MPO processing and causes degradation, highlighting its critical role in cellular defense mechanisms.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Myeloperoxidase (MPO) is crucial for neutrophil microbicidal activity.
- MPO undergoes proteolytic processing from pro-MPO to mature forms.
- The MPO propeptide's role in processing, folding, and granule targeting is unclear.
Purpose of the Study:
- To investigate the role of the MPO propeptide in protein processing, heme incorporation, and granule targeting.
- To determine if propeptide deletion affects MPO maturation and stability.
- To elucidate the propeptide's function in oligosaccharide modification.
Main Methods:
- Constructed a propeptide-deleted MPO mutant (MPODeltapro).
- Transfected full-length and mutant MPO cDNA into myeloid 32D cells.
- Analyzed MPO processing, heme incorporation, degradation pathways, and oligosaccharide side chains using techniques like endoglycosidase H treatment.
Main Results:
- Full-length MPO processed and targeted correctly; MPODeltapro showed lower incorporation efficiency.
- Propeptide deletion prevented MPO maturation into two-chain forms, leading to degradation or secretion.
- Heme incorporation occurred in both pro-MPO and MPODeltapro, indicating the propeptide is not essential for this.
- MPODeltapro degradation was intracellular, not granule-dependent.
- Propeptide deletion altered oligosaccharide processing, with MPODeltapro exhibiting complex chains while secreted pro-MPO retained high mannose chains.
Conclusions:
- The MPO propeptide is critical for the late-stage processing and granule targeting of MPO.
- Elimination of the propeptide blocks MPO maturation and prevents its accumulation in granules.
- The propeptide plays a significant role in guiding MPO through its maturation pathway and into cellular storage sites.