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

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 (MPO⌬pro) was constructed to determine if deletion of the propeptide interferes with processing and targeting after transfection to the myeloid 32D cell line. Transfection of fulllength cDNA for human MPO results in normal processing and targeting of MPO to cytoplasmic dense organelles. Although the efficiency of incorporation was lower for MPO⌬pro, both pro-MPO and MPO⌬pro 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 MPO⌬pro 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 MPO⌬pro 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.

Impact of Two Novel Mutations on the Structure and Function of Human Myeloperoxidase

“…2), suggesting that normal amounts of the G501S precursor entered the secretory pathway despite the failure of proteolytic maturation to proceed. As reported previously for normal MPO biosynthesis (20), ϳ10% of MPO precursor is secreted constitutively and undergoes limited modification of its oligosaccharide side chains during transit through the Golgi, whereby the secreted species is partially resistant to endoglycosidase H digestion (39). To determine whether the secreted MPO precursor from transfectants expressing G501S underwent oligosaccharide modification, the lysates of pulselabeled cells and supernatants conditioned by the 20-h chase period were recovered from each cell line, immunoprecipitated with MPO antiserum, and subjected to digestion with endoglycosidase H or N-glycanase to cleave high mannose or both high mannose and complex mannose side chains, respectively.…”

“…Heme acquisition was necessary for exit from the ER and for proteolytic processing to mature subunits, with the latter event inhibited by disruption of the Golgi by treatment with brefeldin A (data not shown). Approximately 10% of MPO precursor entered the secretory pathway and underwent limited modification of its oligosaccharide side chains, as seen with MPO endogenously produced by cultured promyelocytes (39). The spectral properties of MPO-enriched pellets recovered from stable transfectants were identical to those of normal MPO (see below), with a max of 430 nm for the oxidized sample and a Soret band at 473 nm for the reduced minus oxidized spectroscopy (41).…”

“…Glycosidase Susceptibility of MPO-related Proteins-Modifications of the carbohydrate side chains on MPO-related proteins were assessed by their relative susceptibility to endoglycosidases as done previously with MPO endogenously expressed in myeloid precursors (31,39). MPO-related proteins were immunoprecipitated from cell lysates or culture medium at specified time points and treated with endoglycosidase H or N-glycanase to digest high mannose or complex carbohydrate side chains, respectively.…”

A Redundant Role of Human Thyroid Peroxidase Propeptide for Cellular, Enzymatic, and Immunological Activity

“…Wild-type MPO is targeted for storage in granules by a regulated pathway, or secreted outside the cell by a constitutive pathway (43). Detailed studies on the role of the propeptide in maturation and sorting of MPO in myeloid 32D cell line revealed that MPODpro was effectively transported to the culture medium by the secretory pathway, but the intracellular trafficking to the storage granules was completely blocked (48). Therefore, the propeptide is likely to have an important role in the proper cellular sorting of MPO.…”

Neutrophil granules: heterogeneity of their contents reflects targeting by timing

“…The pro-regions of neutrophil elastase and cathepsin G are not critical for sorting nascent protein to the azurophilic granule when expressed in cultured rat or murine cell lines [14,15]. On the other hand, the pro-region of myeloperoxidase directs a glycosylated construct of lysozyme to lysosomes when transfected into Chinese hamster ovary cells [16], and constructs with the pro-region deleted fail to undergo normal proteolytic maturation and targeting when expressed in murine cells [17]. The extent to which the experimental system, viz.…”