T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation.

Richard P. Laura,Wanda F. Reynolds,David Dong,Richard A. Maki,Luis Eduardo Soares Netto

doi:10.1371/journal.pone.0149391

Abstract

Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO’s single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO’s unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO.

Highlights

The enzyme myeloperoxidase (MPO) catalyzes the formation of hypochlorous acid (HOCl) from the substrates hydrogen peroxide (H2O2) and chloride and is one of several offensive weapons deployed by activated neutrophils
MPO Processing Examined Using T47D cells in hopes of identifying a recombinant cell model that more closely replicates the biosynthetic characteristics of MPO in promyelocytes and HL60 cells than previously reported expression systems
Only two related breast cancer cell lines, T47D and MCF7, were able to efficiently process proMPO into the mature heterotetramer and for T47D cells those levels nearly approach the efficiency of processing that occur for endogenous MPO in HL60 cells (Fig 2A, left panel)

Summary

Introduction

The enzyme myeloperoxidase (MPO) catalyzes the formation of hypochlorous acid (HOCl) from the substrates hydrogen peroxide (H2O2) and chloride and is one of several offensive weapons deployed by activated neutrophils. MPO is stored in an inactive state in the azurophil granules of neutrophils, a modified type of lysosome, and becomes catalytically productive only when a neutrophil is stimulated and turns on the enzyme nicotinamide adenine dinucleotide phosphate-oxidase [1, 2]. Nicotinamide adenine dinucleotide phosphate-oxidase provides high local concentrations of the otherwise limiting substrate H2O2 for the synthesis of HOCl by MPO. The chemical HOCl is a potent reactive oxygen species. The large number of circulating neutrophils combined with a high content of MPO creates the potential for significant reactive oxygen species-mediated damage to invading organisms and to host proteins, lipids and DNA during an inflammatory response. Under conditions of chronic inflammation, especially in older individuals where cell repair pathways begin to fail, accumulated oxidative damage may lead to cell dysfunction and disease [3]

Methods

Results

Conclusion