title: 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.
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.
authors.affiliation: Torrey Pines Pharmaceuticals, Del Mar, California, United States of America.
authors.forename: Richard P
authors.initials: RP
authors.lastname: Laura
authors.affiliation: Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, California, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Dong
authors.affiliation: Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, California, United States of America.
authors.forename: Wanda F
authors.initials: WF
authors.lastname: Reynolds
authors.affiliation: Torrey Pines Pharmaceuticals, Del Mar, California, United States of America.|Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, California, United States of America.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Maki
mesh_terms: D002403:Cathepsins / Q000037:antagonists & inhibitors; D002460:Cell Line; D045744:Cell Line, Tumor; D003553:Cystine / Q000378:metabolism; D004220:Disulfides; D004721:Endoplasmic Reticulum; D004789:Enzyme Activation; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006031:Glycosylation; D006801:Humans; D042541:Intracellular Space; D008247:Lysosomes / Q000378:metabolism*; D008360:Mannosephosphates / Q000378:metabolism; D009154:Mutation; D009195:Peroxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D059748:Proteolysis; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D004220:Disulfides; D008360:Mannosephosphates; D034741:RNA, Small Interfering; C027693:mannose-6-phosphate; D003553:Cystine; D009195:Peroxidase; D002403:Cathepsins