Calreticulin Functions as a Molecular Chaperone in the Biosynthesis of Myeloperoxidase

Myeloperoxidase (MPO), a lysosomal heme protein found exclusively in neutrophils and monocytes, is necessary for efficient oxygen-dependent microbicidal activity. Acquisition of heme by the heme-free MPO precursor apopro-MPO appears to be a prerequisite for its subsequent proteolytic processing and advancement along the biosynthetic pathway to mature MPO. We present data indicating that calreticulin (CRT), a high capacity calcium-binding protein residing in the lumen of the endoplasmic reticulum of a wide variety of cells, interacts specifically with fully glycosylated apopro-MPO. Biosynthetically radiolabeled CRT (60 kDa) and apopro-MPO (90 kDa) were coprecipitated from PLB 985 cells by monospecific antiserum against CRT when the immunoprecipitations were performed either under nondenaturing conditions or following reversible cross-linking. Nonglycosylated MPO precursors synthesized in the presence of tunicamycin did not interact with CRT. The CRT-apopro-MPO interaction was restricted to an early phase of MPO biosynthesis, and CRT did not interact with the later appearing, heme-containing species of MPO, i.e. pro-MPO or the heavy subunit of mature MPO. These data show that CRT participates in the posttranslational processing of MPO, perhaps by maintaining apopro-MPO in a conformation competent to accommodate insertion of the heme group. In this general way, CRT shares certain functional properties with the structurally homologous transmembrane calcium-binding endoplasmic reticulum protein calnexin. Both interact with glycosylated biosynthetic precursors of proteins selectively expressed in specialized cells. Myeloperoxidase (MPO), a lysosomal heme protein found exclusively in neutrophils and monocytes, is necessary for efficient oxygen-dependent microbicidal activity. Acquisition of heme by the heme-free MPO precursor apopro-MPO appears to be a prerequisite for its subsequent proteolytic processing and advancement along the biosynthetic pathway to mature MPO. We present data indicating that calreticulin (CRT), a high capacity calcium-binding protein residing in the lumen of the endoplasmic reticulum of a wide variety of cells, interacts specifically with fully glycosylated apopro-MPO. Biosynthetically radiolabeled CRT (60 kDa) and apopro-MPO (90 kDa) were coprecipitated from PLB 985 cells by monospecific antiserum against CRT when the immunoprecipitations were performed either under nondenaturing conditions or following reversible cross-linking. Nonglycosylated MPO precursors synthesized in the presence of tunicamycin did not interact with CRT. The CRT-apopro-MPO interaction was restricted to an early phase of MPO biosynthesis, and CRT did not interact with the later appearing, heme-containing species of MPO, i.e. pro-MPO or the heavy subunit of mature MPO. These data show that CRT participates in the posttranslational processing of MPO, perhaps by maintaining apopro-MPO in a conformation competent to accommodate insertion of the heme group. In this general way, CRT shares certain functional properties with the structurally homologous transmembrane calcium-binding endoplasmic reticulum protein calnexin. Both interact with glycosylated biosynthetic precursors of proteins selectively expressed in specialized cells. INTRODUCTIONPolymorphonuclear granulocytes (PMNs) 1The abbreviations used are: PMNspolymorphonuclear neutrophilsδ-ALAδ-aminolevulinic acidCRTcalreticulinDTSSP3,3′-dithiobis(sulfosuccinimidyl proprionate)DTTdithiothreitolGRP94glucose-regulated protein, 94 kDahsp90heat shock protein, 90 kDaIEFisoelectric focusingMPOmyeloperoxidaseNEPHGEnon-equilibrium pH gel electrophoresisPAGEpolyacrylamide gel electrophoresisTBStris-buffered salineTMtunicamycinERendoplasmic reticulum. are critical elements in human host defense against invading microorganisms(1Ezekowitz R.A.B. Curr. Top. Microbiol. Immunol. 1992; 181: 283-292PubMed Google Scholar, 2Dinauer M.C. Orkin S.H. Annu. Rev. Med. 1992; 43: 117-124Crossref PubMed Scopus (88) Google Scholar). The most efficient microbicidal system employed by PMNs depends on two elements: reactive oxygen species generated by the NADPH-dependent oxidase (3Clark R.A. J. Infect. Dis. 1990; 161: 1140-1147Crossref PubMed Scopus (177) Google Scholar) and myeloperoxidase (MPO), a heme-containing protein present in the azurophilic granules of PMNs(4Klebanoff S.J. Science. 1970; 169: 1095-1097Crossref PubMed Scopus (221) Google Scholar, 5Klebanoff S.J. Everse J. Everse K. Grisham M. Peroxidases in Chemistry and Biology. CRC Press, Boca Raton, FL1991: 1-36Google Scholar). Within the phagolysosome of the activated PMN, these species interact to produce HOCl and exert microbicidal activity(5Klebanoff S.J. Everse J. Everse K. Grisham M. Peroxidases in Chemistry and Biology. CRC Press, Boca Raton, FL1991: 1-36Google Scholar).MPO (donor:H2O2 oxidoreductase, EC 1.11.1.7) has a molecular mass of 150 kDa and is composed of a pair of heavy-light protomers, each containing a glycosylated 59-kDa heavy subunit as well as a nonglycosylated 13.5-kDa light subunit(5Klebanoff S.J. Everse J. Everse K. Grisham M. Peroxidases in Chemistry and Biology. CRC Press, Boca Raton, FL1991: 1-36Google Scholar, 6Nauseef W.M. Hematol. Pathol. 1990; 4: 165-178PubMed Google Scholar, 7Johnson K.R. Nauseef W.M. Everse J. Everse K. Grisham M. Peroxidases in Chemistry and Biology. CRC Press, Boca Raton, FL1991: 63-82Google Scholar). Each molecule of MPO contains two heme groups, associated with the heavy subunit in each pair of heavy-light protomers.Studies of the biosynthesis of MPO by cultured promyelocytic cell lines have demonstrated that MPO is the product of a single gene. The primary translation product undergoes cotranslational N-linked glycosylation to generate a 90-kDa heme-free precursor, apopro-MPO. The heme group is inserted into apopro-MPO to produce the 90-kDa enzymatically active precursor, pro-MPO. Subsequently, pro-MPO undergoes proteolytic maturation, whereby the 125-amino acid pro-sequence is removed, the remaining peptide is cleaved into heavy and light subunits, the heavy-light protomers are paired into the dimeric mature MPO, and the mature protein is targeted to the lysosome (7Johnson K.R. Nauseef W.M. Everse J. Everse K. Grisham M. Peroxidases in Chemistry and Biology. CRC Press, Boca Raton, FL1991: 63-82Google Scholar, 8Nauseef W.M. McCormick S. Yi H. Blood. 1992; 80: 2622-2633Crossref PubMed Google Scholar, 9Nauseef W.M. Blood. 1986; 67: 865-872Crossref PubMed Google Scholar, 10Nauseef W.M. Blood. 1987; 70: 1143-1150Crossref PubMed Google Scholar, 11Akin D.T. Kinkade Jr., J.M. Arch. Biochem. Biophys. 1987; 255: 428-436Crossref PubMed Scopus (14) Google Scholar, 12Akin D.T. Kinkade Jr., J.M. J. Biol. Chem. 1986; 261: 8370-8375Abstract Full Text PubMed Google Scholar, Kinkade Jr., J.M. 1990; PubMed Scopus Google Scholar, J. M. Blood. PubMed Google Scholar, S.J. H. M. J. Biol. Chem. Full Text PubMed Google Scholar, M. J. Biol. Chem. Full Text PubMed Google Scholar, K. J. Biol. Chem. 1987; Full Text PubMed Google Scholar, K. Biochem. J. PubMed Scopus Google Scholar, K. J. Biol. 1986; Google Scholar, Kinkade Jr., J.M. Hematol. 1992; Google of MPO biosynthesis these the processing of MPO precursor to mature MPO is as as to from the precursor into mature W.M. McCormick S. Yi H. Blood. 1992; 80: 2622-2633Crossref PubMed Google Scholar). the processing and proteolytic of MPO precursor to MPO is when cells are cultured in the presence of an of heme W.M. McCormick S. Yi H. Blood. 1992; 80: 2622-2633Crossref PubMed Google Scholar, Kinkade Jr., J.M. Hematol. 1992; Google Scholar, Kinkade Jr., J.M. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). on these that heme insertion into apopro-MPO be a in the of MPO precursor in the to the of mature MPO in the a molecular in the critical of apopro-MPO to accommodate insertion of heme and subsequent to present data that calreticulin (CRT), a calcium-binding protein present in the of a wide variety of cells in S. Nauseef W.M. R.A. Biol. PubMed Scopus Google coprecipitated with the glycosylated heme-free precursor of MPO. In CRT and apopro-MPO be selectively and that CRT with the MPO precursor early in biosynthesis and that this interaction was these data that the structurally protein as a molecular These the in CRT human cell PLB 985 was from and in with and with were of was from the of biosynthetic was used to was with and and acid were from and was from against and were from 90 was against human and and from and cells. was from and radiolabeled with a the of and were from 985 cells were in an of in the biosynthetic cells were in with for was and the cells were cultured for the the of the cells were by for subsequent as W.M. Blood. 1986; 67: 865-872Crossref PubMed Google Scholar). In cells were in of and 150 of the cell cell were used in a In the of cells were in with and for the In tunicamycin cells were in containing a of for to of to in of promyelocytic cells and of MPO K.R. Nauseef W.M. Everse J. Everse K. Grisham M. Peroxidases in Chemistry and Biology. CRC Press, Boca Raton, FL1991: 63-82Google Scholar). In cells were in for to biosynthetic cells were under these cell was was to of the The of lysosomal and were in W.M. McCormick S. Yi H. Blood. 1992; 80: 2622-2633Crossref PubMed Google were under either or nondenaturing of cells or of were used for immunoprecipitations as W.M. Blood. 1986; 67: 865-872Crossref PubMed Google Scholar). were with for by a of protein to the of radiolabeled with the The was with primary CRT was a monospecific against human CRT expressed in S. Nauseef W.M. R.A. Biol. PubMed Scopus Google Scholar). were a monospecific to human W.M. J. PubMed Scopus Google Scholar). of and the was used as the primary by as the The were with protein S. and the were with each of in pH with 150 in in in in and the the was from protein by for in the presence of pH immunoprecipitations were under the was by the and to for to the of the primary the of the was to by the of pH and and on In immunoprecipitations under nondenaturing conditions the of and and with of the primary to the as immunoprecipitations were performed in two cells were a of biosynthetic with and with antiserum to as The CRT were in the presence of and to the in the and the The were and with for a of and The were under conditions against and MPO, were with to the PLB 985 cells were in and in with and in for the was by the of the and by on for were to as the proteins were to in of were by gel were to gel was the was in and into to the of J. Biol. Chem. Full Text Google Scholar). in the was performed by the and the pH gel The or gel was in containing and into a as W.M. J. PubMed Scopus Google Scholar, W.M. R.A. Blood. 1990; PubMed Google Scholar). The were in Biochem. PubMed Scopus Google and to In the of in the of proteins were on a CRT as a molecular proteins that coprecipitated with CRT its PLB 985 cells or cells not indicating the were not to PLB 985 cells were of cultured human promyelocytic cells. PLB 985 cells as a single protein S. Nauseef W.M. R.A. Biol. PubMed Scopus Google Scholar). the proteins and associated molecular are and Biochem. Full Text PubMed Scopus Google are that immunoprecipitations performed under conditions and nondenaturing conditions were PLB 985 cells were for and with monospecific antiserum against CRT the CRT was the protein of a 90-kDa protein, as a was coprecipitated under these when was performed under nondenaturing CRT and a of the 90-kDa protein were of CRT and the 90-kDa protein was demonstrated by the of a reversible of associated proteins immunoprecipitations to be under and the be by the with to cells were with the and under conditions with antiserum to CRT. proteins as high molecular kDa) when the CRT was under conditions In when the was to the high molecular species CRT and the associated 90-kDa protein PLB 985 cells are active and a of proteins that in the of 90 and the glycosylated precursors of MPO, and pro-MPO. the of the 90-kDa protein, performed immunoprecipitations antiserum to CRT in the by to and MPO in the PLB 985 cells were with and under conditions with antiserum to CRT. the was by under conditions most of the proteins associated with in high molecular mass proteins and kDa In to the high molecular CRT and an protein were by under The in was to the and the with to or MPO an the to CRT CRT when used as the the to that to a radiolabeled protein when used as the in the In the MPO a 90-kDa protein from the when used as the and in under proteins from radiolabeled cells when used as the in the that the to and in the the of these proteins from the CRT the of the cells to the proteins or of the to MPO contains J. J. Biol. 1992; PubMed Scopus Google and the precursor as a 90-kDa protein under conditions as an protein under M. The presence of a of the protein in the that of the coprecipitated MPO precursor was not under these These data that antiserum to CRT selectively the 90-kDa precursor of coprecipitated with the 90-kDa precursor of cells were as in with and with antiserum to CRT. The proteins were by under conditions In the the was and the were with the against or MPO. The the high molecular mass protein and a protein of The proteins in the were for CRT and for MPO. in the MPO precursor to under (90 kDa) and kDa) under the conditions PLB 985 cells proteins and MPO, each of was from cells by the The proteins by CRT antiserum were and to the was by and by in the by CRT into the gel in the the 90-kDa protein as a single protein into the indicating was The of this protein under these conditions was to that of the MPO precursor that the 90-kDa protein coprecipitated with CRT was to MPO and that 90-kDa proteins were the proteins by the CRT antiserum were to the and to in The proteins were in the by or by and the gel was to In the of the 90-kDa protein coprecipitated with CRT under indicating its The of this protein under these conditions was to that of the 90-kDa precursor of MPO In CRT into the as its 90-kDa protein was PLB 985 cells were with MPO antiserum under nondenaturing radiolabeled proteins to MPO were CRT has a in of S. Nauseef W.M. R.A. Biol. PubMed Scopus Google and that this of CRT in CRT of biosynthetic cells were with MPO antiserum and the were with CRT of the CRT was in the not These are the as for and its associated PubMed Scopus Google Scholar, S. J. Biol. Chem. Full Text PubMed Google Scholar, M. J. Biol. Chem. Full Text PubMed Google Scholar, Science. PubMed Scopus Google the glycosylation of the MPO precursor its interaction with the of on the of the precursor In conditions that produce proteins the of molecular M. K. J. PubMed Scopus Google Scholar, J. S. PubMed Scopus Google Scholar, K. S. PubMed Scopus Google Scholar). In the presence of an that N-linked Annu. Rev. Biochem. 1987; PubMed Google the nonglycosylated proteins to and presence in the the of molecular M. K. J. PubMed Scopus Google Scholar, J. S. PubMed Scopus Google Scholar, K. S. PubMed Scopus Google Scholar, J. Full Text PubMed Scopus Google Scholar). In to its on M. Nauseef and S. J. did not the of CRT biosynthesis In antiserum to CRT to 90-kDa protein from cells in the presence of and The of the 90-kDa protein to with CRT that either the of the 90-kDa protein or its with CRT. K.R. Nauseef W.M. Everse J. Everse K. Grisham M. Peroxidases in Chemistry and Biology. CRC Press, Boca Raton, FL1991: 63-82Google PLB 985 cells the nonglycosylated of the MPO precursor These data that CRT associated specifically with the glycosylated of the MPO precursor and not with the nonglycosylated associated with the glycosylated of MPO. radiolabeled PLB 985 cells in the or presence of were with or to with CRT under In the presence of MPO precursor of MPO was associated with CRT cross-linking. PLB 985 cells radiolabeled in the presence of or and with antiserum for MPO under conditions were In the of the fully glycosylated 90-kDa MPO precursor was In of N-linked glycosylation by in of the nonglycosylated that CRT were as a molecular associated with the MPO precursor and a its the of the CRT and the MPO precursor, PLB 985 cells were for and for or the of each cells were with or and with to CRT the of the of MPO precursor and CRT were the of MPO precursor was of 90-kDa and with of for in MPO precursor is the processing of MPO W.M. McCormick S. Yi H. Blood. 1992; 80: 2622-2633Crossref PubMed Google Scholar, K. J. Biol. Chem. 1987; Full Text PubMed Google Scholar, D.T. Kinkade Jr., J.M. Arch. Biochem. Biophys. 1987; PubMed Scopus Google Scholar). These data that the CRT and the MPO precursor was restricted to a early in MPO associated with an MPO precursor early in MPO PLB 985 cells were with for and or for and cells were with or and with antiserum against CRT. the of the of CRT and 90-kDa MPO precursor In the of MPO precursor associated with CRT for and to of the MPO precursor of are of the heme-free precursor, and the enzymatically active heme-containing precursor, are 90-kDa W.M. McCormick S. Yi H. Blood. 1992; 80: 2622-2633Crossref PubMed Google Scholar, 9Nauseef W.M. Blood. 1986; 67: 865-872Crossref PubMed Google Scholar, K. J. Biol. Chem. 1987; Full Text PubMed Google Scholar). and K. J. Biol. Chem. 1987; Full Text PubMed Google Scholar) demonstrated that acid a precursor in heme is into the 90-kDa precursor of MPO of that of are for to the heme-containing 59-kDa heavy subunit of mature MPO. on the of the interaction CRT and the MPO precursor that CRT not with heme-containing species of MPO. CRT associated with of the heme-containing of MPO, PLB 985 cells were for with these is into the heme in pro-MPO and in the heavy subunit of mature MPO K. J. Biol. Chem. 1987; Full Text PubMed Google Scholar). a was used to of into species and to that the heme in the heavy subunit of the mature protein was is to cells with for with is not the of protein by the cells. this the early and in biosynthesis, coprecipitated with CRT two and a cells were with and with antiserum to either CRT or MPO the cells that were not the 90-kDa of MPO precursor was by MPO antiserum cells for the antiserum to MPO the 90-kDa precursors as well as the and 13.5-kDa of mature MPO cells with MPO antiserum the heme-containing pro-MPO and the 59-kDa heavy subunit of mature MPO The of radiolabeled proteins with CRT antiserum from that when MPO antiserum was used to cells, CRT antiserum CRT and a 90-kDa MPO precursor In to the in the of a or MPO precursor coprecipitated with CRT the of with In CRT antiserum to proteins with of the heme-containing pro-MPO and mature MPO, was with CRT indicating the protein coprecipitated with CRT was of i.e. associated with apopro-MPO. PLB 985 cells were with for and for or or for with with and with antiserum to CRT or to MPO. The MPO antiserum radiolabeled 90-kDa MPO precursors from cells and precursor and the and 13.5-kDa of mature MPO from cells that were heme is inserted into the MPO precursor to generate in pro-MPO and the 59-kDa heavy CRT antiserum coprecipitated of the radiolabeled 90-kDa MPO precursor the and to heme-containing species with that CRT associated with conditions that the of heme-free precursor in an in the of precursor associated with CRT. have that of heme in cells cultured in selectively the processing of MPO W.M. McCormick S. Yi H. Blood. 1992; 80: 2622-2633Crossref PubMed Google Scholar). these apopro-MPO is to mature in the biosynthetic is a in proteolytic processing of MPO precursor to mature MPO, a of and a in the of MPO precursor W.M. McCormick S. Yi H. Blood. 1992; 80: 2622-2633Crossref PubMed Google Scholar). the of on the CRT-apopro-MPO cells were for and for in the or presence of cells were with and with antiserum to MPO or to CRT In the presence of the of MPO precursor to of CRT or MPO each to of of heme with the of MPO precursor associated with CRT. PLB 985 cells cultured for in the or presence of were with and for the cell with to with antiserum against MPO or CRT. In the presence of the of MPO precursor coprecipitated with CRT was that the MPO species that associated with CRT was the enzymatically apopro-MPO. these data that CRT associated with apopro-MPO biosynthesis of MPO in the human cell PLB The interaction CRT and apopro-MPO early in MPO biosynthesis and of heme into the protein CRT was not associated with pro-MPO or with the of mature MPO. the CRT and the MPO precursor was restricted to an interaction with fully glycosylated CRT did not interact with nonglycosylated MPO is a high calcium-binding protein that in the in M. K. M. Biochem. J. 1992; PubMed Scopus Google Scholar). most of CRT have on its in that participates in a wide of M. M. Biochem. PubMed Scopus Google from with in the granules of cells M. J. J. Med. PubMed Scopus Google Scholar) to K. M. M. PubMed Scopus Google Scholar, S. M. H. J. H. PubMed Scopus Google most cells, CRT in the by the a S. 1987; Full Text PubMed Scopus Google Scholar, Annu. Rev. Biol. PubMed Scopus Google Scholar). The lumen of the is the of a by molecular J. 1992; PubMed Scopus Google Scholar, Annu. Rev. Biochem. PubMed Google Scholar, Curr. Biol. 1992; 4: PubMed Scopus Google Scholar, Biochem. 1992; Full Text PubMed Scopus Google Scholar, Annu. Rev. Biol. PubMed Scopus Google Scholar). CRT proteins and the by the In CRT in biosynthesis of proteins as are in the M. K. M. Biochem. J. 1992; PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). The for CRT and the molecular and in the J. J. Biol. Chem. 1992; Full Text PubMed Google be and the the and with high capacity and S. 1987; Full Text PubMed Scopus Google Scholar, 1986; PubMed Scopus Google Scholar). S. J. Biol. Chem. Full Text PubMed Google Scholar) have found and CRT the proteins in of specifically by from a protein In of a for CRT in with proteins in the CRT and were from that CRT as a for S. PubMed Scopus Google data that the of CRT as a molecular is to that for PubMed Scopus Google Scholar, S. J. Biol. Chem. Full Text PubMed Google Scholar, M. J. Biol. Chem. Full Text PubMed Google Scholar, S. S. 1992; PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar, J. Biol. Chem. 1992; Full Text PubMed Google Scholar, R.A. J. Biol. Chem. Full Text PubMed Google Scholar, J. Biol. Chem. Full Text PubMed Google Scholar, M.C. J. Biol. Chem. Full Text PubMed Google Scholar, S. PubMed Scopus Google Scholar) and from that of and molecular with glycosylated of nonglycosylated in the presence of not interact with PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). Nonglycosylated proteins synthesized under these conditions are and interact with or molecular M. K. J. PubMed Scopus Google Scholar, J. S. PubMed Scopus Google Scholar, K. S. PubMed Scopus Google Scholar, J. Full Text PubMed Scopus Google Scholar). In a to that of CRT with glycosylated apopro-MPO not with the nonglycosylated MPO precursors synthesized in the presence of In coprecipitated with the nonglycosylated of MPO precursor synthesized in the presence of CRT is a protein the lumen of the M. K. M. Biochem. J. 1992; PubMed Scopus Google is by a transmembrane the of that are homologous to CRT the J. Biol. Chem. Full Text PubMed Google Scholar, Biochem. Full Text PubMed Scopus Google that these in proteins as a molecular in the CRT a the species of the and the calnexin. In the interaction of CRT with apopro-MPO that in cells CRT to apopro-MPO. as to and in a competent for into the functional M. J. Biol. Chem. Full Text PubMed Google CRT apopro-MPO in a conformation that insertion of heme into its and of precursor to the enzymatically active MPO be the of an interaction CRT and a of heme insertion into MPO a for a molecular is that CRT and be of a of molecular that interact with proteins in is that the insertion of heme into apopro-MPO is the for of the precursor data not CRT-apopro-MPO and of the necessary for protein insertion of a functional heme and of synthesized to be INTRODUCTIONPolymorphonuclear granulocytes (PMNs) 1The abbreviations used are: PMNspolymorphonuclear neutrophilsδ-ALAδ-aminolevulinic acidCRTcalreticulinDTSSP3,3′-dithiobis(sulfosuccinimidyl proprionate)DTTdithiothreitolGRP94glucose-regulated protein, 94 kDahsp90heat shock protein, 90 kDaIEFisoelectric focusingMPOmyeloperoxidaseNEPHGEnon-equilibrium pH gel electrophoresisPAGEpolyacrylamide gel electrophoresisTBStris-buffered salineTMtunicamycinERendoplasmic reticulum. are critical elements in human host defense against invading microorganisms(1Ezekowitz R.A.B. Curr. Top. Microbiol. Immunol. 1992; 181: 283-292PubMed Google Scholar, 2Dinauer M.C. Orkin S.H. Annu. Rev. Med. 1992; 43: 117-124Crossref PubMed Scopus (88) Google Scholar). The most efficient microbicidal system employed by PMNs depends on two elements: reactive oxygen species generated by the NADPH-dependent oxidase (3Clark R.A. J. Infect. Dis. 1990; 161: 1140-1147Crossref PubMed Scopus (177) Google Scholar) and myeloperoxidase (MPO), a heme-containing protein present in the azurophilic granules of PMNs(4Klebanoff S.J. Science. 1970; 169: 1095-1097Crossref PubMed Scopus (221) Google Scholar, 5Klebanoff S.J. Everse J. Everse K. Grisham M. Peroxidases in Chemistry and Biology. CRC Press, Boca Raton, FL1991: 1-36Google Scholar). Within the phagolysosome of the activated PMN, these species interact to produce HOCl and exert microbicidal activity(5Klebanoff S.J. Everse J. Everse K. Grisham M. Peroxidases in Chemistry and Biology. CRC Press, Boca Raton, FL1991: 1-36Google Scholar).MPO (donor:H2O2 oxidoreductase, EC 1.11.1.7) has a molecular mass of 150 kDa and is composed of a pair of heavy-light protomers, each containing a glycosylated 59-kDa heavy subunit as well as a nonglycosylated 13.5-kDa light subunit(5Klebanoff S.J. Everse J. Everse K. Grisham M. Peroxidases in Chemistry and Biology. CRC Press, Boca Raton, FL1991: 1-36Google Scholar, 6Nauseef W.M. Hematol. Pathol. 1990; 4: 165-178PubMed Google Scholar, 7Johnson K.R. Nauseef W.M. Everse J. Everse K. Grisham M. Peroxidases in Chemistry and Biology. CRC Press, Boca Raton, FL1991: 63-82Google Scholar). Each molecule of MPO contains two heme groups, associated with the heavy subunit in each pair of heavy-light protomers.Studies of the biosynthesis of MPO by cultured promyelocytic cell lines have demonstrated that MPO is the product of a single gene. The primary translation product undergoes cotranslational N-linked glycosylation to generate a 90-kDa heme-free precursor, apopro-MPO. The heme group is inserted into apopro-MPO to produce the 90-kDa enzymatically active precursor, pro-MPO. Subsequently, pro-MPO undergoes proteolytic maturation, whereby the 125-amino acid pro-sequence is removed, the remaining peptide is cleaved into heavy and light subunits, the heavy-light protomers are paired into the dimeric mature MPO, and the mature protein is targeted to the lysosome (7Johnson K.R. Nauseef W.M. Everse J. Everse K. Grisham M. Peroxidases in Chemistry and Biology. CRC Press, Boca Raton, FL1991: 63-82Google Scholar, 8Nauseef W.M. McCormick S. Yi H. Blood. 1992; 80: 2622-2633Crossref PubMed Google Scholar, 9Nauseef W.M. Blood. 1986; 67: 865-872Crossref PubMed Google Scholar, 10Nauseef W.M. Blood. 1987; 70: 1143-1150Crossref PubMed Google Scholar, 11Akin D.T. Kinkade Jr., J.M. Arch. Biochem. Biophys. 1987; 255: 428-436Crossref PubMed Scopus (14) Google Scholar, 12Akin D.T. Kinkade Jr., J.M. J. Biol. Chem. 1986; 261: 8370-8375Abstract Full Text PubMed Google Scholar, Kinkade Jr., J.M. 1990; PubMed Scopus Google Scholar, J. M. Blood. PubMed Google Scholar, S.J. H. M. J. Biol. Chem. Full Text PubMed Google Scholar, M. J. Biol. Chem. Full Text PubMed Google Scholar, K. J. Biol. Chem. 1987; Full Text PubMed Google Scholar, K. Biochem. J. PubMed Scopus Google Scholar, K. J. Biol. 1986; Google Scholar, Kinkade Jr., J.M. Hematol. 1992; Google of MPO biosynthesis these the processing of MPO precursor to mature MPO is as as to from the precursor into mature W.M. McCormick S. Yi H. Blood. 1992; 80: 2622-2633Crossref PubMed Google Scholar). the processing and proteolytic of MPO precursor to MPO is when cells are cultured in the presence of an of heme W.M. McCormick S. Yi H. Blood. 1992; 80: 2622-2633Crossref PubMed Google Scholar, Kinkade Jr., J.M. Hematol. 1992; Google Scholar, Kinkade Jr., J.M. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). on these that heme insertion into apopro-MPO be a in the of MPO precursor in the to the of mature MPO in the a molecular in the critical of apopro-MPO to accommodate insertion of heme and subsequent to present data that calreticulin (CRT), a calcium-binding protein present in the of a wide variety of cells in S. Nauseef W.M. R.A. Biol. PubMed Scopus Google coprecipitated with the glycosylated heme-free precursor of MPO. In CRT and apopro-MPO be selectively and that CRT with the MPO precursor early in biosynthesis and that this interaction was these data that the structurally protein as a molecular These the in CRT