Biochem. J. (1984) 223, 911-920 911Printed in Great Britain

Biosynthesis, transport and processing of myeloperoxidase in the humanleukaemic promyelocytic cell line HL-60 and normal marrow cells

Inge OLSSON, Ann-Maj PERSSON and Kristina STROMBERGDivision of Hematology, Department of Internal Medicine, University ofLund, S-221 85 Lund, Sweden

(Received 11 June 1984/Accepted 24 July 1984)

The processing and intracellular transport of myeloperoxidase were studied in the hu-man promyelocytic leukaemia cell line HL-60 and in normal marrow cells labelledwith [35S]methionine or (14C]leucine. Myeloperoxidase was precipitated with anti-myeloperoxidase serum; the immunoprecipitates were subjected to sodium dodecylsulphate/polyacrylamide-gel electrophoresis and radiolabelled myeloperoxidasevisualized by fluorography. During a 1 h pulse, myeloperoxidase was labelled in achain of apparent Mr 90000. With a subsequent chase, the Mr 90000 polypeptide dis-appeared and was replaced by chains of Mr 62000 and 12400 corresponding roughlyto the size of neutrophil myeloperoxidase subunits. The identification of the radio-active polypeptides as different forms of myeloperoxidase was established also by thesimilarity in patterns generated by partial proteolysis with V8 proteinase fromStaphylococcus aureus. Processing of myeloperoxidase in HL-60 was slow; maturepolypeptides were significantly increased only after 6h. Another myeloperoxidasechain of apparent M, 82000 was an intermediate precursor or degradation form.Pulse-chase experiments in combination with sucrose-density-gradient separationsof homogenates showed that the Mr 90000 precursor was located in light density or-ganelles only and not in granule fractions, whereas the M, 82000 precursor was lo-cated only in intermediate density organelles, suggesting that the latter is a product ofthe former. Processed mature myeloperoxidase was concentrated in the granulefraction, but some occurred in lower density organelles, which may indicateprocessing during intracellular transport. Only the Mr 90000 polypeptide wassecreted into the culture medium; this was also the only form found in the cytosolfraction.

During maturation of neutrophil precursor cellsspecific proteins are synthesized, transported tothe Golgi apparatus for sorting and packaging intomembrane-bound granules. Two distinct classes ofgranules are formed at subsequent stages ofmaturation (Bainton & Farquar, 1970; Baintonet al., 1971). Azurophil (primary) lysosomal-likegranules are formed in promyelocytes, whereasspecific (secondary) granules are produced inmyelocytes. Myeloperoxidase is a unique constitu-ent of azurophil granules (Agner, 1941; Schultz &Kaminker, 1962; Bretz & Baggiolini, 1974; Spitz-nagel et al., 1974), which also contain a number ofcationic proteinases (Olsson & Venge, 1980). Themechanisms by which neutrophil granule en-zymes are synthesized, processed and stored arelargely unknown. Some lysosomal enzymes are

Abbreviation used: SDS, sodium dodecyl sulphate.

synthesized as precursors and modified during orsubsequent to storage in lysosomes (Hasilik &Neufeld, 1980a; Erickson et al., 1981; Skudlarek &Swank, 1981).To elucidate the subcellular progression and

processing ofnewly synthesized neutrophil granuleenzymes, we undertook to radiolabel them bio-synthetically, to isolate them by immuno-precipitation followed by polyacrylamide-gelelectrophoresis and fluorography. We have chosento study a major constituent of azurophil granules,myeloperoxidase, to which rabbit antibodies havebeen generated (Olsson et al., 1972). For biosyn-thetic labelling the human promyelocytic leukae-mia cell line HL-60 (Gallagher et al., 1979) wasemployed as well as marrow cells from healthyindividuals. Our results demonstrate that myelo-peroxidase is synthesized as larger precursors andshortened to the size of granule-bound myeloper-

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I. Olsson, A.-M. Persson and K. Stromberg

oxidase prior to or subsequent to storage inazurophil granules.

Experimental

ChemicalsL-[35S]Methionine (93Ci/mmol) was from The

Radiochemical Centre, Amersham. Sodium [35S]-sulphate (0.5 Ci/mmol). [3H]uridine (7.1 Ci/mmol),[3H]arginine (15 Ci/mmol), L-[14C]leucine (342mCi/mmol) and En3Hance were from New EnglandNuclear. Protein A-Sepharose CL-4B was fromPharmacia, Uppsala, Sweden. Endo-N-acetyl-glucosaminidase H (endoglycosidase H) was fromMiles Laboratories Inc., Rehovot, Israel. Phenyl-methanesulphonyl fluoride, monensin, and V8 pro-teinase from Staphylococcus aureus were fromSigma Chemical Co. Acrylamide/bisacrylamide(29:1) was from Bio-Rad.

Cell cultureHL-60 cells (passages 26-40) were maintained in

RPMI 1640 medium supplemented with 10% (v/v)heat-inactivated foetal bovine serum. Cells wereincubated at 37°C in a humidified atmosphereof air/CO2 (19:1). All studies were made withexponentially growing cells with 95-100% viabilityjudging from Trypan Blue exclusion.

Isolation of marrow cellsBone marrow cells from healthy individuals

were collected in heparinized McCoy's mediumand separated on Isopaque/Ficoll (1.077g/ml). Thecells from the interphase were washed and used forbiosynthetic labelling.

Labelling of cellsBiosynthetic labelling of myeloperoxidase. HL-60

or marrow cells were resuspended in methionine-free minimum essential medium (Eagle) with 10%foetal bovine serum and incubated at 37°C for60min to allow the depletion of the intracellularmethionine pool. The labelling medium was thesame deficient medium; the addition of 10% foetalbovine serum resulted in optimal biosyntheticprotein labelling judging from overall incorpora-tion of 35S into trichloroacetic acid precipitablemacromolecules. The cells, (2-3) x 106/ml, wereincubated with 5OpCi of [35S]methionine/ml. Inchase experiments, cells pulsed with [35S]-methionine for 1-4h were washed with andresuspended in RPMI 1640 medium with 10%foetal bovine serum at a density of 106 cells/ml, andincubated for 3-18h.

Identification ofsubcellular sitesfor newly synthe-sizedprotein. Cells (lOml, 3 x 106/ml) were incubat-ed at 37°C for 10min with lOO1Ci of [35S]-

methionine/ml in methionine-free minimumessential medium (Eagle) containing 10% foetal-bovine serum, followed by the addition of cyclo-heximide, 200pg/ml, which was also added to thehomogenization medium. For identification of theintracellular site of sulphation, presumed to belocalized to the Golgi apparatus (Young, 1973), cells(lOml, 3 x 106/ml) were incubated at 37°C for 3minwith 50uCi of [35S]sulphate/ml in Dulbecco'smedium. Substrates for sulphation in the form ofglycosaminoglycans are produced in immaturemyeloid cells (Olsson, 1969). Ribosomal RNA wasidentified by incubation at 37°C for 3 h of 8 ml ofcells (106/ml) with lOO4Ci of [3H]uridine/ml ofRPMI 1640 medium with 10% foetal-bovineserum. Homogenates ofthe labelled cells were usedfor preparation of subcellular fractions by differen-tial centrifugation or were analysed by sucrose-density-gradient centrifugation as described be-low. The distribution of macromolecular incor-poration of radioisotope-labelled precursor wasdetermined by trichloroacetic acid precipitation ofaliquots from subcellular fractions followed byliquid-scintillation counting. f-Hexosaminidaseand a-mannosidase were determined as describedby Hultberg et al. (1976).

Subcellular fractionationBefore homogenization of labelled cells, 5 x 107

unlabelled carrier cells were added. Homogen-ization was at a concentration of 108 cells/ml in0.34M-sucrose/5mM-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid] (pH 7.3)/0.5mM-EDTA with 40 strokes of a Dounce glass homo-genizer. The homogenate was diluted with thesame solution and unbroken cells and nuclei wererecovered by centrifugation at 700g for 10min,after which the granule fraction was collected at8000g for 15min. The microsomal fraction wasobtained by centrifugation of the post-granulesupernatant at 200000g for 2h. The resultingsupernatant was the cytosol. The granule andmicrosomal fractions are crude subcellular frac-tions which are to some degree cross-contaminated(see Table 1).

Subcellular organelles were also separated in asucrose-density gradient. The 700g supernatantwas layered on 10ml continuous sucrose gradienits(density range 1.10-1.30g/ml). Centrifugation wasperformed at 650OOg for 16h in rotor model SW25.3 (Beckman Instruments, Bromma, Sweden).Fractions (1 ml) were collected with a peristatticpump. Density was read in a refractometer. Thedistribution in the gradient of Golgi elements andprotein synthesis sites was determined by isotopelabelling of cells as described above. Immuno-chemical determination of the total myeloper-oxidase content of the fractions by single radial

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immunodiffusion (Odeberg et al., 1976) was usedfor localization of the azurophil granules.

Extraction of cells and subcellular fractionsExtraction was with a radioimmunoprecipita-

tion assay (RIPA) buffer consisting of 0.15 M-NaCl/30mM-Hepes (pH7.3)/l% (v/v) Triton X-100/1% (w/v) sodium deoxycholate/0.1% (w/v)SDS. Approx. 1 ml of RIPA buffer containing1 mM-phenylmethanesulphonyl fluoride was used/107 cells. The extracts were kept on ice for 1 hand centrifuged at 4°C at 32000g for 2h. Theclear supernatant was stored frozen until usedfor immunoprecipitation. Sucrose-density-gradientfractions were supplemented with 1/10 the volumeof a 10-fold concentrated RIPA buffer anddialyzed against RIPA buffer to remove sucrose.The fractions were then clarified by centrifugationat 4°C at 32000g for 2h and the supernatants wereused for immunoprecipitation.

Immunoprecipitation of radioactive myeloperoxidaseAnti-myeloperoxidase serum was produced by

immunization of rabbits (Olsson et al., 1972).Adsorbed anti-myeloperoxidase was obtained byadding myeloperoxidase to the antiserum followedby centrifugation to remove the precipitate.RIPA buffer extracts, 100-300pl, of whole cells

and subcellular fractions or the RIPA bufferextract of sucrose-density-gradient fractions (1.0-1.5ml) were mixed with 15pl of anti-myeloper-oxidase. Up to 3ml of culture medium or cytosolwas used for immunoprecipitation with the addi-tion of 50pd of anti-myeloperoxidase. After beingleft on ice for at least 60min, 40,l of a Protein A-Sepharose (200mg/ml) solution in RIPA bufferwas added for collection of the immunoprecipitateby rotation at 4°C for 30-60min. The Protein A-Sepharose was washed five times with RIPAbuffer and the supernatant was carefully removed.The pellet was resuspended in 50pl of water plus15MI of electrophoresis sample buffer [0.4M-Tris/HCl (pH6.8)/50% (v/v) glycerol/10% (w/v)SDS/5% (w/v) mercaptoethanol], boiled for 5minand used for electrophoresis. In some experimentsthe Protein A-Sepharose was extracted with 0.5 mlof 0.5M-NaOH and counted for radioactivity afteraddition of 10ml of Aquasol for determinationof the total 35S activity of immunoprecipitatedmyeloperoxidase.

SDS/polyacrylamide-gel electrophoresis andfluorography

SDS/polyacrylamide-gel electrophoresis wasperformed on slab gels 18cm long, 1.5mm thick,and 16cm wide, with ten wells, in an LKB 2001vertical electrophoresis unit (LKB Products,Bromma, Sweden). The gels and the electrode

buffers were made according to Laemmli (1970).Samples were loaded on a linear gradient of 5-20%polyacrylamide gel with a 3% stacking gel. Theelectrophoresis was run at 25mA/gel withoutcooling. After electrophoresis, gels were fixed in10% (w/v) trichloroacetic acid/10% aceticacid/30% (v/v) methanol for 1 h and then treatedwith En3Hance for 1 h and water for 1 h. Gels weredried on filter paper, and exposed to X-ray film(Kodak X-Omat S) at -80°C for 2-3 days.Apparent M, values were determined by use of

the following [14C]methylated standards (NewEngland Nuclear): cytochrome c, (M, 12300),carbonic anhydrase (Mr 30000), ovalbumin (Mr46000), bovine serum albumin (Mr 69000) andphosphorylase b, (Mr 97400).To quantify radioactivity, individual bands,

localized on the dried gel by fluorography, wereexcised and treated overnight at 37°C in 1 ml of asolution containing 30% H202/30% NH3 (19:1,v/v); 15 ml of scintillation liquid was added forcounting.

Peptide mappingFor peptide mapping HL-60 cells were labelled

with [14C]leucine. Cells were starved in leucine-free minimum essential medium (Eagle) with 1%foetal bovine serum for 60min at 37°C. Incubationwas for 16h in the same deficient medium with 5%foetal bovine serum with 3 x 106 cells/ml and10yCi of leucine/ml. Cell extraction, immunopre-cipitation, and electrophoresis was as describedabove. Regions of the gel corresponding to myelo-peroxidase species were cut out, equilibrated for30min in buffer [0.125-Tris/HCl (pH6.8)/10mM-dithiothreitol/1% SDS) and packed in sample wellscontaining the same buffer (Myerowitz & Neufeld,1981). Digestion with V8 proteinase from S. aureusin gel slices was as described by Clevelandet al. (1977) using a linear gradient of5-20% polyacrylamide gel with 3% stacking gel(3 cm). Buffer (20 u1) containing 10% (v/v) glycerolwith 5 Mug of V8 proteinase was layered over theslices in each well. Electrophoresis was performedat room temperature at 15mA/gel and was inter-rupted for 30min when the dye marker was close tothe bottom of the stacking gel. Electrophoresis wascontinued at 25 mA/gel without cooling followedby fluorography.

Results

Our results (Figs. 1-4) show that myeloperoxid-ase is synthesized as a precursor polypeptide withan apparent Mr of 89700 + 1200 (S.D.). An inter-mediate form with an Mr of 81 500 + 900 (S.D.) wasalso found. The mature myeloperoxidase stored inthe azurophil granules consisted of two polypep-

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I. Olsson, A.-M. Persson and K. Str6mberg

MediLum

-1

46.0-

30.0-

1 3 6

Puilse (h)

9 24 3 6 9 241

Pulse (h.)

(h)

1-

_

>2

.e-

J,

0

~--X3-- ------10

Iclub itio ln ti a tCl- IulsC (li)20

Fig. 1. Pulse-chase labelling with [35S]methionine of myeloperoxidase in HL-60 cells(a) HL-60 cells were pulse-labelled with [35S]methionine for 60min and the label was chased for 3, 6, 9 and 24h.Extraction, immunoprecipitation with anti-myeloperoxidase serum, SDS/polyacrylamide-gel electrophophoresisand fluorography were performed as described in the text. The positions of the two myeloperoxidase precursor poly-.peptides (1 and 2) and the two mature myeloperoxidase polypeptides (3 and 4) are indicated to the right; Mr markers(X 10-3) are shown to the left. (b) The individual bands corresponding to the M, 90000 (1) and 82000 (2) precursor

polypeptides (0), the M, 62000 (3) (@) and M, 12400 (4) (A) mature polypeptides were localized on the dried gelusing fluorography, excised and counted by liquid-scintillation. Data are also given for the Mr 90000 polypeptide ofthe medium (O----O), which was the only species of myeloperoxidase detected extracellularly.

1984

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(a)

10- ,M.

97.4-

Cells

12.3- * - 4

Biosynthesis of myeloperoxidase

tides with Mr values of 61670+2400 (S.D.) and12375 + 100 (S.D.). In addition, bands were usuallyseen on the gels corresponding to Mr values ofapprox. 40000-45000. The latter componentsare regarded as degradation products of myeloper-oxidase.

Biosynthesis of myeloperoxidase in HL-60A pulse-chase experiment with [35S]methionine

is shown in Fig. 1. After 1 h pulse, the major bio-synthetically labelled myeloperoxidase consists ofan Mr 90000 polypeptide (component 1); label isalso associated with polypeptides with Mr values of82000 (component 2), 62000 (component 3) and12400 (component 4). During a subsequent 24hchase, radioactivity of components 1 and 2decreases steadily (after an initial increase) with aconcomitant increase of components 3 and 4.Significant increases in components 3 and 4 do not,however, occur until after 6h of chase. Theseobservations indicate that myeloperoxidase issynthesized as precursor polypeptides with Mrvalues of 90000 and 82000. Furthermore it isshown in Fig. 1 that the only form ofmyeloperoxid-ase which is released to the culture medium is theMr 90000 precursor polypeptide (component 1).Release to the culture medium occurs at a slowrate during the whole chase period, but only smallamounts are released.

1 h PuLlse 4 h Chase

Preincubation of HL-60 cells for 30min withcycloheximide (10g/ml) followed by [35S]-methionine labelling for 4h abolished all myelo-peroxidase biosynthesis (results not shown). Speci-ficity controls showed that myeloperoxid-ase-absorbed anti-myeloperoxidase serum did notprecipitate any labelled myeloperoxidase (resultsnot shown).

Subcellular processing of newly synthesized myelo-peroxidase in HL-60 cells

HL-60 cells were pulsed with [35S]methioninefor 1 h and the label was chased for 4, 8 and 18 h.Microsomal, granule and cytosol fractions wereprepared, extracted and immunoprecipitated withanti-myeloperoxidase serum. Fluorograms ob-tained after SDS/polyacrylamide-gel electro-phoresis are shown in Fig. 2. 35S-labelled Mr 90000polypeptide (component 1) is formed in pulse-labelled cells present both in crude microsomal,granule and cytosol fractions. Approximately thesame pattern is seen after a 4h chase. However,significant changes occur during an 8 h chase, withoccurrence of the Mr 62000 and M, 12400 poly-peptides in both microsome and granule fractions.In addition the M, 82000 polypeptide is visible at8 h. Its presence in the granule fraction is interest-ing because component 1 is faint in that fractionbut predominant in the microsomal fraction.

8 h Chase 18 h Chase

10 3 Mr

69.0

46 0

30.0

1 2.3

K SP -.,ePRR:e4: ' x i*m_

2

3

4

M G C M G C M G C M G C CM

Fig. 2. Pulse-chase labelling with [35S]nethionine ofmyeloperoxidase in HL-60 cells and distribution of labelled myeloper-oxidase in subcellular fractions

HL-60 cells were pulse-labelled with [35S]methionine for 60min and the label was chased for 4, 6 and 18h. Crudesubcellular microsome (M), granule (G) and cytosol (C) fractions were prepared. The analysis result for 18 h chaseculture medium (CM) is also given. Extraction, immunoprecipitation with anti-myeloperoxidase serum,SDS/polyacrylamide-gel electrophoresis and fluorography were performed as described in the text. The positions ofthe two myeloperoxidase precursor polypeptides, M, 90000 (1) and M, 82000 (2), as well as the two mature myeloper-oxidase polypeptides, Mr 62000 (3) and Mr 12400 (4), are indicated to the right; M, markers (x 1o3) are shown tothe left.

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10 3XMr

97.4-

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I. Olsson, A.-M. Persson and K. Strdmberg

(a)

N 2

3

46.0 -

30.0-

12.3- - 4

97.4

69.0

46.0-

30.0 -

12.3-

20 -

_ 15-

-1

10

-5

5-

(b)

*-~ 1

2_ 3

4

(c)

-1.26

- 1.22 --

E

-1.18 >~ca)

1.148

- 1.10

5Fraction no.

Fig. 3. Pulse-chase labelling with [35S]methionine ofmyeloperoxidase in HL-60 cells and distribution oflabelled myeloper-oxidase in subcellular fractions

HL-60 cells were pulse-labelled with [35S]methionine for 4h and the label was chased for 18 h. The post-nuclearsupernatants obtained after pulse (a) and 18 h chase (b) were centrifuged in a sucrose density gradient for separationof subcellular organelles. Extraction, immunoprecipitation with anti-myeloperoxidase serum, SDS/polyacrylamide-gel electrophoresis and fluorography were performed as described in the text. The positions ofthe two myeloperoxid-ase precursor polypeptides, Mr 90000 (1) and Mr 82000 (2), as well as the two mature myeloperoxidase polypeptides,Mr 62000 (3) and M, 12400 (4), are indicated to the right; M, markers are shown to the left. Also shown (c) are the

1984

Biosynthesis of myeloperoxidase

During an 18 h chase most of the radioactivity ofthe M, 90000 and 82000 components disappearedwith a concomitant increase of M, 62000 and12400 components. The results of Fig. 2 also showthat only the M, 90000 precursor form is releasedto the cytosol. Furthermore, this was the only formof myeloperoxidase which was released to theculture medium.

In a separate experiment, repeated twice, HL-60cells were pulse-labelled with [35S]methionine for90min and the label was chased for 4, 8 and 18 h.Thetotal 35S activityofimmunoprecipitated myelo-peroxidase was determined in culture medium,microsomal, granule and cytosol fractions. Com-pared with the 35S-labelled myeloperoxidase ofthe culture medium present after the pulse anincrease by 63% was seen during the chase period,after which labelled myeloperoxidase amounted to2.1% of the total labelled protein of the medium.Similarly, microsomal myeloperoxidase decreasedby 29% during the chase and granule myeloper-oxidase increased by 59%, while cytosol myeloper-oxidase decreased by 38%. Microsomal, granuleand cytosol [35S]myeloperoxidase amounted to0.31%, 0.63% and 0.13%, respectively, of totallabelled protein after 18 h chase. These data aremean values from two separate experiments.

Interpretation of the data of Fig. 2 is compli-cated by the fact that cross-contamination existsbetween the granule and microsomal fractions.This is obvious from Table 1, showing results fromlysosomal enzyme assays as well as distribution ofprotein synthesis sites and Golgi elements. Lyso-somal enzymes are, however, concentrated in the

granule fraction, whereas protein synthesis sitesand Golgi elements are concentrated in themicrosomal fraction. The cross-contaminationbetween the granule and microsomal fractions mayexplain the presence of Mr 90000 precursor myelo-peroxidase also in the granule fraction after 1 hpulse labelling (Fig. 2). Therefore attempts weremade to separate these organelles by using sucrose-density-gradient centrifugation of post-nuclearsupernatant (Fig. 3). Newly synthesized protein,identified by a brief pulse-labelling with [35S]-methionine, banded at a density of 1.14-1.17g/ml;[3H]uridine incorporation sites into RNA bandedat an identical density; Golgi-derived elements,identified by a 3min pulse with [35S]sulphate,banded at a density of 1. 14g/ml. Thus anyseparation between endoplasmic reticulum andGolgi elements was not accomplished. However, acomplete separation was achieved between theseorganelles and granules identified by the distribu-tion in the gradient of total myeloperoxidase(density 1.19-1.23g/ml), which is a specific markerfor azurophil granules.HL-60 cells were pulse-labelled for 4h with

[35S]methionine and the label was chased for18 h. Post-nuclear supernatants were analysed bysucrose-density-gradient centrifugation (Fig. 3).The density gradient fractions were extracted andimmunoprecipitated with anti-myeloperoxidaseserum. The fluorograms obtained upon electro-phoresis are shown in Fig. 3. In pulse-labelled cells,the Mr 90000 precursor polypeptide was located insubcellular fractions with a density of 1.14-1. 17g/ml and in the cytosol, represented by the top

Table 1. Distribution ofa-mannosidase and P-hexosaminidase in subcellularfractions obtained by differential centrifugation ofan HL-60 cell homogenate

Enzyme activity is expressed as units (1 ymol of substrate split/min) per 109 cell equivalents. Data are also given forthe percentage distribution of 35S-labelled macromolecules after a 3min pulse with [35S]sulphate (identifying Golgielements) and the distribution of [35S]methionine-labelled macromolecules after a 10min pulse (identifying sites fornewly synthesized protein; endoplasmic reticulum).

a-Mannosidasea-Mannosidase,B-Hexosaminidase[35S]Sulphate (Golgi)[35S]Methionine (endoplasmic

reticulum)

WholepH homogenate4.5 0.725.5 0.564.5 1.34

78%98%

Nuclearfraction0.110.110.105.5%5.2%

Granule Microsomalfraction fraction0.250.160.5215.0%23.9%

0.190.080.2057.5%46.3%

density gradient (----), the distribution of 35S-labelled macromolecules after a 3min pulse with [35S]sulphate (0)identifying Golgi elements, the distribution of [35S]methionine-labelled macromolecules after 1Omin pulse labelling( x ) identifying sites for newly synthetized protein and the distribution of [3H]uridine incorporation into RNA after3h labelling (@). Immunochemical determination of total myeloperoxidase content (marker for azurophil granules)of the fractions is also shown (U).

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I. Olsson, A.-M. Persson and K. Stromberg

fractions of the gradient; virtually no Mr 90000polypeptide was associated with the dense granule-containing fractions. During an 18 h chase, signifi-cant changes occurred; the Mr 90000 polypeptideis still visible in fractions with a density of 1.15-1.18g/ml and in the cytosol, the Mr 82000 poly-peptide is present with a peak density of 1. 18 g/ml,but most of the radioactivity of the M, 90000 poly-peptide has now disappeared, resulting in anincrease of the mature myeloperoxidase poly-peptides with M, values of 62000 and 12400. Thelatter were concentrated in the fractions contain-ing azurophil granules, but some was also presentin organelles with a lower density. By excising thegel and counting the 35S activity associated withprecursor and mature myeloperoxidase forms,about 70% transfer of radiolabel to the matureform was demonstrated to take place during thechase period.

Synthesis ofmyeloperoxidase by normal marrow cellsExperiments with normal marrow cells from

healthy individuals gave similar results as forHL-60. Marrow cells were labelled with [35S]-methionine for 18 h and crude subcellular fractionswere prepared, extracted and analysed bySDS/polyacrylamide-gel electrophoresis (Fig. 4).The microsomal fraction contained the myeloper-oxidase precursor polypeptides as well as themature polypeptides. The granule fraction lackedthe Mr 90000 polypeptide but contained the othermyeloperoxidase forms. As for HL-60 only the Mr90000 polypeptide was detected in the cytosol. Theculture medium from incubations with marrowcells was difficult to analyse by immuno-precipitation, because unspecific precipitatesoccurred also with non-immune serum, but theresults indicated secretion exclusively of the M,90000 polypeptide (results not shown).

Peptide mappingPartial proteolysis of the [14C]leucine-labelled

Mr 90000, 82000 and 62000 myeloperoxidasepeptide forms, as well as the presumed degradationforms with Mr values of 40000-45000, is shown inFig. 5. When the major precursor (Mr 90000) andmature form (Mr 62000) were compared the onlydifference was in the relative intensity of somepeptide bands in the M, range 30000-40000. Thusthese two forms have profound structural similari-ties. Partial proteolysis of the M, 12400 subunitgave very small fragments and the data aretherefore not included in Fig. 5. Regarding the M,82000 component, the track in Fig. 5 is faintbecause of lack of labelled material; the patternseems, however, to be identical with that of the Mr90000 polypeptide. Also the track in Fig. 5 for thepresumed degradation forms is faint but the

10 3,m,

97.4---2

30.0

1 2.3 4

M G C

Fig. 4. [35S]Methionine labelling of myeloperoxidase inhuman marrow cells and distribution of labelled myeloper-

oxidase in subcellular fractionsMarrow cells were labelled with [35S]methionine for18 h. Crude subcellular microsome (M), granule (G)and cytosol (C) fractions were prepared. Extraction,immunoprecipitation with anti-myeloperoxidaseserum, SDS/polyacrylamide-gel electrophoresis andfluorography were performed as described in thetext. The positions of the two myeloperoxidase pre-cursor polypeptides, M, 90000 (1) and Mr 82000 (2)as well as the two mature myeloperoxidase poly-peptides, M, 62000 (3) and M, 12000 (4) areindicated.

pattern is clearly related to that for the myeloper-oxidase forms.

Discussion

Studies of biosynthesis and processing of neutro-phil granule proteins require cells with activegranulogenesis. The promyelocytic HL-60 cell line(Gallagher et al., 1979) is useful because itproduces azurophil granules with a high myeloper-oxidase content. The present results indicate thatmyeloperoxidase is synthesized as Mr 90000 andMr 82000 precursor polypeptides, which aretrimmed to Mr 62000 and 12400 polypeptides,

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Biosynthesis of myeloperoxidase

10 3\, Mr

69.0

46.0. _Www

30.0 --- _KU

(a) (b) Icl ((]I

Fig. 5. Peptide maps of ['4Clleucine-labelled myeloper-oxidase

HL-60 cells were labelled with [14C]leucine for 16h,extracted and immunoprecipitated with anti-myelo-peroxidase followed by SDS/polyacrylamide-gelelectrophoresis. Regions of the gel corresponding tothe M, 90000 (a); 82000 (b), 62000 (c) and 40000-45000 (d) myeloperoxidase species were localizedand cut out. Electrophoresis was carried out asdescribed in the text.

corresponding to the size of chains found inmature neutrophils (Andrews & Krinsky, 1981).The data confirm some results from a recent reporton myeloperoxidase synthesis in HL-60 cells(Yamada, 1982).The polypeptide chains of higher Mr were

identified as precursors to those of lower Mrbecause they were precipitated by antibodiesraised against neutrophil myeloperoxidase andbecause the large chains decreased during pulse-chase experiments with the appearance of maturemyeloperoxidase polypeptide chains. Thus thedifferent forms are not synthesized independently.The peptide patterns generated by partial proteoly-sis with V8 proteinase also supported a precursor-product relationship. Native myeloperoxidase con-tains two heavy-light protomers, which are joinedby a single disulphide bond between the heavy

subunits (Andrews & Krinsky, 1981). The M,reported for the two subunits is 57500 and 14000(Andrews & Krinsky, 1981) roughly correspondingto the apparent M, values of 62000 and 12400 thatwe found for fully processed myeloperoxidaselocalized in the granule fractions (see Fig. 3). Thespecies seen with M, values of 40000-45 000 wereregarded as degradation products because they donot correspond to any known form of myeloper-oxidase; they may represent intracellular degrada-tion of newly synthesized myeloperoxidase. Sincethe apparent M, of the major precursor form was90000, the conversion to Mr 62000 and 12400subunits most likely occurs by proteolytic cleavage.It is unlikely that this conversion is a result ofmodification of carbohydrate side chains only. Therelationship between the Mr 90000 and 82000precursor polypeptides of myeloperoxidase is notclear. Our data from subcellular fractionation ofHL-60 cells suggest that the Mr 82000 form is anintermediate or degradation product; it is localizedin a subcellular organelle which is distinct fromthat holding most of the Mr 90000 precursor ormature myeloperoxidase. The subcellular site forthe M, 82000 peptide may be a pregranulestructure.

Subcellular fractionation showed that com-pletely processed myeloperoxidase is concentratedin the granule fraction, which is the storage sitefor myeloperoxidase (Bainton & Farquar, 1970;Bainton et al., 1971; Bretz & Baggiolini, 1974;Spitznagel et al., 1974). The finding of someprocessed myeloperoxidase also in fractions withlower density than granules either indicates con-tamination with granule material, release of somegranular myeloperoxidase during sedimentation inthe gradient or processing during intracellularprogression of newly synthesized material. Thesucrose gradient system used did not resolve clearlyendoplasmic reticulum and Golgi elements, judgedfrom brief pulse-labelling experiments to localizesubcellular sites for protein synthesis and sulpha-tion, respectively. Processing of myeloperoxidasein HL-60 cells is relatively slow; mature myeloper-oxidase polypeptides were significantly producedonly after 4-8 h of chase with [35S]methionine.The finding of the myeloperoxidase precursors

mainly associated with microsomal elements andthe mature polypeptides with granules is incon-sistent with results from a recent report, whichshowed that myeloperoxidase precursors werepresent exclusively in the soluble fraction of thecells, but the matured polypeptide chains were inboth the soluble and 'granule' fractions (Yamada,1982). However, in the latter report the homo-genate was treated with a detergent, which couldhave solubilized precursor myeloperoxidase andsome granule myeloperoxidase.

Vol. 223

919

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The M, 90000 precursor was the only myeloper-oxidase form detectable in cytosol and extracellu-lar medium. It cannot be ruled out that cytosolmyeloperoxidase originated from solubilizationduring the homogenization procedure of, e.g.,vesicles involved in transport ofthe precursor poly-peptide. Further work is needed to clarify thispossibility. Despite the, fact that considerableleakage occurred of both lysosomal and micro-somal contents to the cytosol (Table 1), the latterwas totally devoid of labelled mature myeloper-oxidase. Thus mature myeloperoxidase is tightlybound to subcellular sites. The exclusive secretionof the M, 90000 precursor form of myeloperoxi-dase is of interest because myeloperoxidase isdetectable in plasma (Olofsson et al., 1977; Olssonet al., 1979). It remains to be determined if it iscirculating in precursor form and has peroxidaseactivity.

Biosynthesis of lysosomal enzymes differs fromthat of secretory proteins because high-mannoseoligosaccharide side chains of lysosomal enzymesare modified by phosphorylation (Hasilik &Neufeld, 1980b; Neufeld, 1981). A receptor formannose 6-phosphate may therefore play animportant role in directing acid hydrolases tolysosomes (Kaplan et al., 1977; Sahagian et al.,1981). Both the precursor polypeptides of myelo-peroxidase, as well as the mature Mr 62000 poly-peptide, were susceptible to digestion with endo-glycosidase H indicating that they have high-mannose oligosaccharide side chains (I. Olsson,A.-M. Persson& K. Stromberg, unpublished work).It remains, however, to be determined if myelo-peroxidase and other products of the lysosomal-like azurophil granules of neutrophils such asneutral proteinases (Olsson & Venge, 1980) con-tain phosphomannosyl residues to establish thatthe mannose 6-phosphate receptor is part of ageneral pathway for lysosomal protein segregation.

Several lysosomal enzymes have now beenshown to have precursor forms (Hasilik & Neufeld,1980a; Erickson et al., 1981; Skudlarek & Swank,1981), which may be a prerequisite for transport tothe lysosomes or protection against destructiveenzymes. Lysosomal enzymes of fibroblasts arepresent in the extracellular medium in precursorform (Neufeld, 1981) and may be recaptured by thecell through a receptor for mannose-6-phosphate(Kaplan et al., 1977) and inserted into lysosomes.Further studies on the processing and intracellulartransport, storage and secretion of neutrophilgranule proteins are now feasible using the systemsand techniques employed in the present work.

This work was supported by the Swedish CancerSociety, Alfred Osterlund Foundation, and the MedicalFaculty of Lund.

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1984