Vol.

187,

No.

September

BIOCHEMICAL

3, 1992

30,

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1992

EXPRESSION

Kathryn

OF RECOMBINANT MYELOPEROXIDASE EXPRESSION SYSTEM

L. Taylor,

David.

August

13,

J. Uhlinger

1572-1578

USING A BACULOVIRUS

and Joseph

M. Kinkade,

Department of Biochemistry School of Medicine, Atlanta,

Emory University Received

AND

Jr.

GA 30322-3050

1992

SUMMARY: Myeloperoxidase (MPO) is a glycosylated heme-containing enzyme present in the azurophilic granules of normal human polymorphonuclear neutrophils. This enzyme plays a major role in the microbicidal activity of the host defense system by catalyzing the formation of the potent oxidant, hypochlorous acid. Although the amino acid sequence of MPO has been deduced from the cDNA, the structural basis for the observed heterogeneity of this enzyme is not known. Furthermore, the nature of the prosthetic group and its mode of linkage to the apoprotein has To address questions regarding the structural features of not been determined. MPO, which arise during the complex posttranslational processing of this enzyme, we utilized a baculovirus system to express MPO in Sf9 insect cells. Two glycosylated, single-chain precursor species of MPO were observed: an 84 kDa species that was secreted and a 74 kDa species that was cell-associated. This is the first report of an expression system in which a cell-associated MPO precursor undergoes posttranslationalproteolytic pr0cessing.o 1992 AcademicPRESS,lnc.

Myeloperoxidase PMNS (1).

(MPO)

In response

formation

of

chloride

ion

potent Thus,

MPO plays

other

important

of

MPO

into

AG

glycosylated this

precursor

trimming)

through

monomeric

form

other

MPO is

agent, a major

constituent

roles

occurs

during

of complex,

preproprotein undergoes single

cell

the line

chain

of the enzyme consisting

the

and acid,

catalyzes using

defense

(3).

promyelocytic

AG of human

shown the

kDa (5,6). (bothproteolytic

intermediates of one heavy

(-80 (-60

the

HzOz and

system

The

of the

synthesis stage

posttranslational have

of -89-90 processing

released

in the host

physiological

HL-60

of

hypochlorous

role

a series

the human myeloid

shown that

protein

stimuli,

microbicidal

and involves

using

of a large

to various

the

differentiation Studies

a major

(2).

PMN, and may have packaging

is

and of

events initial

PMN (4).

synthesis

We and others

have

andcarbohydrate kDa and -74

kDa)

kDa) and one light

to a (-15

ABBREVIATIONS: AG, azurophilic granules; BME, beta-mercaptoethanol; CEC, cation exchange chromatography; CTAB, cetyltrimethylammonium bromide; Endo H, Endoglycosidase H; FPLC, fast protein liquid chromatography; HPLC, high pressure liquid chromatography; MPO, myeloperoxidase; PMN, polymorphonuclear neutrophils; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Sf9, Spodoptera frugiperda cells ; TFA, trifluoroacetic acid. 0006-291X/92 Copyright All rights

$4.00

0 1992 by Academic Press. Inc. of reproduction in any form reserved.

1572

Vol.

187,

kDa)

No.

subunit

which

chromatographic cited

BIOCHEMICAL

3, 1992

subsequently

dimerizes

RESEARCH

to form mature

isoforms

of mature

sequence

of the MPO gene

has been

reported

sequence

by several

be confirmed

MPO have

heterogeneity addition,

The amino

(10). groups

been

COMMUNICATIONS

MPO (5-8).

identified

is

also

is

linkage Cully

al.

Three

major

(9 and references

in BHK cells

was slowly

released

proteolytic

prosthetic

have

We now report

not the

the

endoplasmic

Moguilevsky active

secreted

recombinant

MPO in Sf9 insect 84 kDa species,

cells.

Using

system

posttranslational

MATERIALS

to MPO MPO

(9).

In

per 150 kDa dimer)

precursor

not

described this

cells for

species

undergo the

was secreted

system,

and an intracellular

undergone

(two

and did have

although

this

of MPO has yet

This

by HL-60

the use of a baculovirus

cDNA

of an 85 kDa glycosylated

that

was not observed

the

determined.

(15)

al.

from

glycosylation

group

reticulum

et

deduced

structure

in

been

and 11 introns

The structural basis for recently demonstrated that

expression

84 kDa MPO species

89 kDa species

to have

12 exons

transfectedwithhumanMPO-cDNA.

glycosylated appeared

have

reported

processing

to the

primary enzyme.

of the heme-like

from

of an enzymatically proteolytic

the

we have

processing.

be similar

but

of

has been

differences

solely

apoprotein

(14)

MPO species

sequence

to

due

to the

et

acid

although

unclear,

not

consisting

of the purified

the structure

and its

(11-13)

by sequencing

heterogeneity

that

BIOPHYSICAL

therein). A partial

Again,

AND

further

expression

by CHO cells.

species

appeared

to

(16). the expression we observed

glycosylated proteolytic

of human a secreted,

74 kDa species processing.

AND METHODS

Sf9 cells were obtained from Invitrogen and grown in TMN-FH media supplemented with 10% fetal calf serum and 10 ml/L Fungizone (Gibco-BRL)(17). Gene Clean was from RPI, restriction enzymes and Lipofectin reagent were from Gibco-BRL. Endo H and Glycopeptidase F were from Boehringer Mannheim.

Construction and Orientation of the Baculovirus Expression Vector. The full length cDNA for human MPO (3.2 kb) was obtained by EcoR I treatment of the plasmid MP-H17 (12), and was purified by electrophoresis on a 0.8% agarose gel (18) using GeneClean. The cDNA was ligated into the EcoR I site of the baculovirus plasmid vector, pSynXIV VI+X3 (19), and the resulting plasmid was used to transform E. coli. Plasmids were isolated from selected colonies and screened for orientation of the MPO gene using the unique restriction sites Nde I and Xba I. The plasmid containing the MPO gene in the correct orientation was designated pSynMPO4. Transfection. Sf9 cells were transfected with pSynMPO4 and wildtype viral DNA using the Lipofectin reagent according to the manufacturer's suggested protocol. Screening for recombinants was performed by plaque assay as previously described (17). MPO species were identified by SDS-PAGE on 13% polyacrylamide minigels followed by Western blotting and development with a monospecific rabbit polyclonal antibody to human MPO (8). Expression and Purification of Recombinant MPO Species. Sf9 cells were infected using a viral stock solution and grown in Excel1 401media (JRL) with or without After 4-7 days, the hemin supplementation (10 pM, added 24 hrs post infection). medium and cells were separated by centrifugation at 1400 rpm x 10 min and the

1573

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3, 1992

BIOCHEMICAL

AND

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RESEARCH

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The cells were medium was treated with 1 mM PMSF and stored at -2O'C until used. extracted using 0.3% CTAB in 10 mM potassium phosphate containing 150 mM NaCl, 2 mM PMSF, 2 mM dithiodipyridine and 2 mM EDTA, pH 7.4. The extract was subjected to one freeze-thaw (-2O'C) followed by vortexing and removal of insoluble material by centrifugation at 40,000 g x 15 min. The cell extract or medium was adjusted to pH 6.0 with 1 M KOH, diluted with one volume of distilled water and recentrifuged. The supernatant was loaded on a 5 ml CM-cartridge (Biorad) preequilibrated in 50 mM potassium phosphate, pH 6.0 (Buffer A). The column was washed (1 ml/min, 50 ml) with the same buffer using a Pharmacia FPLC system and protein was eluted with a 50 ml linear gradient of O-O.5 M NaCl in Buffer A. Column fractions (1 ml) were monitored using immunodot blotting procedures and selected fractions combined, diluted with one volume of 20 mM sodium borate, pH 8.4 (Buffer B), and loaded on a Pharmacia Mono S column (0.5 The column was washed with Buffer B (0.5 ml/min, 5 ml) and then eluted x 5 cm). with a 35 ml linear gradient of O-O.7 M NaCl in Buffer B. Fractions (1 ml) were monitoredby immunodotblotand Westernblotting procedures. Peroxidase activity was measured using the guaiacol assay (20). Reverse Phase HPLC ofRecombinantMP0 Snecies. Immunoreactive fractions from the Mono S column were concentrated to approximately 125 pl in a Centricon 30 microconcentrator (Amicon) and 375 ~1 8 M guanidine HCl in 50 mM Tris HCl containing 1 mM EDTA, pH 7.5, were added. BME (100 mM) was then added, followed after 1 hr at room temperature by the addition of 167 ~1 of 0.5 M iodoacetamide. The sample was incubated at 4OC overnight, then 100 ~1 of 1% TFA was added and the volume brought to 1.05 ml with water. The sample (1 ml) was subjected to HPLC and fractions were collected for analysis by SDS-PAGE and Western blotting.

RESULTS AND DISCUSSION The

construction

pSynMPO4,

is

recombinant cells,

virus

prepared

expressed

MPO species (lanes

1 and

4),

activity

low

levels

of activity

al.

(21)

increase

addition

increase

extracts

kDa species

system

enzymatic

Treatment intracellular

cells,

kDa obtained

observed in

the

in

our

as well

cell

74 kDa (Figure

to

Sf9

system

did

is

2, lanes

7). very

presently with

Asseffa

protein.

result

in

et

However,

a significant both

the

appeared

observed

in

crude

species

cellto be cell found

unknown. Endo H demonstrated

2 and 5) and the

secreted

oligosaccharides

Mr of the cell-associated 1.574

(lane

in a dramatic

cell-associated

mannose

extracts while

P-450,

resulted

84 kDa species

initially

species

cell

media

purification,

secreted

of which

recombinant

cells,

active not

further

as the

culture

infect

extract.

kDa immunoreactive

species (lane 8) containedN-linkedhigh of human MPO (22). The apparent

the

human cytochrome

The activity

of the recombinantMP0

The

immunoreactive

from

from infected

to the system

Upon

the nature

2, two major

of enzymatically

to

activity.

may be due to an -36

of -74

of hemin

activity.

utilized

vector,

section.

human MPO was used to detect

to express

addition

plasmid

and Methods

was then

in the media

detected

10 PM hemin

74 kDa species of

baculovirus

As shown in Figure

of expression

peroxidase

in uninfected the

the

level

of in

associated devoid

that

in the

against

was undetectible were

the

plasmid

a species

the baculovirus

found

this

system.

and -84

Peroxidase Using

antibody

expressed,

of

1 and the Materials

using

by this

were

orientation

in Figure

and a polyclonal

protein

the

and

described

that

both

84 kDa MPO

characteristic species

decreased

to

Vol.

187,

No.

BIOCHEMICAL

3, 1992

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

POLYHEDRON

pSYN

Dlasn MPO_ I

gene

Fin. figure described I, were panel): standards; same as contains correct

-21 5.0 4.2 3.5

kb kb kp kb

2.0 .1.9 1.5 1.3

kb kb kb kb

Construction of a Baculovirus Vector containing the MPO Gene. The top shows a map of the expression vector, pSynMP0, which was constructed as in Materials and Methods. The unique restriction sites, Nde I and Xba used to determine the orientation of the MPO gene as follows (lower Lane 1 contains the 3.2 kb MPO gene and EcoR I digested pSynXIV VI-3 as lanes 2-4 contain EcoR I plasmid digests of pSynMP0, lanes 5-7 are the 2-4, respectively, except digested with Nde I and Xba I, and lane 8 Mr standards. Lanes 5 and 7 contain pSynMP0 with the MPO gene in the orientation whereas pSynMP0 in lane 6 has the incorrect orientation.

Cells +Hemin

Cells -Hemin Lane

1

2

3

4

5

Media 6

7

0

Mr 9

x 1c3 f97 4-76 4-66 4-45 4-36

Fiz. 2. Western Blot Analysis of Recombinant IlPO. Infected Sf9 cells were grown for 4 days in the presence or absence of 10 pM hemin. Samples were prepared by adding 2 ~1 2X sample buffer (125 mM Tris HCl, pH 6.8, containing 2% SDS, 20% glycerol, 10% BME and 0.025% bromphenol blue) to 12 ~1 of media or 5 ~1 cell After 15 min at room temperature, the extract in a total volume of 15 pl. samples were boiled for 5 min, cooled and brought to a final volume of 40 11 containing 62.5 mM Tris HCl, pH 6.8, with the following additions: lanes 1,4,7, no additions; lanes 2,5,8, Endo H (0.8 mu); and lanes 3,6,9, Glycopeptidase F (0.8 U). Samples were incubated overnight at 37°C and driedusing a Savant Speed Vat . After reconstitution in 15 ~1 1X sample buffer, the samples were subjected to SDS-PAGE and Western blotting. 1575

Vol.

187,

-70

kDa after

vitro

No.

3, 1992

Endo H or

translation

these

BIOCHEMICAL

suggested

oligosaccharides.

74 kDa MPO species of the

is

The

apparent

after

Mr

to -79.5

Glycopeptidase

kDa species

the

difference the idea

the

level

that

is

not

Moguilevsky

et

clear

recombinant

work,

These

how

confirm

the

the recombinant

that

has undergone processing

was kDa

indicated

that

due

the 84

oligosaccharides.

indicated solely

observations

to also

from proteolytic

beyond

with

and

secreted

that

the

Mr

differences provided

in support

processing

of the

The reason(s) from

cells

beyond

increased

to

expression

attributed

the

(89 kDa)

extracts from cells of heminmay result Glycopeptidase

difference in

the

of

the

74 kDa

products

of cell 1owerMr

immunoreactive

1576

cells.

(24)

that

89 kDa precursor, but

sequence.

and

et al.

not

(15)

proteolytic

Interestingly, our for proteolytic

be a prerequisite

of MPO. size

or

kDa)

observed

absence

The latter species.

extracts

seen

85 kDa

by HL-60

by Moguilevsky

trimming,

different in cells

(45-66

the

the 84 kDa

and Olsson

the

observed

in band

presence

to

secreted

of

between hemin

is

the not

74 kDa clear.

protein loading on the gel, or supplemented with hemin could possibility

might

be due

to

However, immunoreactive bands appeared to be more prominent in

grown in the absence of hemin, suggesting in a 74 kDa species which is less susceptible

Ftreatment

an additional

level

by

to have the same N-terminal

by Arnljots

signal

form

related Nevertheless,

was found

report

MPO at

observation. of

to degradation

(14).

of heme may not

the

this

et

al.

observed

as the 84 kDa species

may be

89 kDa precursor

of the putative

proprotein for

(15)

al.

structure these

oligosaccharide

addition

grown

of

recoveries during extraction, amounts of the 74 kDa species

contribute

84 kDa MPO species

84 kDa species

cleavage that

recombinant

earlier

posttranslational

suggest

processing

produced

processing

MPO species

cell-associated

not

resulted

active

associated

all

was

by Cully et

enzymatically

indicate

Different increased

complex

8) and to -76.5

type

glycosidase

These

either

observed

heme becomes

species

either

the

Moguilevsky

as the

observations

results

results mannose

the

has the same primary or

MPO species

observedby

sequence

whether

(15)

al.

present

species

had

recombinant

via

kDa

2, lane

These

between

74 kDa species

contain this

derived 84

(Figure

as high

species

species

(7).

9).

3 and 9).

not

b

kDa (23),

of the proprotein.

It the

two

lanes

the

Mr

with

these

(compare

for

(lane in

did

kDa

whether

recombinant

as well

treatment

between

glycosylation

secreted

complex

but

Since

of-77

74

74 kDa intermediate

Endo H treatment

difference

following

species

the

F treatment

contained

Furthermore,

of

2 and 3).

cell-associated

cells

COMMUNICATIONS

species

processing

in HL-60

kDa after

(lanes

to be determined

to the

89 kDa MPO precursor

decreased

the

remains

similar

RESEARCH

F treatment

proteolytic It

BIOPHYSICAL

a nonglycosylated

that

posttranslational

N-linked

in

Glycopeptidase

of MPO mRNA produced

observations

undergone

AND

that the presence to &gra&tion.

fromhemin-supplemented band

(Figure

2, lane

cultures

also

6) suggesting

Vol.

187,

No.

MtStds3

3, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

,2,3,4

1

x10

MPO -

2

0.02

-

0.01

-JJWk

=: z

03

04

5

0

10

15

20

ELUTION

25

30

VOLUME

35

40

.

(ml)

Fin. 3. SDS-PAGE Analysis of Purified Recombinant MPO. Immunoreactive fractions from the Mono S column were combined, concentrated using a Centricon 30 microconcentrator and subjected to SDS-PAGE as described in the Materials and Methods section. The gel was stained for protein using 0.15% Coomassie Blue in acetic acid/methanol/water (l/4/5). Lane 1 contains Mr standards, lane 2 contains the secreted recombinant MPO species, lane 3 contains the cellassociated recombinant MPO species and lane 4 contains purified human MPO. Fig. 4. Reverse Phase HPLC of Cell-Associated Recombinant MPO. The 74 kDa cell-associated MPO species previously isolated by CEC was reduced and alkylated as described in the Materials and Methods section. The sample was subjected to chromatography on a Beckman RPC318 column (0.5 x 20 cm) preequilibratedwith 0.1% TFA in water at 1 ml/min. After5 minunder isocratic conditions, the column was eluted with a 50 ml linear gradient of O-100% acetonitrile containing 0.1% TFA. The column effluent was monitored at 280 nm and 1 ml fractions werecollected.

thathemin

supplementationmay

of the 74 kDa species, The manner

to qualitative

throughhemin

to

Using

that

described

CEC, mature

MPO species

for

recombinant

MPO has been

was also

Mr impurities shown) known as

(Figure

indicated

the multiple whether

has been

recombinant

during

peaks

this

heterogeneity

suggested

we have utilized human

oligosaccharides,

but

posttranslational

proteolytic

the

between the

30-35 multiple

a 74

kDa

exhibited

74 kDa species processing

of the

volume.

cell-associated

form

posttranslational the first

is not

moieties

MPO (25).

to obtain and

processing

not with

It

of normal

low

(data

associated

system

as

74 kDa

to remove

in carbohydrate isoforms

expression

represents

as well

by SDS-PAGE

ml elution

isoforms

recombinant

material

a

(Figure

major

step

of an MPO precursor 1577

CHO cells

of three

fractions

a baculovirus

MPO,

by CEC in

HPLC purification

of column

for

purified

of the precursor

Heterogeneity

is due to differences

to account of

level

a fraction processing.

from

NPO

band of immunoreactive

observed

Both MPO species

at the a final

Analysis broad

elution

species form.

4).

a single,

In summary, secreted

observed

in at least and further

were

shown to consist

is evident (9)) and this heterogeneity the mature forms of the enzyme (7,8). MPO species

changes

association

74 kDa and 84 kDa recombinant

similar

3)(15).

lead

possible

an

84

two kDa

of their

instance

in which

has been

observed

Vol.

in

187,

No.

BIOCHEMICAL

3, 1992

an expression

system.

species

suggests

that

involve

an as yet

undefined

AND

Furthermore,

structural

BIOPHYSICAL

the

differences

targeting

selective between

RESEARCH

COMMUNICATIONS

secretion these

of two

the

84 kDa

MPO forms

may

signal.

ACKNOWLEDGMENTS We thank Dr. Michiyuki Yamada for providing the MP-H17 plasmid containing the We also thank Dr. Lois Miller for her generous gift of the human MPO gene. pSynXIV VI%3, and the complementary wildtype viral DNA, as baculovirus vector, well as for advice concerning the establishment and screening of recombinants This work was supported in part by the U. S. utilizing the baculovirus system. Public Health Service, NIH grants ROl GA-22294 (National Cancer Institute to J.M.K.) and F32 DE-05541 (National Institute of Dental Research to K.L.T.). REFERENCES 1.

2. 3. 4. 5. 6. 7.

8. 9. 10

11 12 13

14

15 16 17

18 19

20 21 22 23 24 25

Schultz, J. and Kaminiker, K. (1962) Arch. Biochem. Biophys. 96, 465-467 Klebanoff, S. J. and Clark, R. A. (1978) The Neutrophil: Functional and Clinical Disorders, North Holland, Amsterdam S. W. and Swan, T. F. (1986) Biochem. J. 237, 601-604 Edwards, Bainton, D. F., Ullyot, J. L. and Farquhar, M. G. (1971) J. Exp. Med. 134, 907-934 Olsson, I., Persson, A. and Stromberg, K. (1984) Biochem. J. 223, 911-920 Hasilik, A., Pohlmann, R., Olsen, R. L. and von Figura, K. (1984) EMBO J. 3, 2671-2676 Akin, D. T. and Kinkade, J. M., Jr. (1986) J. Biol. Chem, 261, 8370-8375 Taylor, K. L., Guzman, G. S., Burgess, C. A. and Kinkade, J. M., Jr. (1990) Biochemistry 29, 1533-1539 Taylor, K. L., Guzman, G. S., Pohl, J. and Kinkade, J. M., Jr. (1990) J. Biol. Chem. 265, 15938-15946 , Morishita, K., Tsuchiya, M., Asano, S., Kaziro, Y. and Nagata, S. (1987) J. Biol. Chem. 262, 15208-15213 . Morishita, K., Kubota, N., Asano, S., Kaziro, Y. and Nagata, S. (1987) J. Biol. Chem. 262, 3844-3851 . Hashinaka, K., Nishio, C., Hur, S., Sakiyama, F., Tsunasawa, S. and Yamada, M. (1988) Biochemistry 27, 5906-5914 . Johnson, K. R., Nauseef, W. M., Care, A., Wheelock, M. J., Shane, S., Hudson, S ., Koeffler, H. P., Selsted, M., Miller, C. and Rovera, G. (1987) Nucleic Acids Res. 15, 2013-2028 . Cully, J., Harrach, B., Hauser, H., Harth, N., Robenek, H., Nagata, S. and Hasilik, A. (1989) Exp. Cell Res. 180, 440-450 . Moguilevsky, N., Garcia-Quintana, L., Jacquet, A., Tournay, C., Fabry, L., Pierard, L. and Bollen, A. (1991) Eur. J. Biochem. 197, 605-614 . Hur, S.-J., Toda, H. and Yamada, M. (1989) J. Biol. Chem. 264, 8542-8548 . Summers, M. D. and Smith, G. E. (1987) A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures. Tex. A & M Univ. Agric. Exp. Sta. Bull. No. 1555, pp. l-57 . Current Protocols in Molecular Biology, (Ausubel, F. M. et al., eds.) (1989) Vol. 1, John Wiley & Sons, NY . Wang, X., Ooi, B. G. and Miller, L. K. (1991) Gene 100, 131-137 . Chance, B. and Maehly, A. C. (1955) Meth. Enzymol. 2, 764-775 . Asseffa, A., Smith, S. J., Nagata, K., Gillette, J., Gelboin, H. V. and Gonzalez, F. J. (1989) Arch. Biochem. Biophys. 274, 481-490 . Nauseef, W. M. (1987) Blood 70, 1143-1150 . Koeffler, H. P., Ranyard, J. and Pertcheck, M. (1985) Blood 65, 484-491 . Arnljots, K. and Olsson, I. (1987) J. Biol. Chem. 262, 10430-10433 . Yamada, M., Mori, M. and Sugimura, T. (1981) Biochemistry 20, 766-771

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