Purification of a human alternative complement pathway inhibitor from hemolymph of larval fall armyworm (Spodoptera frugiperda)

Vol. 122, No. 3, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

August 16, 1984

Pages

1426-1433

PURIFICATION OF A HUMAN ALTERNATIVE COMPLEMENT PATHWAY INHIBITOR HEMOLYMPH OF LARVAL FALL ARMYWORM (SPODOPTERA FRUGIPERDA) Osmond Cancer

Received

Research

July

J.M.

D’Cruz

Program, Oklahoma

13,

and

Noorbibi

K.

Oklahoma Medical City, Oklahoma

FROM

Day

Research 73104

Foundation,

1984

Larval Spodoptera frugiperda hemolymph contains a specific inhibitor of the alternative pathway of human complement. This inhibitor was purified from larval hemolymph (HL) by 50% (NH4)2SO4 precipitation, DEAE-Sephacel chromatography and sequential gel-filtration on Bio-Gel 1.5m, 0.5m and Sephacryl S-200. Purified HL protein (M, = 110,000) was composed of two M, 55,000 polypeptide chains. Addition of purified HL protein to human complement resulted in a dose-dependent inhibition of RaRBC lysis and clumping of cells. The protein inhibitor provides a new tool for investigating the regulation of human alternative complement pathway.

The specific

vertebrate

complement

immunity

considered

to

and have

immunoglobulin

evolved

vertebrate

been

demonstrable

from

some

venom

in

and

erythrocytes

in

More which

(3).

can

recent

in

larval

cells titrations,

potentiate

the

presence

HL of and (RaRBC)

we

either

inhibitor

by

human

us

have

C by

reported

to

shown

Using

the

the

mori

specific activity serum

(1,4).

interact of

insect

Using

both

the

presence

of

not HL

(5).

presence lysis

kinetic a classical

we detected of

of and

factors

C

C assay,

an

rabbit fixed C pathway

the

lowest

cobra

HL contain with

a kinetic

(6).

has

C components

interaction

ACP-mediated

the

sheep

terminal

the

of

purified

unsensitized

L)

of

However,

with

that

is

C reaction

(HL)

specific

(Bombyx for

serum

C-like

vertebrate

C pathway.

an

scale, in

of

reactions of

type

lysis

amplifier

immune

detected

shown

an

development

vertebrate

the

silkworm

also

been

been

of by

the

hemolymph

mammalian

of

activator

to

studies

component(s) the

has

as

of

evolutionary

A true

has

regulate

to

invertebrate

invertebrates

factor

the

(ACP)

both

effector

prior On

C pathway

primitive

system

non-specific

(1,2).

alternative

(C)

red point

yet

Vol.

122,

No.

modulator In

BIOCHEMICAL

3, 1984

in

this

the

paper

zation

of

discuss

the the

MATERIALS

HL

of

we

describe

HL

inhibitor

larval

AND

fall the

phylogenetic

AND

BIOPHYSICAL

armyworm

RESEARCH

(Spodoptera

purification

and

specific

for

the

implication

of

this

frugiperda)

preliminary

ACP novel

of

COMMUNICATIONS

human

(7).

characteriserum

and

xenogeneic

reaction.

METHODS

Smith) larvae mass reared on Insects: Spodoptera frugiperda (J.E. artificial diet (8) were obtained from the USDA Plant Sciences Research HL was collected from adult larvae in chilled Facility, Stillwater, OK. The cell-free HL tubes containing a few crystals of phenylthiourea. obtained after centrifugation (2000 rpm/lO min, 4°C) was used for fractionation of the ACP inhibitor. Assay of ACP inhibitor: One ml of a l:3 dilution of normal human serum (NHS) in G-VBS-8mM EGTAZmM Mg++ buffer, pH 7.3 (9) was preincubated at 37°C for 60 min with dilutions of HL fractions (100 ~1) or with PBS as control. 200 pl of rabbit red ceI1 suspension in G-VBS-EGTA-Mg buffer was then added to the reaction mixture (i: 0.7 at 700 nm) and the incubation continued at 37°C for 60 min. The degree of inhibition of hemolysis was monitored by recording the turbidity of the reaction mixture at 700 nm (IO). Isolation of the ACP inhibitor: 90 ml of cell-free HL obtained from nearly 3000 adult larvae was made to 50% (NH4)2SO4 fraction. The dissolved precipitate after dialysis was applied on a DEAE-Sephacel column (1.5 x 60 cm) equilibrated with 1OOmM sodium phosphate buffer, pH 7.2. Five ml fractions were collected. The bound proteins were eluted with a linear NaCl gradient (O-500mM). Aliquots (100 ~1) of the dialyzed fractions were tested for the classical C pathway inhibitor (7) as well as for the inhibition of RaRBC lysis in human serum. The ACP inhibitor fractions obtained from DEAE-Sephacel step were then sequentially gel-filtered on Bio-Gel 1.5m (2 x 150 cm), Bio-Gel 0.5m (2 x 150 cm) and Sephacryl S-200 (0.5 x 100 cm) in 1OOmM sodium phosphate buffer (pH 7.2) in presence of 150mM NaCl. Low molecular weight contaminants were removed by dialysis (MW cut off 60,000). Gradient PAGE: The active fractions obtained from each chromatographic step were tested on a 4-30% gradient PAGE run exactly as specified by the manufacturer's instructions (Pharmacia Fine Chemicals, Piscataway, NJ). Standard proteins used as markers included: thyroglobulin 669K, ferritin 440K, catalase 232K and bovine serum albumin 66K. SDS-PAGE: SDS-polyacrylamide gel electrophoresis was carried out on 10% slab gels according to the method of Laemmli (11). Reduction of desulfide bonds was achieved with 5% (v/v) 2-mercaptoethanol in the sample buffer. Molecular weight was estimated with a Bio-Rad (Richmond, CA) HMW kit consisting of myosin 2OOK, B-galactosidase ll6K, phosphorylase s92K, bovine serum albumin 66K and ovalbumin 45K as standards. Amino acid analysis: 100 ug of the purified protein was hydrolyzed in vacua with 200 ul of 6 N HCl and heated at 108°C for 24 hrs. Amino acid analysis was performed on a Durrum Model D-500 amino acid analyzer.

RESULTS DEAE-Sephacel Spodoptera their

HL charge

chromatography resulted

difference

in and

the

of

50%

separation further

(NH4)2SO4 of

purified 1427

two

fraction C inhibitors

independently

of based (Fig.

on la).

Vol.

122,

No.

3, 1984

BIOCHEMICAL

AND

BIOPHYSICAL

FRACTION

FRACTION

Pig.

NO.

of ACP gradient ACP inhibitor tion, II. Bio-Gel 1.5 m elution Bio-Gel 0.5 m elution. Sephacryl S-200 elution performed as outlined 280 nm. Active fractions

DEAE-Sephacel

(B)

CC) (D)

The

classical

was

obtained

C pathway in

DEAE-Sephacel of peak

this

gave

was

active from

Subsequent the

the

inhibitor

the

high

a homogeneous

the

on

active

peak

of 1.5m

weight

anionic

Bio-Gel

0.5m

Additional

the

The

range

Bio-Gel

molecular

1~).

by

fractions.

in on

gel-filtration (Fig.

detected

flowthrough

fraction

C inhibitor

NO

frac-

Assays for ACP inhibitor activitv were profile. Fraction size 5 ml, (0) absorbance at the text. were pooled as indicated by the horizontal bars.

inhibitor

eluted

FRACTION

NO.

inhibitor from Spodoptera HL elution profile: I. Classical fraction. profile of ACP inhibitor.

in

COMMUNICATIONS

NUMBER

FRACTION

Purification

(A)

RESEARCH

Id).

1428

ACP

lOO-3OOmM resolved

resulted

C2 assay

inhibitor

the

bound

minor

inhibitor

(Fig.

lb).

in

the on

(7) to

Gel-filtration

NaCl.

proteins

gel-filtration (Fig.

limited

enrichment

Sephacryl

of S-200

Vol.

122,

No.

3,

BIOCHEMICAL

1984

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

2OOK 1161 -* 92K e

t

66K

b

a

c

d

e

4 5K

-

669k

-

uok

-

232k

-

wk

4t

nrrg

*

03

02 Fig. 2 Track a, Track b, Track c, Track d, Track e,

4-30X gradient DEAE-Sephacel Rio-Gel 1.5 m Bio-Gel 0.5 m Sephacryl S-200 Marker proteins

PAGE of ACP inhibitor active fraction; active fraction; active fraction: eluted fraction: of known molecular

Fig. 3 SDS polyacryiamide Marker proteins Track A Unreduced protein Track B Track C Reduced protein SDS-PAGE was on a 10% slab R 250.

Fig.

2 shows

obtained (c)

and

by

Sephacryl apparent

daltons

on

SDS-lo%

mobility single

varied

on at subunit

PAGE

pH. Mr

and

staining

(Fig.

3,

track

gels

due

Following

= 55,000

of

was

ACP (a),

when

weight

was with

of

DEAE-Sephacel

columns

gradient

alkaline of

(d)

molecular

4-30%

gel

ACP inhibitor

enrichment on

S-ZOO

The

of

stepwise

chromatography

PAGE.

but

the

gel

weight.

B) to

1429

on

ACP under

1.5m

a 4-30%

(b),

0.5m

gradient was

non-reducing

110,000 conditions

electrophoretic

with (Fig.

blue

fraction

inhibitor

differential

reduction obtained

inhibitor Bio-Gel

tested the

Coomassie

2-mercaptoethanol, 3,

lane

C).

a Thus,

Vol.

122,

No.

3, 1984

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

-l

0

I

3

2 ACP Inhibitor

4

5

(Ng)

Fig. 4 Inhibition of ACP mediated lysis of RaRBC by HL protein 1 ml of a 1:12 dilution of NHS in G-VBS-EGTA-Mg buffer pH 7.3 was incubated with purified HL protein (0.5-5 up) at 37"C/60 min. Next, 200 ~1 of RaRBC suspension was added to the reaction mixture and incubation continued for another 60 min The turbidity of the reaction mixture was monitored at 700 "Ill. 1 ml of a I:12 dilution of NHS served as control.

the

110,000

species

disulfide

consists

bonds.

conditions (Fig.

3,

The

on

SDS-lo%

track

B)

dilution

of

inhibition ml

of

of

a 1:12

This

NHS

HL protein

also

The presented

layer

in

Table

residues.

The

noteworthy

is

I.

than

virtual

cell

of

acidic lack

the

and

with

a I:12

ug

of

of

RaRBC

protein

to

protein

is

chelated

(data

not

a

NHS with shown).

the

formation

of

ACP

inhibitor

is

a

clumps.

purified aspartic

basic

of

methionine

acid

residues

comprise

24%

1.8:1.

Also

is and

low

1

hemolysis.

HL

EGTA

toward

protein.

a dose-dependent

2.5

of of

by

35,000

native

vg)

of

M,

Spodoptera

to

1430

the

inhibition

purified

a tendency

Glutamine of

>90%

of

about

Preincubation

composition

ratio the

showed

of

(0.5-5

conglutination

rather

acid

the

band

brought

together

non-reducing

additional

Addition in

held

under

product

4).

ACP. in

RaRBC

amino

an

buffer

that

resulted

cell

inhibitor

resulted

human

polypeptides

HL protein

(Fig.

dilution

55,000

a degraded

purified

lysis

of

agglutinated

continuous

is

indicates

inhibitor

showed

G-VBS-EGTA-Mg

RaRBC

experiment

potent

The

in

two ACP

PAGE

of

NHS

purified

which

Preincubation

of

cysteine

of

content.

the

Vol.

122,

No.

BIOCHEMICAL

3, 1984

AND

Table

Amino

Acid

Composition

Amino

of

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

I

ACP Inhibitor

Isolated

from

Spodoptera

Amino Acid (residues/100

4cid

HL

Composition residues) 13.5 7.5 5.0 II .o 5.6 8.3 7.8 trace 7.2

Asp Thr Ser GlU Pro GAY Ala CYS Val net Ill.? LeLl TY~ Phe His LYS *G? Total acidics Total basics Acidicslbasics

4.5 8.2 2.2 4.5 2.0 6.7 4.7 24.5 13.4 1.82

DISCUSSION

In larval

this

paper

Spodoptera

inhibitor assay the

we

(a

HL.

In

the

classical product

human

C showed

a single

molecular

The

weight

HL

were

inhibitor

of

inhibitor

is From

isolated. human

to

converting

clumping

of

cells

lacked

the

ACP

by

adsorption ability

band

on

110,000

of

a reaction

to

hemagglutinating

a limited

C2

separated

of

lysis

PAGE

3 mg of

protein

conglutinin

by

with

an

PAGE.

two

inactive to

The

RaRBC

HL protein HL

from

DEAE-Sephacel.

a subunit

HL,

activity. 1431

C

SDS-lo%

revealed

Spodoptera

comparable

classical

gradient

on

cell-free

hemolytically

the

inhibit

a 4-30%

of

from

was to

composed

Preincubation

inhibitor

using

daltons

therefore

ACP

inhibitor

conditions

90 ml

RaRBC --

protein)

the

Purified

ACP.

addition

itself

of

reducing

chains.

inhibitor

on

stained

under

polypeptide

inhibitor

study,

based

of isolated

specific

inhibitor

enriched

Electrophoresis 55,000.

present

purification

previously

larval

C pathway

final

the

We have

C4-binding

(7).

apparent

describe

of

identical pure is

with also

M,

ACP a potent

human

C in

brought

about

(12).

The

Vol.

122,

No.

3,

The

1984

amino

inhibitors

acid

of

suggests

such

the

insect

control

polypeptide human

nature inactivator.

Three

probable

protein

of

the the

the

product

indicator

for

human

the

context

From inhibitors

of

The

fact

of

it

C3b

degradation.

of

C in

insect

HL

mammalian

C pathway

suggests

operative

in

HL,

assays.

Since

homologies highly in

insect C3

which

is

probable

for

C-like

reaction

existed

prior

inhibitors

components

molecules

mammalian

C in

insect

that

the

to

the have

appearance had

interact may

the

the

regulators of

a common

a C-type origin

structural (15),

it

C may

also

presence of

mammalian

of

other

inhibitors,

any

case,

a thorough

their

specificity

characterization and

mechanism 1432

of of

the action,

exist for

specific

C perhaps of

important

HL complement and

these metabolic

pathways. In

is

a detectable

Some

reaction. with

appropriate

substrates

absence of

either

be

require and

the in

with also

natural

Alternatively, HL

speculations:

vertebrate

be

a

specific

duplication

of

could

ii)

suggests

might

key

RaRBC.

specific

several

might

gene

the

with

two

sequence

with

from

C3b

and

of

which

HL

result

selectively

reactions of

insect

on

of

selectively

considerable

protein.

i.nhibitor

inhibitors

These

HL.

react

HL raises

detection

consistent

product

presence

similar

the

C3 convertase.

a sensitive

can

with

sites the

early

can

the

that

C4 have

these

an

two

could

acceptor C3 for

and

which

inhibition

or

provide

proteins

by

the

Spodoptera

the

also

that

Spodoptera

either

and

similarities

expected

which

from

shows

with

phylogeny,

mammalian

that

could

different

protein

protein

C3b

is

mammalian (14)

weight

with

inhibitor

inactivator

molecular

receptor

react

C3b

COMMUNICATIONS

from

the

be

compete

could

If

and

First,

C3b

could

degraded

i)

the

inhibitor

degradation.

can ACP.

differs

protein

HL

RESEARCH

inhibitor

inhibitor

mechanisms

inhibitor

ACP

H (13)

Spodoptera

either

BIOPHYSICAL

However,

mammalian

blocking

Third,

Factor ACP

of

inhibits

Second,

as

of

proteins.

C3b

AND

composition

ACP

that

mammalian

the

BIOCHEMICAL

their

Vol.

122,

No.

relationship

to

understand geneic

BIOCHEMICAL

3, 1984

the

insect significance

AND

metamorphotic of

BIOPHYSICAL

events these

(6)

phylogenetically

RESEARCH

is

required conserved

COMMUNICATIONS

to xeno-

reactions.

ACKNOWLEDGEMENTS This work was supported by grants Foundation (#PCM-8216557) and American thank Patricia Cook for her technical for typing the manuscript.

from the National Science Cancer Society (#IM-298). We assistance and Karen Deatherage

REFERENCES

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Gewurz, H., Finstad, J., Muschel, L.M., and Good, R.A. (1966) In: Phylogeny of Immunity, Eds. Smith, R.T., Miescher, P.A., and G%, 105-117, University of Florida Press, Gainvesville, FL. R.A., pp. Porter, R.R., and Reid, K.B.M. (1978) Nature 275, 699-704. Fujii, T., and Murakawa, S. (1981) Dev. Comp. immunol. 5, 251-258. Day, N.K., Gewurz, H., Johannsen, R., Finstad, J., and Good, R.A. (1970) J. Exp. Med. 132, 941-948. Day, N., Geiger, H., Finstad, J., and Good, R.A. (1972) J. Immunol. 109, 164-167. D'Cruz, O.J.M. (1983) Fed. Proc. 42, 1237. D'Cruz, O.J.M., and Day, N.K. (1984) Fed. Proc. 43, 1764. Burton, R.L. (1967) USDA Bulletin ARS 33-117. Platts-Mills, T.A.E., and Ishizaka, K. (1976) J. Immunol. 113, 348-358. Polhill, R.B., Pruitt, K.M., and Johnston, Jr., R.B. (1978) J. Tmmunol. 121, 363-370. Laemmli, U.K. (1970) Nature (London) 227, 680-685. Linscott, W.D., Raymond, R., and Triglia, R.P. (1978) J. Immunol. 121, 658-664. Sim, R.B., and Discipio, R.G. (1982) Biochem. J. 205, 285-293. Hsiung Li-min, A., Barclay, N., Brandon, M.R., Sim, E., and Porter, R.R. (1982) Biochem. J. 203, 293-298. Sackstein, R., Colton, H.R., and Woods, D.E. (1983) J. Biol. Chem. 258, 14693-14697.

1433