Conformational changes and complement activation induced upon antigen binding to antibodies

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CONFORMATIONAL CHANGES AND COMPLEMENT ACTIVATION

INDUCED

UPON ANTIGEN BINDING TO ANTIBODIES

I. Departments

Pecht,

of Chemical

B. Ehrenberg,

Immunology of Science,

Received

September

E. Calef

and Chemical

and R. Arnon

Physics,

The Weizmann

Institute

Rehovot , Israel

30,1976

SUMMARY: The interaction between antibodies specific for the loop region of lysozyme and this monovalent antigenic determinant was compared to their interaction with either the dimeric derivative of the loop (bis-loop) or the polyvalent antigen loop-A--L. This comparison was based on complement fixation capacity, and on spectroscopic changes in the circular polarized fluorescence (CPL) of the antibodies. The binding of the loop to its antibodies causes spectroscopic changes, assigned to conformational changes in the Fc region, which are not accompanied by complement activation. However, the binding of the bisloop led to distinctly different CPL changes and concomitantly to complement fixation comparable to that induced upon binding of the loop-A--L. Complement fixation and the CPL changes can be induced in the intact antibodies only; reduction of the interchain disulfide bridges, or cleavage by papain or pepsin digestion abolishes both activities. Three ing

different

antibody

effector

According

(1Jl. thermodynamics body

conformational mission,

changes

in turn,

physicochemical the

circular

such changes It

(4-6).

These

interaction probably

was previously

between

were

observed the

from those a change

Indeed,

in the

with

the whole

conformation

Copyright 0 I977 by Academic Press, Inc. All rights of reproduction in any form reserved.

of Fc. 1302

antirequires mechanism),

and their

(an by trans-

using

several

and measurement

report

antibodies

and their

their

respective

antigens

antigens

Fab fragments

antibody of the

of

on the existence

intact

multivalent

in CPL of the

with

Fab,

investigations

(3))

with

Fc and Fab fragments

mechanism

of detection

in both

(5) that

changes

observed the

recent

interaction

antigen

favorable

The third, CH2 domains. functions are initiated to the

(CPL)

detected

the

of initiat-

to antigen-mediated

The second

the method

of the

with , the

Fab and Fc (“distortive”

at the effector

of luminescence

as a result

leads

by antigen-binding

including

the mechanism

of antibody

phenomena. between

to the Fc region.

for

activation)

which

hinderence that the

induced

methods,

and poly-D&alanine, different

these

steric infers

polarization

Fab fragments

for

relationship

so as to possibly remove “allosteric” mechanism),

interaction

(“associative” attachment,

accounts angular

upon

first,

of multipoint

in the

have been proposed

function

to the

aggregation,

a change

alternatives

were

molecules, specific

With monovalent

of (5,6).

such as RNase markedly indicating

antibody, haptens,

and a rough ISSN

0006291

X

Vol. 74, No. 4, 1977

correlation

between

phosphorylcholine saccharides changes

the

transitions

which

attributed

to the

it

b) It ing

about

dimers

of lysozyme for

neither

but

is

nor

antigen,

reasons:

still

monovalent

and high

affinity

(K = 3 x lo6

The above mentioned changes

with

antigen

complex,

induction

complement.

We wish

body

with

complex

whereas

the

distinctly antibody

in terms M-l)

to report formed This

different

the

for

or of the

which

of changes

prepar-

of a larger

specificity.

d) It heterogeneity

in the CPL which

effector

functions to other

or with

dimeric

lysozyme,

or polyvalent

correlated formed

with with

the

of these

of the anti-loop

in analogy

the complex

MATERIALS

a part

range.

for

and functional

the correlation

loop,

loop-antibody

wavelength

is a significantly loop

this

(9).

that with

for

of aromatic

can be used

constitutes

of the

capacity

activity

It

devoid

relevant but

structural

of one of the with

the monovalent

complexes

complement-fixing.

c)

of restricted observations

namely

in the

lysozyme,

led us to investigate

the

can be

and useful

a) Being

determinant, (8).

namely

antibodies

antibodies

which

convenient

fluoresces

derivatives

specific

its

antibody.

in the conformational

changes

is particularly

the following

absorbs

or polyvalent occurring

molecule,

anti-loop

induced

Fc region.

provokes

free

in the antibody,

antibodies.

lysozyme)

of intact

haptens

and oligo-

respective

of hen egg-white

small

the small

(5)

in their

Fab and the Fc regions

has been shown to be a monovalent

naturally

in

64-80

from

whereas

tetra-i-alanine changes

(residues

brings

peptide

(7) it

spectral

differs

RESEARCH COMMUNICATIONS

in CPL was observed;

any change,

loop

of investigation,

residues

cause limited

to both

that

The loop

not fragment

assignable

AND BIOPHYSICAL

and changes

produced

“loop”

implies

type

size did

(6)

The lysozyme This

BIOCHEMICAL

induces

of the

antibody-

antibody

to fix

systems

(2,11),

did not loop

CPL spectra, the bis-loop

loop

spectroscopic

fix

the anticomplement,

derivatives which

were were

and those

of the

complex.

AND METHODS

AnAn. The loop fragment and its conjugate, loop-A--L, were prepared as described previously (8,9). The synthetic loop, prepared by the solid phase synthesis (12), was described in detail in an earlier publication (13). This peptide comprised residues 64 to 80 in the amino acid sequence of lysozyme and did not contain any fluorescent residues. The loop dimer was prepared by coupling the synthetic loop to each of the using water soluble 1-ethyl-3-(3-diethyltwo amino groups of 1,9-diamino-nonane, aminopropyl) carbodiimide (EDCI) (Ott Chem. Co., Muskagon, Michigan, USA): Fifty mg of loop were reacted with equivalent amount of EDCI (4 mg) for 30 min at room temperature and the slight precipitate formed was centrifuged off. Two mg (half equivalent) of 1,9-diaminononane was added and the reaction mixture was maintained Separation of the dimer from lower molecular at room temperature overnight. weight components was carried out on a Sephadex G-10 column (7.4 x 0.8 cm) in 0.05 molar acetic acid. The fractions under the main peak (monitored at 230 nm) The purity of this were pooled and lyophilized to yield 30 mg of bis-loop. product was assayed by high voltage (3000 volts) electrophoresis on Whatman 3 MM paper at pH 1.9. Only a single spot, corresponding to the bis-loop, was observed.

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Antibodies. Antibodies specific to the loop region of hen egg-white lysozyme were prepared as described elsewhere by immunizing New Zealand rabbits with the loop-A--L conjugate (8). The antibodies were subsequently purified on a lysozyme-Sepharose immunoadsorbent (8,9) . The cleavage of the antibodies was carried out by papain digestion according to Porter (14). (Fab’) dimer fragments of the anti-loop IgG were prepared by pepsin digestion (15). Reduction of the inter-heavy chains disulphide bridge was carried out in a 5 mg/ml solution in PBS with 2.5 mM dithiothreitol (16,17). CPL measurements. The measurements of circular polarization of the emission of the immunoglobulins were carried out with an instrument constructed in the Department of Chemical Physics of this Institute, and described elsewhere (18,19). Sample cells of 1.0 or 2.0 mm light path were used. The circular polarization of luminescence is expressed by the emission anisotropy factor defined as gem = 2 * Af/f, where Af is the intensity of the circularly polarized component of the fluorescence (defined positive for left-handed circular polarization), and f is the total intensity of the fluorescence (3). The exciting light, set at 275 nm (band pass 30 nm), was passed through a chlorine filter (Ophthos Instruments) to remove stray light. The fluorescence was monochromated by a Jarrell-Ash double monochromator (model 82-410). The experimental error part of the emission in the measurement of gem was + 5 x 10V5 in the central band and increased to about f 1 x 10s4 at the red edge of it. Micro-complement fixation assay. Sheep red blood cells were freshly prepared, hemolysin was purchased from Difco Laboratories, Detroit, Mich. and Titration of whole guinea pig serum was used as the source of complement. complement and complement fixation assay were performed according to Wasserman and Levine (20), in a total volume of 1.4 ml, using 0.02 M tris-HCl buffer, pH 7.4, containing 0.14 M NaCl, 0.5 mM Mg2+, 0.15 mM Ca2+ and 1% bovine serum albumin. For measurement of complement fixing activity a series of the antigen dilutions was made in 0.2 ml buffer. A premixed solution (1 ml) of the antibodies in suitable dilution, and complement in an amount that would give 90% lysis, were added to each tube as its contents were rapidly stirred. After equilibration overnight at 4’C, 0.2 ml of a 0.25% suspension of hemolysinsensitized sheep red blood cells was added at O’C and the tubes were incubated at 37°C until the complement controls showed approximately 90% lysis. The tubes were then transferred to O’C, centrifuged and the supernatants were monitored at 410 nm. Appropriate controls of the complement at several dilutions, of the antigen or antibody separately, and of sheep red blood cells without complement were set with each experiment. Ultracentrifugal analysis. Sedimentation velocity measurements were carried out at 25°C and 56,000 rpm with an electronic speed control in a Beckman Spinco Model E analytical ultracentrifuge equipped either with Schlieren optics, or with optical scanner. RESULTS Circular bodies

are shown

throughout of the

polarization the

different

previously antigen

binding

in Figure

emission

1.

band

the binding

of the protein to other

The variation is

tryptophans

(51,

CPL spectrum

of fluorescence. a result

(Fig. rabbit

of anti-loop

in the

anisotropy

of the

contributing of the

The CPL spectra

to this

loop

leads

1).

1304

spectrum.

to a very

In analogy

antibodies

emission

heterogeneity

(5,6),

of the

a marked

factor chirality

As observed

pronounced

to the

anti-

effect shift

change observed towards

in the upon positive

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

: : 1’

t5 -

d t 0 x O-

-5 -

I

30( 3

I

350

400 X (nm)

Fig. 1. Circular polarized fluorescence spectra of anti-loop ml) 0 -0, and it complexes with 1.5 equivalents of loop, loop xx, respectively.

antibodies (6.5 mg/ e-e, and bis-

Q

Fig. 2. The CPL spectra of papain-cleaved anti-loop antibody o-o, and its complexes with 1.5 equivalents of loop o-m, respectively. values

of gem is observed The binding

namely, negative

the than

in the

of the bis-loop

gem values that

of this

of the free

longer leads

complex antibody.

wavelength

half

to an inversion in the range That this 1305

of the of this

(7.0 mg/ml) bis-loop x-x,

emission

band.

spectral

pattern,

340-380 nm is even more effect is due to the rever-

Vol. 74, No. 4, 1977

BIOCHEMICAL

300

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

350

400

A (nm) Fig. 3. Effect of the reduction of the interchain disulphide bridges of antiloop antibodies on the circular polarized fluorescence spectra of the free antibody o-o, complex with the 1.5 equivalents of loop o-o, and bis-loop x-x, respectively.

sible binding of loop or bis-loop was confirmed by addition of an excess loop which resulted in reversal of the CPL pattern to that obtained with the loop. Variation of the equivalent ratio of loop or bis-loop to antibody from 1:l up to 6:l did not cause significant changes in the CPL spectra. Cleavage of the antibodies by papain to Fab and Fc,or mild reduction of their

interchain

disulfide

bond, resulted in a product with CPL spectrum similar

to that of the intact free protein (Fig. 2 and Fig. 3). The binding of either loop or bis-loop to the cleaved or reduced antibody did not cause significant gem changes in the longer wavelength half of the emission band; yet, somediffer ence is found in the blue half of the emission band below 350 nm. A similar behaviour is observed in the CPL spectra of (Fab')2 and of its complexes with the loop and bis-loop, as shown in Fig. 4. Complementfixation. The results of the complement fixation assay are presented in Figure 5 and expressed in terms of the percentage of fixed complement as a function of loop concentration. The homologousmultivalent antigen, loop-A--L, fixed complementreadily. On the other hand, no significant fixation was observed upon interaction of the anti-loop with either the loop or lysozyme, even when added in higher concentration than the antigen (in terms of equivalent weight). The bis-loop led to a level of complement fixation almost as high as the homologous antigen at the sameantibody concentrations.

1306

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

nm Fig. 4. The CPL spectra of complex with 1.5 equivalents respectively.

Sedimentation anti-loop

velocity

antibodies Both

examined

as references. fold

followed

excess

by the

of oligomeric

measurements.

and the bis-loop

centrifuge. to six

(Fab’)z of anti-loop of loop l -•,or

free

antibodies

over

the

Schlieren

species

The complexes

were and the

The bis-loop

studied

sites. system,

formed

formed

with

were

In the

of its

between loop

sedimentation were

were

equivalence pattern,

amounts

complexes

the

ultra-

the

from half

no significant

of the antibody-bis-loop

o-o,and x-x,

in the analytical

complex

concentrations

antibody

optical

antibodies with bis-loop

(less

than

5%)

observed.

DISCUSSION The assumption the effector spectral

functions

changes

biological

that

antibodies

mechanism

upon antigen-antibody

observed

activities

to its

an allosteric

does not

complex lead

the one operative

binding,

in the Fc region

of the

is

should

would

be manifested

as well.

However,

to complement

activation,

but

still

those

a monovalent

conformational

sufficient

antigen

complement in the

for

fixation.

changes functional

the

anti-loop

This

monitored sense

by CPL, though

(1,Z).

1307

On the other

of the it

perhaps hand,

is

in the Fc even a larger

also

in this

incapable system,

necessary, the

loop

does cause

residues comprising

at least

the

in the various

though

antibodies,

means that,

that

the binding

spectroscopic changes which probably stem from tryptophan part of the molecule (5). Furthermore, intact lysozyme, of eliciting

imply

in inducing

polyvalent

are not

BIOCHEMICAL

Vol. 74, No. 4, 1977

I 0.4 Concentral~on

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 0.6 of loop

I 12 tn the ontgen

I 1.6 sdution

I 2.0 (yglmll

Fig. 5. Complement fixation of anti-loop IgG antibodies bis-loop 0 lysozyme AA, and loop-A--L fJ concentration is 4 ngjml.

antigen

loop-A--L,

complement for

to induce

In this

we have fixation

from

that

interaction also

complement

that

binding

production and his

loop

it

only

in a change

dominated

by both of divalent

haptens

colleagues a spacer

at equilibrium seems that

(23,24), of ca.

This (with

it

was a dimer a different

(23).

situation

However,

1308

differing

of the

complex

that

the

of the

it

complex,

unexpected leads

in the

studies

in as

in view of

microscopy

showing to preferential of Schumaker

of a soluble

species

was

efficient

spacing)

with

immuno-

domains,

Moreover, being

upon binding

is prevailing.

of along

the antibody

species

the main

capable

indicates

was somewhat

that

and antibodies

distinctly

though

For example,

i! length,

in

body of

antibodies,

within

appropriate

was found

large

likewise

and electron

dimers. 15-16

the

relationships.

by monomeric

analysis.

effective

behavior loop

complexes,

ultracentrifugation

of antibody-hapten with

obtained

results

by sedimentation studies

hapten

spectroscopic monomeric

are

is

anti-loop

CPL spectra,

antibody

(8),

21,22). is

to the

on the inter-domain

loop o-o, The antibody

of antigens

the bis-loop

This

not

with

(l-3,

with

the bis-loop

fixation,

evidenced several

that

line

the complex

dependent

demonstrated

loop.

in

attachment

on their

the

antibodies

functions

shown that effect

by the

versus

are

upon binding

marked

induced

the bis-loop is

findings

immunoglobulin

study

a concomitant

anti-loop

of multipoint

complement

with

but

These

the requirement

the effector

inducing

precipitates

activation,

evidence

logical

which

with o-o.

of the

in the case of the The bis-loop

bivalent

complex bisprepared

Vol. 74, No. 4, 1977

and used in this nonane

BIOCHEMICAL

investigation

and further and the

terminus

of the synthetic

extended

form

two extra

would,

for

closed with

loop,

antibody It

the

not

with

of the free

that

intactness

of the

the Fab fragment,

can they

be demonstrated

fixing

mechanism

should

are

essential

binding for

which

and that

in the ratio

the observed antibody

changes

of a is

of its

complex

of bis-loop

to

(Fab’)2

or inmildlyreduced

bridges,

mixture

as well

in CPL above

molecule,

its

with

As is

namely

they

Fc fragment;

known

as fragmentation,

340 nm

are not neither

antibodies,

where

(16,17),the

mild

also

abolish

the

of the antibodies. here,

support,

be conceived

for

therefore,

activating

and by cross the transformation

linking

the notion

the

of the IgG class. To a certain extent this allosteric and distortive mechanisms; these by antigen

be the

The formation

CPL spectrum,

antibody

in its

to be

plausibly

(25).

carboxy

spacer

expected

could

observed

bond was disrupted.

activity

described

of this is

or with

in the

of the disulphide

due to the

point.

with

The data

the

by an increase

significance

disulphide

which

line

altered

This

form of complex

that

observed

complement

complex

1, 9 diamino-

at each of the

The length

in

on the

reduction

(13).

of the bonds

present

both

are dependent

the inter-heavy

peptide

equivalence

is of great

consists

amont to Q 36 ii.

is

from

and is

beyond

residues

and dominant

complex

which

and carbon-carbon acid

of an intermal

different

the

loop

favored

monomeric

amino

therefore,

closure

thermodynamically distinctly

has a spacer

16 carbon-nitrogen

cysteine

sufficient

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

effector

the

functions

a unique of antibodies

combines elements of the “simple” conformation changes induced both

the two sites into

that

active

of an IgG molecules state.

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Metzger, H. (1974) Adv. Immunol. 18, 169-207. Hyslop, N.E. Jr., Dourmashkin, R.R., Green, N.M. and Porter, R.R. (1970) J. Exp. Med. 131, 783-802. Steinberg, I.r(l975) In: Concepts in Biochemical Fluorescence, (Chen, R. and Edelhoch, H. eds.) Marcel Dekker, New York. Pilz, I., Kratky, O., Licht, A. and Sela, M. (1973) Biochemistry 12, 49985005. Schlessinger, J., Steinberg, I.Z., Givol, D., Hochman, J. and Pecht, I. (1975) Proc. Natl. Acad. Sci. USA 72, 2775-2779. Jaton, J.-C., Huser, H., Braun, D., Givol, D., Pecht, I. and Schlessinger, J (1975) Biochemistry 2, 5308-5312. Canfield, R.E. (1963) J. Biol. Chem. 238, 2698-2707. Arnon, R. and Sela, M. (1969) Proc. Natl. Acad. Sci. USA 62, 163-170. Maron, E., Shiozawa, C., Arnon, R. and Sela, M. (1971) Biochemistry lo, 763-771. Pecht, I., Maron, E., Arnon, R. and Sela, M. (1971) Eur. J. Biochem. 19, 368-371. V.N., Glovsky, M.M., Rebek, J. and MUllerGoers, J.W., Schumaker, Eberhard, H.J. (1975) J. Biol. Chem. 250, 4918-4925.

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

12. Merrifield, R.B. (1965) Science 150, 178-185. 13. Arnon, R., Maron, E., Sela, M. and Anfinsen, C.B. (1971) Proc. Natl. Acad. Sci. USA 68, 1450-1455. 14. Porter, R.R. (1959) Biochem. J. 73, 119-126. 15. Nissonof, A., Wissler, F.C., Lippman, L.N. and Woernley, D.L. (1960) Arch. Biochem. Biophys. 89, 230. 16. Press, E.M. (1975)Tiochem. J. 149, 285-288. 17. Isenman, D.E., Dorrington, K.J. and Painter, R.H. (1975) J. Immunol. 114, 1726-1729. 18. Steinberg, I.Z. and Gafni, A. (1972) Rev. Sci. Instrum. 43, 409-413. 19. Schlessinger, J. (1974) Ph.D. Dissertation, The Weizmann Institute of Science, Rehovot, Israel. 20. Wasserman,E. and Levine, L. (1960) J. Immunol. 87, 290-295. 21. Landsteiner, K. (1945) The Specificity of Serological Reactions. Harvard University Press. 22. Siraganian, R.P., Hook, W.A. and Levine, B.B. (1975) Immunochemistry 12, 149-157. 23. Wilder, R.L., Green, G. and Schumaker, V.N. (1975) Immunochemistry 12, 39-47. 24. Wilder, R.L., Green, G. and Schtimaker, V.N. (1975) Immunochemistry 12, 49-54. 25. Schumaker, V.N., Green, G. and Wilder, R.L. (1973) Immunochemistry 10, 521528.

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