Vol. 78, No. 4, 1977
Fc
FRAGMENT
OF
INTER-CHAIN
JO&
A.
BIOCHEMICAL
OF HUMAN
IMMUNOGLOBULIN
DISULFIDE
Lopez
de
BONDS
Department
of
Received
August
G:
AND
COMPLEMENT
Vivanco
Fundacidn
Immunology.
REDUCTION
ENHANCES
Fernando
Castro:
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and
Jimenez
CARBOXYMETHYLATION
ACTIVATING
Fernando
Diaz.
CAPACITY
Ortiz Madrid,
Spain.
26,1977
Summary: The complement activating capacity of the FC fragment of human immunoglobulin G increases after selective reduction and carboxymethylation of the inter-chain disulfide bonds. Differences in the circular dichroism spectra of intact and modified fragment suggest that minor conformational changes have occurred. Our results are discussed in relation to the molecular requirements for complement activation.
INTRODUCTION There the
is
strong
IgG
moleculeff
complement
(Cl)
is
still
not
is
located
possible
to
the
acid
residues
complement
effector
for
into
by
abrogates interaction,
fully
has
been for
IgG
that
complement (8)
the
be
measured
also
of
circular
of
polarization
#
disulfide
0 1977 by Academic oj reproduction in any
Press, Inc. iowl reserved.
1319
of
amino that
the residues
af upon of
(2,6). bonds
are
complexes these
bonds
also
antigen-antibody
fluorescence
To whom correspondence should be addressed. Present address: of Biophysics. The Johns Hopkins University School of Medicine, Wolfe Street, Baltimore, Md. 21205 (U.S.A.). ff Abbreviations: IgG: Immunoglobulin G: DTE: Dithioerythritol; Sheep red blood cells; CD: Circular dichroism; W: Ultraviolet.
Copyrighr Ail rights
suggest
chain
antigen-antibody
induced
the
non-consecutive
alkylation Fc
reports
suggesting
by
of it
and
sequence
polypeptide
by and
Some
of
However, groups
a linear
chain
region component
(1,2).
evidence
the
the
first
chemical
determined
activity
changes by
by
inter-heavy
Reduction
.
is
folding
conformational as
effector formed
the
region
site.
binding
molecules
the
the
homology
the is
that
with
the
could
by
studies
characterize
there
complement
shown
CT2
site
However,
of
interaction
the
of
proximity
a number
the
in
binding
(2-S).
site
essential and
in
requirements
that
It
from
involved
conformational
brought
evidence
(9,lO)
Department 725 North SRBC:
(7)
Vol. 78, No. 4, 1977
and
enhances
these
an
the
studies
disulfide
internal
point bonds
integral
part
of
was
undertaken
these
bonds
in
AND
the
of
the
IqG
molecule
the
important
role
of
the
hinge
region,
there
is
evidence
the
controlling
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
flexibility
out
in
study
MATERIALS
BIOCHEMICAL
with
complement the the
binding
aim
of
further
biological
site
(11).
inter-heavy
chain
that
(8,
12).
investigating activity
Although
of
they The the
the
Fc
are
not
present role
of
region.
METHODS
FC fragment was obtained by papain digestion of pooled human IqG in the absence of cysteine as described (13) and purified by DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Neither IgG nor any other serum protein was found in the Fc preparations, but a small amount of Fab appeared sometimes in immunoelectrophoresis as a weak precipitine band. This contamination could not be detected by polyacrylamide gel electrophoresis. Selective reduction and alkylation of inter-chain disulfide bonds was carried out as previously described (14) with some modifications. Aliquots of Fc fragment (1 mg/ml in 10 mM Tris/HCl buffer, 0.5 M NaCl, 2 miY EDTA, pH 7.8) were incubated for two hours at 25OC with dithioerythritol (DTE) at concentrations ranging between 1 and 10 mM. The reduced protein was alkylated with iodoacetamide for 20 minutes in the dark. In other experiments the protein was incubated with 3 mM DTE as described above and alkylation was carried out for 20 minutes in the dark by adding iodoacetamide or iodoacetic Titration of sulfhydryl groups was acid to a final concentration o f40+ M. achieved by alkylation with lC-iodoacetamide as described (15). Nonspecific radioactivity was detected by incubating non-reduced Fc fragment with l- 14C-iodoacetamide under the same conditions. Complement activating capacity was measured by the quantitative compleSerial ment fixation test (16), modified to obtain maximal sensitivity. dilutions of non-aggregated protein were incubated with 1 CH50 unit of human complement for 1 hour at 30°C and then for 18 hours at 4'C, in 2 ml total After incubation, 1 ml of an 0.15% suspension of sheep red blood volume. sensitized with an excess of rabbit anti-Forssmann antibody, cells (SRBC), All tubes were incubated for 1 hour at 30°C. was added to each tube. Spontaneous haemolysis controls (sensitized SRBC+diluent) and complement activity controls (sensitized SRBC + 1 CH50 + diluent) were included in each After incubation, the non-damaged cells were spun down at 350 x experiment. g for 15 minutes at 4Oc and optical densities of the supernatants were Haemolysis inhibition percentages were calculated as measured at 414 run. described (16) and plotted against protein concentration. Circular dichroism (CD) measurements were made at 25°C using a Cary-60 and cells with 1 cm path recording spectropolarimeter with CD attachment, Samples length. Spectra were recorded in the range from 212to 320 nm. contained 100 microg./ml for far-W measurements and 1 mg/ml for near-W. Ellipticity (0) was expressed as molar ellipticity which is defined as ox M x 100 8 = dxC 17 d the path length where M is the molecular weight of FC (52,000), All meters-(d = 1 cm) and C the concentration of protein in g/l. were obtained in triplicate and the arithmetic mean was calculated.
1320
in centithe spectra
Vol. 78, No. 4, 1977
TABLE
BIOCHEMICAL
AND 5lOPHYStCAL
RESEARCH COMMUNICATIONS
I
Titration of sulfhydryl groups of the 3 mM dithioerythritol treated The specific activity ratio of labelled protein FC fragment of human IgG. to radioactive iodoacetamide is equal to the molar ratio of sulfhydryl groups to protein and therefore represents the number of sulfhydryl groups Controls of non-reduced Fc fragment were included in each per molecule. experiment.
Specific bCi/mM Exp. l-l*C-iodoacetamide.
activity
1
Exp.
100
Control
. . . . . . . . . . . .
Reduced
FC
2
Exp.
100 5.26
fragment.
Specific Specific Exp.
1
protein iodac.-
Exp.
2
Exp.
3
100 5.26
431.58
3
act. act.
5.26
431.58
0.052
0.052
0.052
4.3
4.3
5.1
specific
reduction
514.80
RFSULTS In
order
chain
disulfide
added
to
to
find bonds,
identical was
estimated
Sephadex
G-100
column.
efficiency
of
reduction
its
of
to
disulfide
shown
in
bonds
was
number only
Table
In
Protein
carried
out
this
I.
They
papain parallel
for
with
the
percentage
acid.
3 ti bonds
of
The
eluted
results
indicate
that
The
number
of
the
normal
pool
sensitive experiments,
subclasses, samples
three
I-
of
of
1321
of
human
than
90%.
in
these
IgGl
in affect
inter-chain
IgG,
monomeric
accomplish
and intact
are disulfide
with
taking
order
intra-
experiments
agrees
the
dissociated
to
would
different
cleaved
namely
protein
generated
agent
a
and
'* C-iodoacetamide
reduction bonds
through
better
groups
were
of
acid
enough
inter-
10 mM)
protein
of was
reducing
of
extent
a yield
sulfhydryl
of
The
1 M acetic
DTE
with
to
reduced
with
titration
of
BYTE (1
mg/ml).
of
as
concentration
bonds.
(1
samples was
by
of
protein
disulfide
selective.
calculated the
Fc
passing
in
for
concentrations
determination
whether
chain
by
inter-chain
was
test
of
chains
Quantitative conditions
increasing
expressed
polypeptide
reduction
conditions
aliquots
reduction
into
optimal
the into
average account
IgG3. Fc
fragment
and
Vol. 78, No. 4, 1977
BIOCHEMICAL
% Fc
Figure
of
1.
monomeric
Fc
selectively
were
tested
previous in
the
fragment
as
compared
induced
by
spectrum
between
210
ellipticity the
were
of
intact
and
320
spectrum
spectrum
was of
identical
at
in
the
far
W
in
the
215-220
it
in
(Figure
by
within
exhibited nm band
the
(Figure
described
A 5-
to
in
lo-fold
carboxymethylated
detect
any
inter-chain
Fc
not
show
positive
In
the
near
W,
272,
288
292
between
carboxymethylated error
215
increase
of
in
bands
of
the
far
In
nm.
Fc
experimental
bonds.
ellipticity negative
and
band
conformational disulfide
did
a significant
with as
2.
extent by acid.
observed. to
of
a negative an
Figure and
was
attempt
approximately
reduced
former
an
3).
in
reduced Fc
alkylated
capacity
shown
intact
fragment
dominated
the
are
of
and
activating
modification
Fc
selectively
to
that
chemical
appeared
3 mM DTE
selectively
obtained
nm
with
results of
to
of dithioerythritoL on the disulfide bonds, as measured dissociated in 1 M acetic
complement
The
activity
CD spectra
The
reduced for
section.
increase
changes
dtssoclatod
Effect of the concentration of reduction of inter-chain the percentage of Fc fragments
iodoacetate the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and
220
fragment the
W The
run. was
near
negative
W
but
ellipticity
3).
DISCUSSION The index
of
number the
of selectivity
titrated
sulfhydryl of
reduction
groups of
1322
the
per
molecule
inter-chain
Serves disulfide
as
an
bonds
BIOCHEMICAL
Vol. 78, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RO-
60-
Figure
2.
Inhibition of hemolytic activity of complement by intact (a) and selectively reduced and carboxymethylated (0) Fc fragment. The dispersion of data in each group of experiments is represented as the extreme values.
since
these
chain
disulfide
cysteine
bonds
is
percentage
of these
(10.3%).
pool
pool
fragment
This
to subclasses
IqGl
should
be 2.9 since
would agrees
markedly result
in controlling
yield with
reduction enhances points the
reduction
value
number
in our
importance
groups
of the
IgGl
and Fcy3
per molecule and 11 for bonds
of Fc
per molecule.
This
experiments.
of covalent
1323
(89.7%)
Yl
disulfide
activating
of the
intra-
the resultant
bonds
is 2 for
sulfhydryl
the complement
functionality
of IqG,
of inter-chain
obtained
the
Considering
disulfide
and carboxymethylation
out the
(13).
of Fc
chain
their
2 x 2.9 = 5.8 the
pool
approximately
of inter-heavy
than
on IgG in the absence
and IgG3
in the normal
be composed
The selective
Specific bonds
of papain
subclasses
would
to reduction
The action
The mean value
LgG3 (17).
figure
bonds. restricted
Fc fragment
in this
are much more susceptible
effector
of the
inter-chain
capacity bonds site
of Fc fragment. in the
for
disulfide
hinge
complement.
region It
Vol. 78, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(+I
(+)
2-
-4
-2 m k 5 -cl
6.
240
Figure
has
3.
binding
220
shown
that
activity
(la),
binding
events,
240
230
of
probably
this
kind
human
Fc yl
Clg
and
activation
affected
by
with
the
effector.
possibility
that
the
enhancing
modified has
is
and
fragment
been
20),
(19,
It
Fc
then
the
role
of
of Our
260
2?0
2eo
290
300
Clq,
but
spectral
alteration affects
the
changes
in the
studies
(21)
to
overall
at
least
cleavage
of the
in
complement
whatever
bonds
the
the
in covalent
peptide
bonds
chromophores. of
non-covalent
1324
to
out
the
of
the
Cl.
the
IgG
effect
the
For
example,
molecule
activation
is
of
FC
noted
during
than
on is
interpreted
aromatic
absence
the
event,
of of
assymetry
shown
best
bind
Cl
non-equivalent
rule
other
affect been
interactions
can
molecular
be
to
~4
disulfide
has
requirements
components
that,
other
the
due
not
but
we cannot
is
assume
environment have
effect
as
connected
Furthermore,
binding
can
are
3fO
does
However,
molecular
this
some
Cl
different
inter-chain
data
following
of
5 (-I.320 A(m)
) and selectively (--------) of human in the 215-220 nm increase in negative
modification (8).
complement
of
the
chemical fragment
that
reasonable
alkylation
binding
with
demonstrated but
of
of
interaction
it
250
Circular dichroism spectra of intact ( reduced and carboxymethylated Fc fragment IqG. General identity was observed except negative ellipticity band,&ere a significant ellipticity was detected.
been
? 2x
-3
unknown.
reduction
fragment,
not
the
affected. terms
of
interchain with
a conformational bonds
no X-ray
interactions
which
conformational diffraction between
the
Vol. 78, No. 4, 1977
two
cH2
domains.
interactions Both
The
are
and
cH3
upon
by
hardly bands
in
after
reduction
for
the
far
cleavage
of
(CH2-CH3
flexibility)
(21)
could
it
by
steric
could
in
the
are
some
(near in
imposed
Fc
alteration be
of
longitudinal
asymmetry
of
compatible, after
flexibility
absence
the
CH2-CH2 CH2-CH3
individual the
observed
CD
are
fragment
This
by
and
CD spectral
may
the
to
LJV)
conformational
of
the
the
unlikely bonds
alteration
by by
explain
very
range
region.
possible limited
this
Changes
hinge
overall
qualitatively
confor-
flexibility
made
the
model,
on
disulfide
of
restrictions
changes
(22)
Based
This
be
followed
bonds.
domains.
peptide
changes
inter-
bonds, in
the
far
CD spectrum. Thus,
graphic
on and
ccmplement specific
bonds
could
also
basis
our
capacity
more
compatible
and
related idea
to only
direct
experiments
evidence,
reduction
this of
of
immunochemical
be
We favour absence
the
activating
after
are
and
would
independently
results.
internal
be
disulphide
absorption
our
in
inter-chain
inter-chain
existence
bonds
and
produce
thus,
increased
would
actions
are
carboxymethylation. an
non-covalent
structure
groups with
extensive
the
segment.
of
the
disulfide
interactions
W
agreement
by
intra-domain
aromatic
UV indicate
the
lateral
and
in
by
chain
alkylation
the
with
and
folded the
in
and
example,
domains
a loosely
and
expected,
stabilized
polypeptide
affecting
changes
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
is
cH3
each
reduction
spectral
It
in
changes
region
the
separated
mational occur
Fc
between
cHZ
and
BIOCHEMICAL
an as
the
we suggest
which
takes
enhanced a working other present
of
the
the the
for of
crystallo-
increase
of
isolated
Fc
inter-chain
flexibility
hypothesis patterns
the
in of
internal
available
that
place
carboxymethylation
evidence, with
and
of it
structural
is
clear
fragment disulfide
the
protein. that
in
alteration
data.
ACKNOWLEDGEMENTS The authors Junta de Energia circular dichroism University School
wish to thank Dr. C. Davila and Dr. ;. Mingot, from the Nuclear, Madrid, for help and advice in obtaining the spectra. We thank Dr. L.M. Amzel, from the Johns Hopkins of Medicine, for his invaluable criticism and encouragement.
1325
Vol. 78, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Colomb, M. and Porter, R.R. (1975). Biochem. J. 145, 177-183. Yasmeen, D.; Ellerson, J.R.; Dorrington, K.J. and Painter, R.H. (1976). J. Immunol. 116, 518-526. Kehoe, J.M. and Fougereau, M. (1969). Nature (London) 224, 1212-1213. Kehoe, .l.M.; Bourgois, A.; Capra, D.J. and Fougereau, M. (1974). Biochemistry l3, 2499-2504. Isenman, D.E.; Painter, R.H. and Dorrington, K.J. (1975). Proc. Nat. Acad. Sci. U.S. 3 548-552. Schur, P.H. and Christian, G.D. (1964). J. EXP. Med. 120, 531-545. Press, E.M. (1975). Biochem. J. 149, 285-288. Isenman, D.E.; Dorrington, K.J. and Painter, R.H. (1975). J. Immunol. 2, 1726-1729. Schlessinger, J.; Steinberg, 1.2.; Givol, D.; Hochman, J. and Pecht, I. (1975). Proc. Nat. Acad. Sci. U.S.A. 12, 2775-2779. Jaton, J.U.; Huser, H.; Braun, D.G.; Gsol, D.; Pecht, I. and Schlessinger, J. (1975). Biochemistry g, 5312-5315. Chan, L.M. and R.E. Cathou (1977). J. Mol. Biol. l&?, 653-656. Utsumi, S. (1969). Biochem. J. 112, 343-355. Stanworth, D.R. and Turner, M.W. (1973). Handbook of Experimental Immunology, Ed. 2 (Ed. by Weir, D.M.) Vol. 1: Immunochemistry, pp. lo-14 10-15. Blakwell Scientific Corp. Gall, W; Cuningham, B.A.; Waxdall, M.J.; Konigsberg, W.H. and Edelman, Biochemistry z, 1973-1982. G.M. (1968). Haber, E. and Anfinsen, C.B. (1960). J. Biol. Chem. 236, 422-424. Mayer, M.M. (1961). In Kabat, E.A. and Mayer, M.M. Experimental Immunochemistry. Ed. 2, pp. 133-240. Charles C. Thomas Publ., Springfield, Ill. Michaelsen, T.E; Frangione, B. and Franklin, E.C. (1977). J. Biol. Chem. 252, 883-889. Allan, R. and Isliker, H. (1974). Immunochemistry 11, 243-248. Muller-Eberhard, H.J. and Lepow, 1-H. (1965). J. Exp. Med. 121, 819-833, Willoughby, W.F. and Mayer, M.M. (1965). Science 150, 907-908. Huber, R.; Deisenhofer, J.; Colman, P.M. and Matsuxma. (1976). Nature (London) 264, 415-420. Poljak, R.J.; Amzel, L.M.; Avey, H.P.; Chen, B.L.; Phizackerley, R.P. and Saul, F. (1973). Proc. Nat. Acad. Sci. USA 10, 3305-3310.
1326
Fc
FRAGMENT
OF
INTER-CHAIN
JO&
A.
BIOCHEMICAL
OF HUMAN
IMMUNOGLOBULIN
DISULFIDE
Lopez
de
BONDS
Department
of
Received
August
G:
AND
COMPLEMENT
Vivanco
Fundacidn
Immunology.
REDUCTION
ENHANCES
Fernando
Castro:
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and
Jimenez
CARBOXYMETHYLATION
ACTIVATING
Fernando
Diaz.
CAPACITY
Ortiz Madrid,
Spain.
26,1977
Summary: The complement activating capacity of the FC fragment of human immunoglobulin G increases after selective reduction and carboxymethylation of the inter-chain disulfide bonds. Differences in the circular dichroism spectra of intact and modified fragment suggest that minor conformational changes have occurred. Our results are discussed in relation to the molecular requirements for complement activation.
INTRODUCTION There the
is
strong
IgG
moleculeff
complement
(Cl)
is
still
not
is
located
possible
to
the
acid
residues
complement
effector
for
into
by
abrogates interaction,
fully
has
been for
IgG
that
complement (8)
the
be
measured
also
of
circular
of
polarization
#
disulfide
0 1977 by Academic oj reproduction in any
Press, Inc. iowl reserved.
1319
of
amino that
the residues
af upon of
(2,6). bonds
are
complexes these
bonds
also
antigen-antibody
fluorescence
To whom correspondence should be addressed. Present address: of Biophysics. The Johns Hopkins University School of Medicine, Wolfe Street, Baltimore, Md. 21205 (U.S.A.). ff Abbreviations: IgG: Immunoglobulin G: DTE: Dithioerythritol; Sheep red blood cells; CD: Circular dichroism; W: Ultraviolet.
Copyrighr Ail rights
suggest
chain
antigen-antibody
induced
the
non-consecutive
alkylation Fc
reports
suggesting
by
of it
and
sequence
polypeptide
by and
Some
of
However, groups
a linear
chain
region component
(1,2).
evidence
the
the
first
chemical
determined
activity
changes by
by
inter-heavy
Reduction
.
is
folding
conformational as
effector formed
the
region
site.
binding
molecules
the
the
homology
the is
that
with
the
could
by
studies
characterize
there
complement
shown
CT2
site
However,
of
interaction
the
of
proximity
a number
the
in
binding
(2-S).
site
essential and
in
requirements
that
It
from
involved
conformational
brought
evidence
(9,lO)
Department 725 North SRBC:
(7)
Vol. 78, No. 4, 1977
and
enhances
these
an
the
studies
disulfide
internal
point bonds
integral
part
of
was
undertaken
these
bonds
in
AND
the
of
the
IqG
molecule
the
important
role
of
the
hinge
region,
there
is
evidence
the
controlling
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
flexibility
out
in
study
MATERIALS
BIOCHEMICAL
with
complement the the
binding
aim
of
further
biological
site
(11).
inter-heavy
chain
that
(8,
12).
investigating activity
Although
of
they The the
the
Fc
are
not
present role
of
region.
METHODS
FC fragment was obtained by papain digestion of pooled human IqG in the absence of cysteine as described (13) and purified by DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Neither IgG nor any other serum protein was found in the Fc preparations, but a small amount of Fab appeared sometimes in immunoelectrophoresis as a weak precipitine band. This contamination could not be detected by polyacrylamide gel electrophoresis. Selective reduction and alkylation of inter-chain disulfide bonds was carried out as previously described (14) with some modifications. Aliquots of Fc fragment (1 mg/ml in 10 mM Tris/HCl buffer, 0.5 M NaCl, 2 miY EDTA, pH 7.8) were incubated for two hours at 25OC with dithioerythritol (DTE) at concentrations ranging between 1 and 10 mM. The reduced protein was alkylated with iodoacetamide for 20 minutes in the dark. In other experiments the protein was incubated with 3 mM DTE as described above and alkylation was carried out for 20 minutes in the dark by adding iodoacetamide or iodoacetic Titration of sulfhydryl groups was acid to a final concentration o f40+ M. achieved by alkylation with lC-iodoacetamide as described (15). Nonspecific radioactivity was detected by incubating non-reduced Fc fragment with l- 14C-iodoacetamide under the same conditions. Complement activating capacity was measured by the quantitative compleSerial ment fixation test (16), modified to obtain maximal sensitivity. dilutions of non-aggregated protein were incubated with 1 CH50 unit of human complement for 1 hour at 30°C and then for 18 hours at 4'C, in 2 ml total After incubation, 1 ml of an 0.15% suspension of sheep red blood volume. sensitized with an excess of rabbit anti-Forssmann antibody, cells (SRBC), All tubes were incubated for 1 hour at 30°C. was added to each tube. Spontaneous haemolysis controls (sensitized SRBC+diluent) and complement activity controls (sensitized SRBC + 1 CH50 + diluent) were included in each After incubation, the non-damaged cells were spun down at 350 x experiment. g for 15 minutes at 4Oc and optical densities of the supernatants were Haemolysis inhibition percentages were calculated as measured at 414 run. described (16) and plotted against protein concentration. Circular dichroism (CD) measurements were made at 25°C using a Cary-60 and cells with 1 cm path recording spectropolarimeter with CD attachment, Samples length. Spectra were recorded in the range from 212to 320 nm. contained 100 microg./ml for far-W measurements and 1 mg/ml for near-W. Ellipticity (0) was expressed as molar ellipticity which is defined as ox M x 100 8 = dxC 17 d the path length where M is the molecular weight of FC (52,000), All meters-(d = 1 cm) and C the concentration of protein in g/l. were obtained in triplicate and the arithmetic mean was calculated.
1320
in centithe spectra
Vol. 78, No. 4, 1977
TABLE
BIOCHEMICAL
AND 5lOPHYStCAL
RESEARCH COMMUNICATIONS
I
Titration of sulfhydryl groups of the 3 mM dithioerythritol treated The specific activity ratio of labelled protein FC fragment of human IgG. to radioactive iodoacetamide is equal to the molar ratio of sulfhydryl groups to protein and therefore represents the number of sulfhydryl groups Controls of non-reduced Fc fragment were included in each per molecule. experiment.
Specific bCi/mM Exp. l-l*C-iodoacetamide.
activity
1
Exp.
100
Control
. . . . . . . . . . . .
Reduced
FC
2
Exp.
100 5.26
fragment.
Specific Specific Exp.
1
protein iodac.-
Exp.
2
Exp.
3
100 5.26
431.58
3
act. act.
5.26
431.58
0.052
0.052
0.052
4.3
4.3
5.1
specific
reduction
514.80
RFSULTS In
order
chain
disulfide
added
to
to
find bonds,
identical was
estimated
Sephadex
G-100
column.
efficiency
of
reduction
its
of
to
disulfide
shown
in
bonds
was
number only
Table
In
Protein
carried
out
this
I.
They
papain parallel
for
with
the
percentage
acid.
3 ti bonds
of
The
eluted
results
indicate
that
The
number
of
the
normal
pool
sensitive experiments,
subclasses, samples
three
I-
of
of
1321
of
human
than
90%.
in
these
IgGl
in affect
inter-chain
IgG,
monomeric
accomplish
and intact
are disulfide
with
taking
order
intra-
experiments
agrees
the
dissociated
to
would
different
cleaved
namely
protein
generated
agent
a
and
'* C-iodoacetamide
reduction bonds
through
better
groups
were
of
acid
enough
inter-
10 mM)
protein
of was
reducing
of
extent
a yield
sulfhydryl
of
The
1 M acetic
DTE
with
to
reduced
with
titration
of
BYTE (1
mg/ml).
of
as
concentration
bonds.
(1
samples was
by
of
protein
disulfide
selective.
calculated the
Fc
passing
in
for
concentrations
determination
whether
chain
by
inter-chain
was
test
of
chains
Quantitative conditions
increasing
expressed
polypeptide
reduction
conditions
aliquots
reduction
into
optimal
the into
average account
IgG3. Fc
fragment
and
Vol. 78, No. 4, 1977
BIOCHEMICAL
% Fc
Figure
of
1.
monomeric
Fc
selectively
were
tested
previous in
the
fragment
as
compared
induced
by
spectrum
between
210
ellipticity the
were
of
intact
and
320
spectrum
spectrum
was of
identical
at
in
the
far
W
in
the
215-220
it
in
(Figure
by
within
exhibited nm band
the
(Figure
described
A 5-
to
in
lo-fold
carboxymethylated
detect
any
inter-chain
Fc
not
show
positive
In
the
near
W,
272,
288
292
between
carboxymethylated error
215
increase
of
in
bands
of
the
far
In
nm.
Fc
experimental
bonds.
ellipticity negative
and
band
conformational disulfide
did
a significant
with as
2.
extent by acid.
observed. to
of
a negative an
Figure and
was
attempt
approximately
reduced
former
an
3).
in
reduced Fc
alkylated
capacity
shown
intact
fragment
dominated
the
are
of
and
activating
modification
Fc
selectively
to
that
chemical
appeared
3 mM DTE
selectively
obtained
nm
with
results of
to
of dithioerythritoL on the disulfide bonds, as measured dissociated in 1 M acetic
complement
The
activity
CD spectra
The
reduced for
section.
increase
changes
dtssoclatod
Effect of the concentration of reduction of inter-chain the percentage of Fc fragments
iodoacetate the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and
220
fragment the
W The
run. was
near
negative
W
but
ellipticity
3).
DISCUSSION The index
of
number the
of selectivity
titrated
sulfhydryl of
reduction
groups of
1322
the
per
molecule
inter-chain
Serves disulfide
as
an
bonds
BIOCHEMICAL
Vol. 78, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RO-
60-
Figure
2.
Inhibition of hemolytic activity of complement by intact (a) and selectively reduced and carboxymethylated (0) Fc fragment. The dispersion of data in each group of experiments is represented as the extreme values.
since
these
chain
disulfide
cysteine
bonds
is
percentage
of these
(10.3%).
pool
pool
fragment
This
to subclasses
IqGl
should
be 2.9 since
would agrees
markedly result
in controlling
yield with
reduction enhances points the
reduction
value
number
in our
importance
groups
of the
IgGl
and Fcy3
per molecule and 11 for bonds
of Fc
per molecule.
This
experiments.
of covalent
1323
(89.7%)
Yl
disulfide
activating
of the
intra-
the resultant
bonds
is 2 for
sulfhydryl
the complement
functionality
of IqG,
of inter-chain
obtained
the
Considering
disulfide
and carboxymethylation
out the
(13).
of Fc
chain
their
2 x 2.9 = 5.8 the
pool
approximately
of inter-heavy
than
on IgG in the absence
and IgG3
in the normal
be composed
The selective
Specific bonds
of papain
subclasses
would
to reduction
The action
The mean value
LgG3 (17).
figure
bonds. restricted
Fc fragment
in this
are much more susceptible
effector
of the
inter-chain
capacity bonds site
of Fc fragment. in the
for
disulfide
hinge
complement.
region It
Vol. 78, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(+I
(+)
2-
-4
-2 m k 5 -cl
6.
240
Figure
has
3.
binding
220
shown
that
activity
(la),
binding
events,
240
230
of
probably
this
kind
human
Fc yl
Clg
and
activation
affected
by
with
the
effector.
possibility
that
the
enhancing
modified has
is
and
fragment
been
20),
(19,
It
Fc
then
the
role
of
of Our
260
2?0
2eo
290
300
Clq,
but
spectral
alteration affects
the
changes
in the
studies
(21)
to
overall
at
least
cleavage
of the
in
complement
whatever
bonds
the
the
in covalent
peptide
bonds
chromophores. of
non-covalent
1324
to
out
the
of
the
Cl.
the
IgG
effect
the
For
example,
molecule
activation
is
of
FC
noted
during
than
on is
interpreted
aromatic
absence
the
event,
of of
assymetry
shown
best
bind
Cl
non-equivalent
rule
other
affect been
interactions
can
molecular
be
to
~4
disulfide
has
requirements
components
that,
other
the
due
not
but
we cannot
is
assume
environment have
effect
as
connected
Furthermore,
binding
can
are
3fO
does
However,
molecular
this
some
Cl
different
inter-chain
data
following
of
5 (-I.320 A(m)
) and selectively (--------) of human in the 215-220 nm increase in negative
modification (8).
complement
of
the
chemical fragment
that
reasonable
alkylation
binding
with
demonstrated but
of
of
interaction
it
250
Circular dichroism spectra of intact ( reduced and carboxymethylated Fc fragment IqG. General identity was observed except negative ellipticity band,&ere a significant ellipticity was detected.
been
? 2x
-3
unknown.
reduction
fragment,
not
the
affected. terms
of
interchain with
a conformational bonds
no X-ray
interactions
which
conformational diffraction between
the
Vol. 78, No. 4, 1977
two
cH2
domains.
interactions Both
The
are
and
cH3
upon
by
hardly bands
in
after
reduction
for
the
far
cleavage
of
(CH2-CH3
flexibility)
(21)
could
it
by
steric
could
in
the
are
some
(near in
imposed
Fc
alteration be
of
longitudinal
asymmetry
of
compatible, after
flexibility
absence
the
CH2-CH2 CH2-CH3
individual the
observed
CD
are
fragment
This
by
and
CD spectral
may
the
to
LJV)
conformational
of
the
the
unlikely bonds
alteration
by by
explain
very
range
region.
possible limited
this
Changes
hinge
overall
qualitatively
confor-
flexibility
made
the
model,
on
disulfide
of
restrictions
changes
(22)
Based
This
be
followed
bonds.
domains.
peptide
changes
inter-
bonds, in
the
far
CD spectrum. Thus,
graphic
on and
ccmplement specific
bonds
could
also
basis
our
capacity
more
compatible
and
related idea
to only
direct
experiments
evidence,
reduction
this of
of
immunochemical
be
We favour absence
the
activating
after
are
and
would
independently
results.
internal
be
disulphide
absorption
our
in
inter-chain
inter-chain
existence
bonds
and
produce
thus,
increased
would
actions
are
carboxymethylation. an
non-covalent
structure
groups with
extensive
the
segment.
of
the
disulfide
interactions
W
agreement
by
intra-domain
aromatic
UV indicate
the
lateral
and
in
by
chain
alkylation
the
with
and
folded the
in
and
example,
domains
a loosely
and
expected,
stabilized
polypeptide
affecting
changes
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
is
cH3
each
reduction
spectral
It
in
changes
region
the
separated
mational occur
Fc
between
cHZ
and
BIOCHEMICAL
an as
the
we suggest
which
takes
enhanced a working other present
of
the
the the
for of
crystallo-
increase
of
isolated
Fc
inter-chain
flexibility
hypothesis patterns
the
in of
internal
available
that
place
carboxymethylation
evidence, with
and
of it
structural
is
clear
fragment disulfide
the
protein. that
in
alteration
data.
ACKNOWLEDGEMENTS The authors Junta de Energia circular dichroism University School
wish to thank Dr. C. Davila and Dr. ;. Mingot, from the Nuclear, Madrid, for help and advice in obtaining the spectra. We thank Dr. L.M. Amzel, from the Johns Hopkins of Medicine, for his invaluable criticism and encouragement.
1325
Vol. 78, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Colomb, M. and Porter, R.R. (1975). Biochem. J. 145, 177-183. Yasmeen, D.; Ellerson, J.R.; Dorrington, K.J. and Painter, R.H. (1976). J. Immunol. 116, 518-526. Kehoe, J.M. and Fougereau, M. (1969). Nature (London) 224, 1212-1213. Kehoe, .l.M.; Bourgois, A.; Capra, D.J. and Fougereau, M. (1974). Biochemistry l3, 2499-2504. Isenman, D.E.; Painter, R.H. and Dorrington, K.J. (1975). Proc. Nat. Acad. Sci. U.S. 3 548-552. Schur, P.H. and Christian, G.D. (1964). J. EXP. Med. 120, 531-545. Press, E.M. (1975). Biochem. J. 149, 285-288. Isenman, D.E.; Dorrington, K.J. and Painter, R.H. (1975). J. Immunol. 2, 1726-1729. Schlessinger, J.; Steinberg, 1.2.; Givol, D.; Hochman, J. and Pecht, I. (1975). Proc. Nat. Acad. Sci. U.S.A. 12, 2775-2779. Jaton, J.U.; Huser, H.; Braun, D.G.; Gsol, D.; Pecht, I. and Schlessinger, J. (1975). Biochemistry g, 5312-5315. Chan, L.M. and R.E. Cathou (1977). J. Mol. Biol. l&?, 653-656. Utsumi, S. (1969). Biochem. J. 112, 343-355. Stanworth, D.R. and Turner, M.W. (1973). Handbook of Experimental Immunology, Ed. 2 (Ed. by Weir, D.M.) Vol. 1: Immunochemistry, pp. lo-14 10-15. Blakwell Scientific Corp. Gall, W; Cuningham, B.A.; Waxdall, M.J.; Konigsberg, W.H. and Edelman, Biochemistry z, 1973-1982. G.M. (1968). Haber, E. and Anfinsen, C.B. (1960). J. Biol. Chem. 236, 422-424. Mayer, M.M. (1961). In Kabat, E.A. and Mayer, M.M. Experimental Immunochemistry. Ed. 2, pp. 133-240. Charles C. Thomas Publ., Springfield, Ill. Michaelsen, T.E; Frangione, B. and Franklin, E.C. (1977). J. Biol. Chem. 252, 883-889. Allan, R. and Isliker, H. (1974). Immunochemistry 11, 243-248. Muller-Eberhard, H.J. and Lepow, 1-H. (1965). J. Exp. Med. 121, 819-833, Willoughby, W.F. and Mayer, M.M. (1965). Science 150, 907-908. Huber, R.; Deisenhofer, J.; Colman, P.M. and Matsuxma. (1976). Nature (London) 264, 415-420. Poljak, R.J.; Amzel, L.M.; Avey, H.P.; Chen, B.L.; Phizackerley, R.P. and Saul, F. (1973). Proc. Nat. Acad. Sci. USA 10, 3305-3310.
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