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Mouse Lambda-Chain Sequences

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Abstract and Figures

Amino-acid sequences of the variable regions of three lambda chains produced by plasmacytomas of BALB/c mice are compared. Two are almost certainly identical and one differs from these by three amino acids. These findings extend our earlier conclusion on the relative uniformity of sequences in this type of immunoglobulin light chain. With amino-acid sequence data on two additional lambda chains, eight mouse lambda chains studied to date are indistinguishable and four probably differ from these by one, two, or three amino acids.
Content may be subject to copyright.
Proc.
Nat.
Acad.
Sci.
USA
Vol.
70,
No.
7,
pp.
2112-2116,
July
1973
Mouse
Lambda-Chain
Sequences
(variable
regions/plasmocytomas/BALB/c
mice)
ITALO
M.
CESARI*
AND
MARTIN
WEIGERT
The
Salk
Institute
for
Biological
Studies,
San
Diego,
California
92112
Communicated
by
Herman
N.
Eisen,
April
9,
1973
ABSTRACT
Amino-acid
sequences
of
the
variable
regions
of
three
lambda
chains
produced
by
plasma-
cytomas
of
BALB/c
mice
are
compared.
Two
are
almost
certainly
identical
and
one
differs
from
these
by
three
amino
acids.
These
findings
extend
our
earlier
conclu-
sion
on
the
relative
uniformity
of
sequences
in
this
type
of
immunoglobulin
light
chain.
With
amino-acid
sequence
data
on
two
additional
lambda
chains,
eight
mouse
lambda
chains
studied
to
date
are
indistinguish-
able
and
four
probably
differ
from
these
by
one,
two,
or
three
amino
acids.
We
reported
previously
that
six
of
10
lambda
chains
pro-
duced
by
plasmacytomas
of
BALB/c
mice
were
indistinguish-
able
by
partial
sequence
and
composition
analyses
of
peptides
derived
by
enzymatic
digestions
of
these
chains.
Four
of
the
10
lambda
chains
differed
from
these
by
one,
two,
or
three
amino-acid
substitutions.
Thus,
these
lambda
chains
exhibit
a
strikingly
simple
pattern
of
variability
compared
to
human
kappa
and
lambda
chains.
Not
only
has
the
contribution
to
variability
due
to
polymorphism
been
eliminated,
but
BALB/c
mice
appear
to
express
only
one
lambda
variable-
region
subgroup,
which
we
interpret
to
be coded
for
by
a
single
germ-line
lambda
variable-region
gene.
The
simple
pattern
of
variability
suggests
that
we
might
be
analyzing
antibodies
in
the
initial
stages
of
selection
by
antigen
(1).
Two
of
the
indistinguishable
lambda
chains,
J558
and
xS104,
have
now
been
compared
by
further
sequence
analysis
of
their
variable
regions,
and
the
results
strengthen
the
argu-
ment
that
they
are
identical.
The
variant
lambda
chains
differ
from
the
ostensibly
identical
lambda
chains
(1)
only
in
those
regions
that
are
hypervariable
(2).
This
is
con-
firmed
here
by
additional
sequence
data
on
the
variable
re-
gion
of
the
variant
S178.
Further,
two
additional
lambda
chains
have
been
partially
sequenced
and
are
also
indistin-
guishable
from
the
six
apparently
identical
lambda
chains
previously
reported.
MATERIALS
AND
METHODS
The
light
chains
used
in
this
study
are
produced
by
plasma-
cytomas
of
BALB/c
mice.
J558,
W3159,
S104,
and
S178
are
tumors
induced
in
Dr.
Melvin
Cohn's
laboratory
(Hirst,
J.,
Jones,
G.,
Weigert,
Mi.,
and
Cohn,
M.,
unpublished).
M511
was
given
to
us
by
Dr.
Michael
Potter.
TPCK-treated
trypsin
(EC
3.4.4.4)
and
a-chymotrypsin
(EC
3.4.4.5)
were
obtained
from
Worthington
Biochemical
Corp.
(Freehold,
N.J.).
Thermolysin,
grade
B,
was
obtained
from
Calbiochem
(Los
Angeles,
Calif.),
and
was
recrystallized
according
to
the
method
described
by
Matsubara
(3).
Carboxypeptidase-A-
DFP
(EC
3.4.2.1)
was
obtained
from
Sigma
Chemical
Co.
(St.
Louis,
Mo.).
The
myeloma
proteins
produced
by
J558,
W3159,
and
S104
tumors
were
IgA.
These
proteins
were
purified
from
the
sera
of
tumor-bearing
mice
(4),
and
the
light
chains
were
isolated
from
these
proteins
(5).
S178
produces
only
lambda
chain,
which
was
isolated
from
the
urine
of
female
BALB/c
mice.
M511
lambda
chain
was
isolated
from
the
urine
of
female
BALB/c
mice
(4).
xS104
is
a
derivative
of
the
S104
tumor,
which
produces
mainly
lambda
chain
as
well
as
small
amounts
of
IgA.
The
protein
used
here
was
the
urinary
lambda
chain.
The
purified
lambda
chains
were
denatured
by
either
per-
formic-acid
oxidation
(6)
or
complete
reduction
and
S-amino-
ethylation
(7).
Denatured
lambda
chains
were
enzymatically
digested
in
0.05
M
NH3HCO3
for
2
hr
at
370
at
an
enzyme
to
substrate
ratio
of
1:50
w/w
with
the
light
chains
at
concentrations
of
10-20
mg/ml.
Peptides
were
digested
in
0.05
M
NH4HCO3
for
2
hr
at
37°
with
the
enzymes
at
a
0.01%
w/v
concentra-
tion.
Enzymatic
digestions
were
stopped
by
addition
of
an
equal
volume
of
1
N
acetic
acid,
and
the
samples
were
lyo-
philized.
Peptides
resulting
from
enzymatic
digestions
were
purified
by
electrophoresis
on
paper
at
pH
4.7,
by
electrophoresis
followed
by
chromatography
(8),
or
by
gel
filtration
on
Bio-
Gel
P-4
columns
(0.9
X
90
cm)
equilibrated
with
1%
formic
acid.
Peptides
purified
by
electrophoresis
and
chromatog-
raphy
were
detected
by
staining
the
paper
with
0.02%
nin-
hydrin
in
acetone
and
eluted
with
either
50%
pyridine
or
6
N
HCl.
The
amino-acid
sequence
of
peptides
was
determined
by
the
subtractive-Edman
method
(9)
and/or
the
dansyl-Edman
method
(10).
The
positions
of
certain
residues
were
determined
by
carboxypeptidase-A
digestion
of
peptides.
This
enzyme
was
used
at
a
concentration
of
0.01%
in
25
mM
Tris-
HC1
(pH
7.5)-0.5
M
NaCl
(11).
Aliquots
were
withdrawn
at
dif-
ferent
intervals
of
digestion
and
analyzed
for
free
amino
acids.
Complete
acid
hydrolysis
was
done
in
6
N
HCl
for
16
hr
at
1050
in
the
presence
of
4%
thioglycolic
acid
(12).
Partial
acid
hydrolysis
was
done
either
in
6
N
HCl
for
30
min
at
1050
or
in
0.03
N
HC1
at
1050
for
12
hr.
Amino-acid
analyses
were
performed
on
Beckman/Spinco
analyzers,
models
120C
and
121.
2112
*
Present
address:
Centro
de
Microbiologia,
Instituto
Vene-
zolano
de
Investigaciones
Cientificas,
Apartado
1827,
Caracas,
Venezuela.
Mouse
Lambda-Chain
Sequences
2113
TABLE
1.
Tryptic
peptides
from
the
variable
region
of
J558,
xSl04,
and
S178
lambda
chains
Peptide
Residues
Protein
Amino-acid
sequence
T-1
1-23
J558,
xS104,
S178
PCA
(Ala2,
Val3,
Thr6,
Glx3,
Ser2,
Pro,
Gly,
Leu2,
Cys)
Arg
T-2
24-56
J558,xS104
Ser-Ser-Thr
(Gly4,
Ala2,
Val2,
Thr4,
Ser,
Asx5,
Tyr,
Trp,
Glx2,
Lys,
Pro,
His,
Leu2,
Phe,
Ile)
Arg
S178
Ser-Asn-Thr
(Gly3,
Ala2,
Val2,
Thr4,
Ser,
Asx6,
Tyr,
Trp,
Glx2,
Lys,
Pro,
His,
Leu2,
Phe,
Ile)
Arg
T-3
57-63
J558,
xS104,
S178
Ala-Pro-2.y-Val-Pro-Ala-Arg
T-4
64-72
J558,
xS104,
S178
Phe-Ser-Gly-Ser-Leu-Ile-Gly-Asx-Lys
T-5
73-90
J558,
xS104,
S178
(Ala4,
Leu,
Thr3,
Ile2,
Gly,
Glx3,
Asx,
Tyr,
Phe)
Cys
T-6
91-105
J558,xS104
Ala-Leu-Trp-Tyr-Ser-Asx-His
(Trp,
Val,
Phe,
Gly3,
Thr)
Lys
T-6a
91-97
S178
Ala-Leu-Trp-Tyr-Ser-Asx-Arg
T-6b
98-105
S178
Trg-Val-Phe-Sl
-G-G1
-Thr-Lys
T-7
106-113
J558,
xS104,
S178
Leu-Thr-Val-Leu-Gly-Glx-Pro-Lys
(-)
Indicates
subtractive-Edman
and/or
dansyl-Edman.
*
Indicates
that
the
position
of
these
residues
is
inferred
from
the
known
action
of
trypsin
on
lysine,
arginine,
and
aminoethylcysteine
residues.
RESULTS
The
partial
sequences
of
three
lambda
chains,
M511,
W3159,
and
S104,
were
established
by
the
methods
described
(1).
By
these
methods
these
light
chains
are
indistinguishable
from
the
xS104
and
J558
light
chains.
Fig.
1
shows
the
tentative
sequence
of
the
variable
region
of
the
xS104,
J558,
and
S178
lambda
chains.
The
order
of
the
variable-region tryptic
peptides
(Table
1)
was
established
by
certain
chymotrypsin
or
thermolysin
peptides
isolated
by
electrophoresis
and
chromatography
of
digests
of
S-amino-
ethylated
lambda
chains.
The
relevant
peptides
are
the
chymotrypsin
peptides
C-4,
C-9,
C-10,
and
C-20
(Table
2)
and
the
thermolysin
peptide
Th-19
(Table
3).
This
order
of
tryptic
peptides
as
inferred
from
the
composition
of
these
peptides
is
supported
by
the
homology
of
these
sequences
and
the
revised
sequence
of
the
mouse
lambda
chain
MOPC-
104E
(Appella,
personal
communication).
By
a
comparison
with
the
complete
sequence
of
MOPC-104E
(13),
all
but
the
region
between
residues
175
and
183
was
accounted
for
by
the amino-acid
compositions
of
peptides
detected
after
elec-
trophoresis
and
chromatography
of
either
trypsin
or
thermo-
lysin
digests
of
S-aminoethylated
S178.
The
electrophoretic
and
chromatographic
position
of
the
peptides
arising
from
residue
114
to
the
carboxy-terminus
of
xS104,
J558,
and
S178
were
indistinguishable.
Differences
between
these
pro-
teins
in
this
region
that
do
not
alter
the
electrophoretic
and
chromatographic
position
of
a
peptide
or
that
occur
between
residue
175
and
183
(not
detected
by
these
procedures)
cannot
be
excluded.
The
following
methods
were
used
for
sequencing
the
tryptic
peptides
that
could
be
ascribed
to
the
variable
region
by
comparison
with
the
lambda
chains
of
MOPC-104E
(13).
T-1
(residues
1-23)
Two
peptides
could
be
detected
after
electrophoresis
and
chromatography
of
the
tryptic
digest
of
the
S-aminoethylated
S178
protein
only
after
they
were
stained
by
chlorination
(14).
One
of
these
peptides
could
also
be
detected
by
staining
the
paper
for
arginine-containing
peptides
(15).
By
analogy
with
the
results
obtained
with
S-aminoethylated
MOPC-104E
(13),
it
was
assumed
that
these
peptides
represented
residues
1-22
and
1-23
of
the
178
protein.
For
sequence
analysis,
this
pep-
tide
was
obtained
by
electrophoresis
at
pH
4.7 of
tryptic
digests
of
performic
acid-oxidized
protein.
Guide
strips
were
stained
with
ninhydrin
and
for
arginine-containing
peptides.
T-1
was
detected
as
a
ninhydrin-negative,
arginine-positive
peptide.
The
peptide
was
eluted
from
the
unstained
portion
of
the
paper,
lyophilized,
and
digested
with
either
ther-
molysin
or
chymotrypsin.
These
digests
were
subjected
to
electrophoresis
at
pH
4.7,
and
guide
strips
were
stained
with
ninhydrin
and
by
chlorination.
Peptides
that
could
be
de-
tected
by
ninhydrin
staining
were
eluted,
analyzed
for
total
amino-acid
composition,
and
sequenced
by
the
subtractive-
Edman
procedure
and
by
carboxypeptidase-A
digestion.
Thermolysin
peptides
(Table
3)
were
ordered
by
the
peptides
obtained
after
chymotrypsin
digestion
of
T-1
(Table
2).
Peptide
Th-1
resulting
from
thermolysin
digestion
and
C-1
from
chymotrypsin
digestion
of
T-1
could
be
detected
only
by
staining
of
guide
strips
by
chlorination.
The
amino-acid
composition
of
Th-1
was
glutamic
acid
and
alanine.
It
was
assumed
that
this
peptide
represented
the
first
two
amino
acids
of
T-1
and
that
the
first
residue
of
the
lambda
chains
was
pyrrolidone
carboxylic
acid.
T-2
(residues 24-56)
This
peptide
could
be
obtained
in
small
amounts
after
elec-
trophoresis
and
chromatography
of
digests
of
S-aminoeth-
ylated
protein.
In
order
to
obtain
larger
quantities
of
this
peptide,
digests
were
fractionated
by
gel
filtration.
The
ap-
propriate
fraction
was
further
purified
by
electrophoresis
at
pH
4.7.
The
amino-acid
sequence
of
the
first
three
residues
of
this
peptide
was
determined
by
the
dansyl-Edman
method.
Thermolysin
or
chymotrypsin
digests
of
T-2
were
sub-
jected
to
electrophoresis
at
pH
4.7
followed
by
chromatog-
raphy.
The
sequences
of
certain
of
the
peptides
resulting
from
these
digestions
were
determined.
The
chymotrypsin
peptide
C-5
(Table
2)
was
found
to
have
an
NH2-terminal
sequence
Proc.
Nat.
Acad.
Sci.
USA
70
(1973)
2114
Immunology:
Cesari
and
Weigert
J558,
xS104,
and
1
5
10
15
20
25
MOPC-104E
Pca-Ala-Val-Val-Thr-Glx-Glx-Ser
-Ala-Leu-Thr-Thr-Ser
-Pro-Gly-Glx-Thr-Val-Thr-Leu-Thr-Cys-Arg-Ser
-Ser
-
S178
Asn-
J558,
xS104,
and
MOPC-104E
S178
J558,
xS104,
and
MOPC-104E
S178
J558,
xS104,
and
MOPC-104E
S178
J558,
xS104,
and
MOPC-104E
S178
30
35
40
45
50
Thr-Gly-Ala-Val-Thr-Thr-Ser
-Asx-Tyr-Ala-Asx-Trp-Val-Glx-Glx-Lys-Pro-Asp-His-Leu-Phe-Thr-Gly-Leu-Ile
55
60
65
70
75
Gly-Gly-Thr-
Asx-Asx-Arg-Ala-Pro-Gly-Val-Pro-Ala-Arg-Phe-Ser
-Gly-Ser
-Leu-Ile
-Gly-Asx-Lys-Ala-Ala-Leu-
Asn
80
85
90
95
100
Thr-Ile
-Thr-Gly-Ala-Glx-Thr-Glx-Asx-Glx-Ala-Ile
-Tyr-Phe-Cys-Ala-Leu-Trp-Tyr-Ser
-Asx-His-Trp-Val-Phe-
Ar
105
110
Gly-Gly-Gly-Thr-Lys-Leu-Thr-Val-Leu-Gly-Glx-Pro-Lys
FIG.
1.
The
tentative
sequence
of
the
variable
region
of
mouse
lambda
chains.
Equivalent
sequences
were
found
for
lambda
chains
xS104
and
J558.
This
was
also
the
case
for
most
of
the
S178
lambda
chain
as
indicated
by
a
line.
Positions
at
which
S178
differed
in
sequence
from
xS104
and
J558
are
indicated
by
the
residue
involved.
The
revised
sequence
of
the
MOPC-104E
urinary
lambda
chain
is
indistinguishable
from
xS104
and
J558
(Appella,
E.,
personal
communication).
At
several
positions
(designated
Asx
or
Glx)
the
se-
quencing
procedures
used
did
not
permit
the
distinction
between
asparagine
and
aspartic
acid
or
glutamine
and
glutamic
acid.
The
se-
quences
of
the
light
chains
are
assumed
to
be
identical
at
these
positions,
however,
since
the
electrophoretic
mobilities
of
the
corre-
sponding
peptides
from
each
protein,
including
MOPC-104E
(1),
that
contain
these
residues
are
identical.
identical
to
the
NH2-terminal
sequence
of
the
first
three
residues
of
the
T-2
peptide
(Table
1)
and
could
thus
be
placed
at
)position
24-34.
The
order
of
the
thermolysin
and
the
chymotrypsin
peptides
was
established
by
their
respective
overlaps
(Tables
2
and
3).
By
the
subtractive-Edman
pro-
cedure,
it
was
only
possible
to
establish
the
sequence
of
the
first
three
residues
of
the
chymotrypsin
peptide
C-7
(residues
38-46)
or
the
thermolysin
Th-9
(residues
38-45).
The
rest
of
the
sequence
was
established
by
analysis
of
certain
peptides
resulting
from
partial
acid
hydrolysis
of
peptide
Th-9
(Table
4).
The
lysine
at
position
41
is
resistant
to
cleavage
by
trypsin.
S178
differs
from
XS104
and
J558
by
asparagine
at
posi-
tion
25
instead
of
serine,
and
asparagine
at
position
52
in-
stead
of
glycine.
It
is
most
likely
that
the
amino-acid
sub-
stitution
in
both
cases
is
to
asparagine
rather
than
to
as-
partic
acid,
since
the
electrophoretic
mobilities
of
chymo-
trypsin
peptides
C-5
and
C-8
(XS104
and
J558)
are
identical
to
those
of
the
corresponding
chymotrypsin
peptides
C-5
and
C-8
from
the
S178
light
chain.
It
cannot
be
excluded
that
the
substitution
at
52
in
S178
is
to
aspartic
acid
since
a
second
substitution
in
peptide
C-8
from
aspartic
acid
to
asparagine
at
either
position
54
or
55
would
neutralize
the
expected
TABLE2.
Chymotrypsin
peptides
used
to
establish
the
sequence
and
order
of
the
tryptic
peptides
from
the
variable
region
of
J558,
xSl04,
and
S178
lambda
chains
Tryptic
peptide
Peptide
Residues
Protein
Amino-acid
sequence
T-1
C-1
1-10
J558,
xS104,
S178
PCA
(Ala,
Val2
Thr,
Glx2,
Ser,
Leu).
C-2
11-20
J558,
xS104,
S178
Thr-Thr-
er-Pro-G
(Glx,
Thr2,
Val,
Leu)
C-3
21-23
J558,
xS104,
S178
fihr
CsyT
rz
T-1
and
T-2
C-4
21-34
J558,xS104
(Thr4,
Cys,
Arg,
Ser3,
Gly,
Ala,
Val,
Asx,
Tyr)
S178
(Thr4,
Cys,
Arg,
Ser2,
Gly,
Ala,
Val,
Asx2,
Tyr)
C-5
24-34
J558,xS104
Ser-Ser-Thr-Gly-Ala-Val-Thr-Thr-Ser-Asx-Tyr
S178
Te-r-Thsx-fh-l--1a-V-Thr-T
'-Ser-X-Tyr
T-2
C-6
35-37
J558,
xS104,
S178
AlZ-AxT-Trp
C-7
38-46
J558,
xS104,
S178
Val-Glx-Glx
(Lys,
Pro,
Asx,
His,
Leu,
Phe)
C-8
47-56
J558,xS104
wiir-U-Leu-Ile-G24-Glz-Thr
(Asx2,
Arg)
S178
F-h-Leu-Ile-G-Asx-hir
(Asx2,
Arg)
T-2,
T-3
and
T-4
C-9
55-64
J558,
xS104,
S178
(Asx,
Arg2,
Ala2,
Pro2,
Gly,
Val,
Phe)
T-4
and
T-5
C-10
65-75
J558,
xS104,
S178
(Ser2,
Gly2,
Leu2,
Ile,
Asx, Lys,
Ala2)
C-12
73-75
J558,
xS104,
S178
(Ala2,
Leu)
C-13
76-88
J558,
xS104,
S178
Thr-ile-Thr-Gl
(Ala2,
Glx3,
Thr,
Asx,
Ile,
Tyr
C-14
73-90
J558,
xS104,
S178
(Thr
I1e2,2Giy,
Ala2,
Glx3,
Asx,
Tyr,
Phe,
Cys
C-15
91-93
J558,
xS104,
S178
(Ala,
Leu,
Trp)
C-16
94-98
J558,xS104
l-Ser-Asx
(His,
Trp)
C-17
94-97
S178
Tyr-5er-Xs-Arg
C-18
99-100
J558,
xS104,
S178
l7h
T
C-19
101-105
J558,
xS104,
S178
(Gly3
,
Thr,
Lys)
T-6
and
T-7
C-20
101-106
J558,
xS104,
S178
(Gly3,
Thr,
Lys,
Leu)
(-
)
Indicates
subtractive-Edman.
Proc.
Nat.
Acad.
Sci.
USA
70
(1973)
Mouse
Lambda-Chain
Sequences
2115
TABLE
3.
Thermolysin
peptides
used
to
establish
the
sequence
and
order
of
the
tryptic
peptides
from
the
variable
region
of
J558,
xS104,
and
S178
lambda
chains
Tryptic
peptide
Peptide
Residues
Protein
Amino-acid
sequence
Th-l
1-2
J558,
xS104,
S178
(Glx,
Ala)
Th-2
3-9
J558,
xS104,
S178
Val-Val-Thr-Glx-Glx-Ser-Ala
T-1
Th-3
10-17
J558,
xS104,
S178
Leu-Thr-Thr-Ser-Pro-l-x-Thr
Th-4
18-19
J558,
xS104,
S178
Val-Thr
Th-5
20-23
J558,
xS104,
S178
Leu-Thr
(Cys,
Arg)
Th-6
24-28
J558,xS104
Ser-Ser-Thr-Gl-Ala
S178
ei-Asn-Thr-Gr1-A1a
Th-7
29-31
J558,
xS104,
S178
Val
(Thr27
T-2
Th-8
32-37
J558,
xS104,
S178
(Ser,
Asx2,
Tyr,
Ala,
Trp)
Th-9
38-45
J558,
xS104,
S178
Val-Glx-Glx
(Lys,
Pro,
Asx,
His,
Leu)
Th-10
46-48
J558,
xS104,
S178
Fh-e
5;7-
Th-1l
49-56
J558,xS104
Leu-Ile-Gly-Gl-Thr
(Asx2,
Arg)
5178
Leu-Ile-Asn-TYhr
(Asx2,
Arg)
*
Th-12
73-74
J558,
xS104,
S178
Ala-Ala
Th-13
75-76
J558,
xS104,
S178
(Leu,
Thr)
Th-14
77-84
J558,
xS104,
S178
Ile-Thr-Gly-Ala-Glx-Thr-Glx-Asx
T-5
Th-15
85-86
J558,
xS104,
S178 Gix-Ala
Th-16
77-86
J558,
xS104,
S178
(7e,
Thr2,
Gly,
Ala2,
Glx3,
Asx)
Th-17
87-88
J558,
xS104,
S178
Ile-Tyr
Th-18
89-90
J558,
xS104,
S178
Phe-Cys
T-5
and
T-6
Th-19
89-91
J558,
xS104,
S178
Phe
(Cys,
Ala)
Th-20
92-94
J558,
xS104,
S178
(Leu,
Trp,
Tyr)
Th-21
95-98
J558,xS104.
Ser-Asx
(His,
Trp)
T-6
Th-22
95-97
S178
Ser-Asx-Arg
Th-23
99-105
J558,
xS104,
S178
Val-Phe-Gly-Gly-Gl
(Thr,
Lys)
.Liz
()
Indicates
subtractive-Edman,
(-)
carboxypeptidase-A.
*
Ala-Ala
dipeptide
has
a
different
electrophoretic
and
chromatographic
mobility
than
free
alanine.
electrophoretic
difference.
However,
since
the
chymotryptic
peptide,
C-9
(55-64),
is
identical
in
all
three
cases,
such
a
second
substitution
is
unlikely.
T-3
(residues
57-63),
T-4
(residues
64-72),
and
T-7
(residues
106-113)
These
peptides
were
obtained
from
electrophoresis
and
chromatography
of
trypsin
digests
of
S-aminoethylated
protein.
The
peptides
were
eluted
from
paper,
and
the
se-
quence
was
determined
by
the
subtractive-Edman
procedure.
T-5
(residues
73-90)
T-5,
from
tryptic
digests
of
S-aminoethylated
lambda
chain,
was
purified
by
gel
filtration
followed
by
electrophoresis
at
pH
4.7.
The
order
of
the
peptides
resulting
from
thermolysin
digestion
was
established
by
chymotrypsin
peptides.
T-6
(residues
91-105)
This
peptide
was
isolated
by
gel
filtration
of
tryptic
digests
of
the
S-aminoethylated
J558
and
xS104
proteins,
and
sequenced
by
the
subtractive-Edman
procedure.
Thermolysin
or
chymotrypsin
peptides
of
T-6
were
separated
by
electro-
phoresis
at
pH
4.7
and
sequenced
by
the
same
procedure.
The
order
of
the
chymotrypsin
peptides
was
established
by
the
peptides
obtained
by
thermolysin
digestion.
T-6a
(residues
90-97)
and
T-6b
(residues
98-105)
T-6
was
absent
in
the
trypsin
digest
of
S-aminoethylated
S178,
being
replaced
by
T-6a
and
T-6b.
T-6a
and
T-6b
were
isolated
by
gel
filtration,
sequenced
by
the
subtractive-
Edman
procedure,
and
ordered
by
their
homology
with
T-6
from
J558
and
xS104.
TABLE
4.
Partial
acid
hydrolysis
and
amino-acid
sequence
of
thermolysin
peptide
Th-9
(residues
38-45)
of
J558,
xSlO4,
and
S178
lambda
chains
Peptide
Residues
Amino-acid
composition
and
sequence
Amino-acid
sequence
t
A-1
43
Free
aspartic
acid
A-2
38-42
(Vall,
Glx2,
Lys,,
Pro,)
A-3
44-45
His-Leu
Val-Glx-Glx-Lys-Pro-Asp-His-Leu
B-1
43
Free
aspartic acid
B-2
39,40
Free
glutamic
acid
B-3
40-41
(Glx1,
Lys1)
B-4
44-45
(His,,
Leu)
()
Indicates
subtractive-Edman.
*
A-1,
A-2,
and
A-3
are
peptides
derived
from
partial
acid
hydrolysis
in
0.03
N
HCO
at
1050
for
12
hr.
B-1,
B-2,
B-3,
and
B4
are
peptides
derived
from
partial
acid
hydrolysis
in
6
N
HCl
at
i05a
for
30
min.
t
Combined
data
from
Tables
3
and
4.
Proc.
Nat.
Acad.
Sci.
USA
70
(1978)
2116
Immunology:
Cesari
and
Weigert
DISCUSSION
With
the
addition
of
lambda
chains
W3159
and
M511
to
the
previous
study
(1),
eight
of
12
mouse
lambda
chains
com-
pared
are
indistinguishable
in
amino-acid
sequence
of
peptides
that,
on
the
basis
of
comparison
with
MOPC-104E
lambda
chain,
correspond
to
the
entire
variable
segment
(positions
1-113).
This
conclusion,
based
previously
on
partial
sequences
and
amino-acid
compositions
of
peptides,
is
strengthened
by
the
more
extensive
sequence
data
presented
here
on
the
variable
regions
of
three
of
the
repeat
lambda
chains.
Two
of
these,
xS104
and
J558,
(shown
here)
are
identical
to
the
revised
sequence
of
MOPC-104E
lambda
chain
(Appella,
E.,
personal
communication).
The
four
variant
mouse
lambda
chains
are
indistinguishable
from
the
eight
repeat
lambda
chains
in
all
but
the
hypervariable
regions
that
seem
to
determine
the
combining
site.
This
has
been
confirmed
here
for
one
variant
chain
by
the
complete
sequence
of
the
variable
region
of
S178.
We
have
interpreted
these
observations
to
mean
that
the
identical
sequences
are
coded
for
by
a
single
germ-line
vari-
able-region
gene
and
the
variant
lambda
chains,
such
as
S178,
by
somatic
variants
of
this
gene.
The
finding
of
lambda
chains
such
as
RPC-20
and
S176
that
have
one
amino-acid
replacement
(accountable
for
by
single
base
chainges)
im-
plies
that
antigenic
selection
can
act
on
the
products
of
single-
step
mutants
of
a
germ-line
gene.
The
S178
lambda
chain
that
differs
probably
only
by
three
amino-acid
replacements
(accountable
for
by
four
base
changes)
from
the
repeat
sequence
supports
the
idea
that
antigenic
selection
operates
sequentially
on
amino-acid
replacements
in
specificity-de-
termining
regions
(1).
If
myeloma
tumors
arose
from
antibody-producing
cells
previously
selected
for
by
antigen,
the
expression
of
identical
sequences
before
diversification
can
be
explained
if
the
product
of
a
germ-line
variable-region
gene
in
combination
with
an
appropriate
heavy
chain
had
been
selected.
This
possibility
was
suggested
from
the
finding
that
two
of
the
myeloma
proteins
associated
with
lambda
chains,
J558
(Hirst,
J.,
Jones,
G.,
Weigert,
M.,
and
Cohn,
M.,
unpub-
lished)
and
MOPC-104E
(16)
have
specificity
for
the
a-1,3
glucosyl
linkage
in
dextran.
As
shown
in
Fig.
1,
the
X
chain,
isolated
from
the
IgAX,
J558,
is
identical
to
the
urinary
X
chain
produced
by
tumor
MOPC-104E,
which
is
indis-
tinguishable
from
the
X
chain
associated
with
the
IgM
myeloma
protein
also
produced
by
this
tumor
(Appella,
E.,
personal
communication).
That
the
lambda
chain
indeed
contributes
to
the
a-1,3
glucosyl
linkage
specificity
of
these
proteins
is
likely
since
the
anti
a-1,3
antibody
elicited
by
im-
munization
of
BALB/c
mice
with
dextran
is
exclusively
as-
sociated
with
lambda
chains
(17),
even
though
this
light-
chain
class
represents
only
3-5%
of
the
normal
light-chain
population
in
mice
(18).
As
other
myeloma
proteins
associated
with
this
lambda
chain,
such
as
S104
and
W3159
described
here
or
J698
and
H2061
(1),
do
not
have
a-1,3
glucosyl
linkage
specificity,
this
specificity
is
not
solely
due
to
the
germ-line
lambda
chain,
and
must
result
from
a
specific
heavy-
and
light-chain
in-
teraction.
Thus,
the
association
of
the
germ-line
lambda
chain
with
different
heavy
chains
is
likely
to
result
in
antibodies
with
different
specificities.
Selection
for
these
specificities
as
well
as
for
anti-a-1,3
glucosyl
linkage
specificity
could
ex-
plain
the
high
frequency
of
myelomas
with
germ-line
lambda
chains.
This
work
was
supported
by
research
grants
from
the
Na-
tional
Institutes
of
Health
(A-105875)
and
a
Training
Grant
(CA-05213)
to
Dr.
Melvin
Cohn,
and
no.
18263
to
M.W.
I.M.C.
was
supported
by
Fogarty
International
Fellowship
no.
3
F05
TWO160S-01S1,
and
M.W
by
an
American
Cancer
Society
Fac-
ulty
Research
Award
No.
PRA-59.
We
thank
Dr.
Melvin
Cohn
for
his
helpful
discussions
and
advice
and
Mrs.
Cheryl
Look
and
Miss
Shirlee
Yonkovich
for
their
excellent
technical
assistance.
1.
Weigert,
M.
G.,
Cesari,
I.
M.,
Yonkovich,
S.
J.
&
Cohn,
M.
(1970)
Nature
228,
1045-1047.
2.
Wu,
T.
T.
&
Kabat,
E.
(1970)
J.
Exp.
Med.
132,
211-250.
3.
Matsubara,
H.
(1970)
in
Methods
in
Enzymology,
eds.
Perlmann,
G.
E.
&
Lorand,
L.
(Academic
Press,
New
York),
Vol.
XIX,
pp.
646-647.
4.
Potter,
M.
(1970)
Methods
Cancer
Res.
2,
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C.
A.
&
Gray,
H.
M.
(1968)
Biochemistry
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C.
H.
W.
(1956)
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&
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&
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W.
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in
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H.
W.
(Academic
Press,
New
York),
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XI,
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142-
149.
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J.
E.
&
Schirmer,
E.
W.
(1963)
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Biol.
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Matsubara,
H.
&
Lasaki,
R.
M.
(1969)
Biochem.
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Res.
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35,
175-181.
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Appella,
E.
(1971)
Proc.
Nat.
Acad.
Sci.
USA
68,
590-594.
14.
Mazur,
R.
H.,
Ellis,
B.
W.
&
Cammarata,
P.
S.
(1962)
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Biol.
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Yamada,
S.
&
Itano,
H.
A.
(1966)
Biochim.
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130,
538-540.
16.
Leon,
M.
A.,
Young,
N.
M.
&
McIntire,
K.
R.
(1970)
Biochemistry
9,
1023-1030.
17.
Blomberg,
B.,
Geckeler,
W.
R.
&
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M.
(1972)
Science
177,
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McIntire,
K.
R.
&
Rouse,
A.
M.
(1970)
Fed.
Proc.,
29,
704.
Proc.
Nat.
Acad.
Sci.
USA
70
(1973)
... Furthermore, cDNA nucleotide sequence analysis of the entire variable and constant regions for the heavy and light chains of the L8D and 6-19J mAbs revealed a complete identity (data not shown) corresponding to the published nucleotide or amino acid sequences of the 6-19 ␥3 heavy chain and the J558 1 light chain. 11,27,42,43 This indicates that these 2 antibodies are identical in the amino acid sequences of their heavy and light chains Intraperitoneal implantation of L8D cells into (MRL ϫ BALB/ c)F1 mice induced a rapid increase in serum levels of IgG3 within 4 days (1.3 Ϯ 0.8 mg/mL), reaching 3 to 5 mg/mL at approximately 7 days. By day 10, all 8 mice developed severe acute glomerulonephritis similar to that induced by the original 6-19 RF hybridoma cells (Table 1, Figure 2A). ...
... Glycosylation of the IgG3 mAb studied should only occur in the constant region of their heavy chains because of the lack of potential glycosylation sites in their V H and V L regions. 11,42 Notably, in murine IgG3, there is an additional potential glycosylation site in its CH3 domain. 27 The presence of the CH3 oligosaccharides has been suggested by a recent demonstration that the self-associating ability was significantly reduced in a murine IgG3 RF mutant mAb lacking the glycosylation site in the CH3 domain. ...
Article
Cryoglobulin activity associated with murine immunoglobulin G3 (IgG3) has been shown to play a significant role in the development of murine lupuslike glomerulonephritis. A fraction, but not all, IgG3 monoclonal antibodies are capable of inducing a severe acute lupuslike glomerulonephritis as a result of direct localization of IgG3 cryoglobulins, suggesting the importance of qualitative features of cryoglobulins in their nephritogenic activities. Here a remarkable difference is shown in the renal pathogenicity of 2 murine IgG3 monoclonal cryoglobulins, identical in the amino acid sequences of their heavy and light chains but different in galactosylation patterns of oligosaccharide side chains because of their synthesis in different myeloma cells. The antibody lacking the capacity to induce severe glomerulonephritis displayed an increased proportion of galactosylated heavy chains. Changes in conformation, as revealed by gel filtration analysis, reduced cryoglobulin activity, and accelerated clearance could account for the lack of the renal pathogenicity of the more galactosylated variant. This observation provides a direct demonstration for the role of IgG galactosylation in the pathogenic potential of cryoglobulins.
... When 1.9 diazonium groups were put on Hdex 24 in the presence of hapten, the idiotype was lost even though virtually none of the label was found on the L chains ( Table I). Results of amino acid sequence analysis show that )~-type light chains are highly conserved (36) and also argue against their contributing residues that are directly involved in binding to anti-IdI reagents. ...
Article
Two dextran-binding myeloma proteins, J558 and Hdex 24, which possess the same individual idiotype (IdI) were diazotized to low levels (1-3.3 groups per subunit) with 1-[14C]-p-aminobenzoate. Both proteins lost the IdI idiotype under these conditions with most of the label incorporated on the heavy chains of each protein. When the diazotization ws carried out in the presence of the hapten 1-O-methyl-alpha-D-glucopyranoside the loss of idiotypic reactivity could be prevented for J558 but not for Hdex 24. Under these conditions most of the label was incorporated on the light chains of J558, but on the heavy chains of Hdex 24. For J558, these results show that a major determinant of the individual idiotype is within the hypervariable positions of the heavy chain. For Hdex 24 the determinant being modified is on the heavy chain but not involved in hapten binding. These results are consistent with previous work showing that J558 and Hdex 24 differ in amino acid sequence in the D and the J segments of the heavy chain and offer an alternative and complementary strategy for assigning idiotypic determinants.
Chapter
When a mammalian organism is immunized with an antigenic determinant, a highly complex humoral immune response is induced. This consists of a large number of antibodies complementary to the antigenic determinant. Such antibodies may vary in class and subclass. Furthermore, within a single subclass, there are differences in the energy of interaction with which each antibody of the population binds the antigenic determinant. These interactions may be viewed at a single point in time, although the antibodies expressed at any one time in the immune response may be only a fraction of the number that the animal is capable of producing. Certainly, in viewing these antibodies over a period of time, there are shifts in the populations produced after the initial immunization.
Chapter
All immunoglobulins (Ig’s) consist of one or more basic units composed of identical pairs of heavy (H) and light (L) polypeptide chains (Figure 1). Each chain folds into several compact globular domains, connected by relatively narrower but more exposed areas. Each domain is approximately 110 amino acids in length and characterized by an intrachain disulfide bond connecting two cysteine residues approximately 60 amino acid residues apart (Figure 1). All Ig polypeptide chains can be divided into an amino-terminal portion, the variable (V) region, and a carboxylterminal portion, the constant (C) region. The V regions of both H and L chains are equivalent in size to a domain. The C regions of L chains are of similar size, whereas those of H chains are two to four times longer. Based on the degree of amino acid sequence homology, the V regions have been divided into three main groups, Vk , Vλ, and VH. The C regions have been divided into k and λ types (L chains) as well as γ, α, µ, ∈, and δ classes (H chains) according to their antigenic and serological properties. Some of the L-chain types and H-chain classes are further divided into subtypes and subclasses, respectively. Details of Ig structure are given in earlier Chapters. A general review on this subject was given by Gally (1973). This chapter is devoted to the genetic aspects of the synthesis of Ig polypeptide chains.
Chapter
Plasma cells are terminally differentiated cells of the B-cell lineage. They can develop from any of several mature B-cell subsets, including germinal center (GC), memory, marginal zone (MZ), and B1 cells. Plasma-cell tumors of mice, termed plasmacytomas (PCT), are increasingly recognized as sharing many features with the major type of plasma-cell tumors in humans, termed multiple myeloma (MM), an almost uniformly lethal disease. Consequently, an increasing number of mouse models of plasma-cell neoplasia are being developed to dissect the mechanisms underlying the initiation, progression, and maintenance of the transformed phenotype with the long-term goal of improving diagnosis and therapy. Studies of plasma-cell neoplasms in mice and humans have been remarkably informative for identifying genes and signaling pathways critical to normal B-cell differentiation, function, and survival, as well as neoplastic transformation (Fig. 24.1). Examinations of mouse plasma-cell neoplasms initiated in the National Cancer Institute over 50 years ago have been joined synergistically with studies in the National Institute of Allergy and Infectious Diseases, together with their collaborators, of normal and transformed plasma cells from retrovirus-infected and autoimmune mice. Together, they have painted an increasingly rich picture of the later stages of normal B-cell differentiation and the changes that redirect these cells to neoplasia. Other recent publications provide reviews of other plasma-cell tumor models, as well as other aspects of the studies described here [1,2].
Chapter
An immune serum usually consists of a very heterogeneous population of immunoglobulins (Ig’s) the appearance of which in the serum has been induced by antigen. The antigen-ligating function of the Ig molecule (see Figure 1) is confined to the combining regions, which are two symmetrical areas at the solvent-exposed ends of the Fab arms of the Y-shaped Ig molecules. The combining region is situated in the variable (V-region) domain, a compact region consisting of the N-terminal half of the light (L) chain and the N-terminal quarter of the heavy (H) chain that is linked by sulfhydryl bonds. Between the areas of this domain occupied by the L- and H-chain V regions is a cleft exposed to the solvent. Antigens have been shown to bind in, or close to, this cleft (Amzel et al., 1974). An induced antibody population is said to be specific because it usually binds most strongly to the immunizing antigen and with lesser binding energies to certain compounds that resemble the immunogen in structure. Heteroclitic antibodies may be induced that bind more strongly to some determinant other than the immunogen (Mäkelä, 1965). In general, antibody populations show a high degree of specificity, in that they are able to discriminate among chemical compounds differing by as little as a single functional group, between stereoisomers, or between two proteins differing by as little as a single amino acid residue (Reichlin, 1974).
Chapter
It is now well established that the vertebrate immune system is capable of synthesizing an enormous number of distinct antibody variable region structures. In recent years the genetic basis for this ability in the mouse has been elucidated. Diversity of the V region arises from four sources: (a) diversity encoded directly in the germ-line genome in the form of V region heterogeneous multigene families; (b) diversity created by the somatic rearrangement of different combinations of gene segments to form functional V region genes, and the association of different V, and V, polypeptides to form the heterodimeric V domain (combinatorial diversity); (c) diversity created at the junctions of V gene segments due to apparent addition and deletion of nucleotides during segment joining (junctional diversity); and, finally, (d) diversity created by somatic replacement of nucleotides in expressed V, and V, genes (somatic mutation). What now remains to be determined is the manner in which this potential for generating diversity is utilized toward the formation of antibody specificity and immunity. This chapter reviews the genetic mechanisms that create diversity and discusses recent experiments that provide insights into the question of how this diversity is utilized during an immune response.
Article
A group of eight IgM hybridoma proteins induced with beta(1,6)-D-galactan-containing antigens has been characterized in terms of primary amino acid sequence and idiotype expression. The H chain amino acid sequences reveal very strong homology in the VH segment although several substitutions are seen that suggest the occurrence of somatic mutation in these IgM molecules. Significant sequence variation was observed in CDR-3, the region generated by the D segment, and the two recombination events, VH-D and D-JH. The number of amino acids in this region contributed by the D segment was found to vary from two to six, yet the overall length of CDR-3 was precisely maintained by the addition of amino acids on either side of D during the recombination processes. These additional amino acids are suggested to result from nucleotide addition by repair enzymes. Idiotypic analysis of these proteins, in conjunction with an assessment of the H chain sequences, has permitted an identification of the molecular basis of both cross-reacting and unique idiotypic determinants expressed by these molecules.
Article
To determine the chromosomal localization of murine lambda light (L) chain structural genes, DNA from a panel of 11 mouse x hamster somatic cell hybrids was scored for the presence of sequences homologous to cloned lambda DNA probe molecules. Six of the hybrids had detectable lambda I and lambda II gene sequences. In all six, the full complement of murine sequences was present, and in its germline configuration. The remaining hybrids lacked any detectable murine lambda L chain gene sequences. The only mouse chromosome present in all of the positive hybrids and absent from the negative ones was number 16, allowing the assignment of lambda L chain structural genes to this chromosome. Together with the previous assignments of the kappa L chain genes to chromosome 6 and heavy chain genes to chromosome 12, this finding completes the mapping of Ig structural genes in the mouse at the chromosomal level.
Article
In an attempt to account for antibody specificity and complementarity in terms of structure, human κ-, human λ-, and mouse κ-Bence Jones proteins and light chains are considered as a single population and the variable and constant regions are compared using the sequence data available. Statistical criteria are used in evaluating each position in the sequence as to whether it is essentially invariant or group-specific, subgroup-specific, species-specific, etc. Examination of the invariant residues of the variable and constant regions confirms the existence of a large number of invariant glycines, no invariant valine, lysine, and histidine, and only one invariant leucine and alanine in the variable region, as compared with the absence of invariant glycines and presence of three each of invariant alanine, leucine, and valine and two each of invariant lysine and histidine in the constant region. The unique role of glycine in the variable region is emphasized. Hydrophobicity of the invariant residues of the two regions is also evaluated. A parameter termed variability is defined and plotted against the position for the 107 residues of the variable region. Three stretches of unusually high variability are noted at residues 24–34, 50–56, and 89–97; variations in length have been found in the first and third of these. It is hypothesized that positions 24–34 and 89–97 contain the complementarity-determining residues of the light chain—those which make contact with the antigenic determinant. The heavy chain also has been reported to have a similar region of very high variability which would also participate in forming the antibody-combining site. It is postulated that the information for site complementarity is contained in some extrachromosomal DNA such as an episome and is incorporated by insertion into the DNA of the structural genes for the variable region of short linear sequences of nucleotides. The advantages and disadvantages of this hypothesis are discussed.
Article
The simple pattern of variability in mouse lambda chains suggests that diversity is generated by somatic spontaneous mutation and by sequential selection by antigen of single step mutants.
Article
The immune response to dextran having the α-1,3 linkage may be under the control of antibody structural genes. Mice that respond well to this antigen produce a