Vol.70,No.2,
1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
PARTIAL AMINO ACID SEQUENCE OF MOUSE B,-MICROGLOBULIN Appella,
E.,
Tanigaki,
N.,
Natori,
T. and Pressman,
Laboratory of Cell Biology, National Cancer Institute, Maryland 20014 and Department of Immunology Research, Park Memorial Institute, Buffalo, New York 14263
Received
March
D. Bethesda, Roswell
17,1976
SUMMARY: A highly purified preparation of mouse B,-microglobulin has been obtained from sodium thiocyanate extracts of liver cell membranes of A/J strain mice and the amino acid sequence has been partially determined. The first 40 residues have been assigned except for position 34. In the sequence, the mouse protein differs only at 9 positions from human f+-microglobulin at 8 positions from the dog homologue and at 11 positions from the rabbit homologue. The sequence has also a homology to the constant regions of mouse yG most closely to the C 3 region. These data support the conclusion that t i i'mouse protein is indee L! the mouse homologue of human Bz-microglobulin. Human e2-microglobulin and is
present
types
in various
as well
(2).
homology Recent
work
body fluids
The amino
to the
constant
acid
has demonstrated
in other
that
amino
species
acid
sequences
A mouse protein globulin
in molecular
purified
from
this protein
cell
communication, and show it
appears
to be similar
kidney
determined
and the
has been pointed
out
is associated
The major to contain
cell
(3).
with
histocompatibility a human B,-microglobulin
have been isolated
and the partial
(5,6).
to be the mouse homologue acid
of spleen the
with
human b,-microglobulin
amino
we report
of patients
has been totally
(4).
11,800
of various
sequence
have been reported
membranes
urine
weight
surface
from
in dog and rabbit
weight,
cell
isolated
have been found
that
of a molecular
and on the
antigens
The homologues
homologue.
protein
of IgG heavy chain
regions
human HLA histocompatibility antigens
a simple
It was first
(1).
malfunction
is
composition and liver
partial
amino
of human 8,-micro-
and antigenicity of A/J
acid
strain
mice
sequence
to human 8,-microglobulin
has been (7,8).
of this and the
mouse homologues
in dog and rabbit. MATERIALS AND METHODS: cell
membranes
The mouse s2-microglobulin
of A/J strain
mice
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
by sodium 425
was extracted
thiocyanate
from
In
liver
(NaSCN) and purified
Vol.70,No.2,1976
by gel
BIOCHEMICAL
filtration,
described
ion-exchange
previously
chloride
method
sulphate.
Automatic by using
Sequencer
with
acids
polyamide graphy
chromatography
NH2-terminal
according
performed
amino
(5).
to Gray
sequence
program
(9)
identified
layers
(10).
on columns
and by hydrolysis
buffer
in nanomoles, degradation with
of the
data
obtained
permitted residue
the
in a nuclear
purified from
sequence
qualitative
assessment.
differences When aligned appears
there
of the amino
34.
(11)
with
rabbit
only
The extent
obtained
differences
and the homology
the corresponding regions
between regions
portions
the mouse protein
is
have No
at position
from
the
acids
(Fig. the from
1).
depended with
upon
the
Of the
human protein, the rabbit
39 eight
homologue.
of 8,-microglobulin any of the other
NH2-terminal
of mouse IgG is
IgG (Table
After
is compared
of human and rabbit
of human and rabbit
in conjunction
40 residues.
of IgG than
the CH3 region
of homology
by
chromatography
of PTH-amino
the mouse homologue
resemble
data
of cysteine
and dog homologue
nine
recovered,
human B,-microglobulin.
identification sequence
by
obtained
layer
The assignment
homology
The partial
are
and thin
maximum homology,
that
on
to be Ile
acids
These
for
and the homology the conclusion
was found
and 11 differences
regions.
between
residue
the dog homologue,
mouse BP-microglobulin found
gas chromatograph
from
to more closely
homology
PTH-
was made by gas chromato-
of 39 of the first
on the
and the
compared,
Ca.).
phenylthiohydantions
gas-chromatography
low and the
human 8,-microglobulin residues
Alto,
890 B
chromatography
mouse B,-microglobulin.
at position
were
mg) was
Chicago
amounts
of the
determination
was recovered
30, yields
terminal
1 shows the
HI hydrolysis
25 is at the moment based step
(1.3
HI (12).
Table
after
Palo
identification
RESULTS AND DISCUSSION: The amino method.
dodecyl
in the Beckman Model
by thin-layer
Quantitative
with
protein
as
by the dansyl-
of 2% sodium
native
Instruments,
qualitatively
electrophoresis
was determined
presence
of the
(Beckman
of 2% OV-275
acid
in the
analysis
111314
and column
amino
dimethylallylamine
were
the danysl
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2).
the mouse homologue
portion similar
of to that
8,-microglobulins The data
support
of human &-micro-
Vol. 70, No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Automated Degradation
1
Amino
of Mouse
Terminal 62-Microglobulin Cycle
Residue
57.1 53.2
26
Tw
4.0
27
Val
8.2
LYS Thr
43.2
28
Thr
5.8
45.9
29
Glu
4.4
Pro
44.5
30
Phe
3.0
Glu
40.8
7
Ile
36.6
8
Glu
33.0
9
Val
31.9
10
Tyr Ser
21.0
Arg His
C
14 15
Pro
27.2
Pro
26.0
16
Glu
17.3
17
Cycle
1 2 3 4 5 6
11
12 13
Residue
Yielda
Ile Glu
8.3
b
Asp
9.6
18
GUY
12.9
19
LYS Pro
6.7
20 21 22 23 24 25
Yielda
9.5 6.4
Asp Ile
8.0
Leu
6.5
Asp
5.0
CYS
aThe yields (nmoles) for the major amino acid phenylthiohydantoins identified at each cycle were determined by HI hydrolysis and were not corrected for either overlap or background. bThe phenylthiohydantoin Pauly reaction.
derivative
of histidine
'The phenylthiohydantoin phenanthrenequinone
derivative reaction.
of arginine
427
was identified was identified
by the by the
VoL70,No.
2, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5
6
7
8
9
10
15
16
17
18
19
20
Human WI
Vai
/Gin 1
His
Pro
Rabbit
Val
M
Arg
Ala
11
12
13
14
bbuse
SW
Arg
His
Pro
Hlmwl
SW
Arg
His
PI-0
wi
Ser
Arg
His
PI-0
Rabbit
Ser
Am
His
Pro
21
22
23
24
Pro
1 Ala
Ala
9
25
Glu
AM
GUY
LYS
PI-0
Glu
Asn
GUY
LYS
Ser
Glu
Asn
GUY
Lys
Pro
IGlu
Am
Gly
Lysl
Asp
27
28
29
30
26
kJUS.?
Human Dog Rabbit
MWSe
Human Dog Rabbit
Figure 1 - Comparison of the amino acid sequences of mouse, human, dog and rabbit B2-microglobulin. The data for the human protein are from Smithies and Poulik (21) and from Peterson et al. (3); for the dog protei! from Smithies and Poulik -7 (13); for the rabbit protein from Cunningham and Beggard (6).
Table Identical Mouse
Residues
2 in
the Sequences
BP-Microglobulin
f32m
and Mouse YG2a1
cH1
cH2
B2m
-
CHl
11
cH2
5
B
cH3
13
13
10
CL
7
8
10
1 Residues used for comparison mouse yG2a 173 (22).
of
have
428
been taken
BH3
DL
15 from
the
sequence
of
Vol.70,No.2,1976
globulin
BIOCHEMICAL
and indicate
mouse are closely Since domains
the
protein
work
was found
(15)
component
(16)
is
coded coded
for for
microglobulin
production
by the T/t
rabbit
and
histocompatibility have,been
B,-microglobulin
the major
complex,
appear
TL antigens
a possible
the
capacity
culture
is
that
Bn-microanti-human
of a T-cell
factor
Thus,
Bz-microglobulin
(18).
to have an important
equivalent Effect
to contain
a report
role
and Ss and with
embryonic Allogeneic
has been reported there
(13,14) complex
Furthermore,
In humans,
blocks
with
or a B,-micro-
histocompatibility
complex.
in in vitro
substances
that
by the major
(17).
antiserum
B2-
to stimulate or the
in the mechanism
of cell
interaction.
We have found serum raised
that
against
J2,000-dalton
our
preparation
of H-2 antigens,
the
subunits
small
identical
to mouse B,-microglobulin. to mouse 8,-microglobulin subunit
on the evolutional found
in various
reported
in function
for they
react
with
and biological
immunologically
these
substances
may perhaps
well
are not
still
be very
Isolation
of a
and T-cell
structure
significance important
binds
anti-
mouse 82-microglobulin
Fg antigen
of chemical
rabbit
that
and structure.
Ss protein,
by the analysis
relationship
i.e.
However,
from TL antigen,
when accompanied
do not
of mouse B,-microglobulin
component
Therefore,
dalton
and Fg antigen
Ss protein
(19,20).
subunits
dog,
to imnunoglobulin
of the major
associated
immune mediator,
determinants
analogous
present
histocompatibility
a soluble
globulin
factors
human,
significance
in mouse has revealed
both
of the major
12,000
from
to have a homology
and immunobiological
substance
Fg antigen
similar
proteins
related.
genetic
globulin-like
to the
four
to be elucidated.
Recent
to cell
the
and to be a fundamental
problem
antibody
all
8,-microglobulin
antigens,
Factor,
that
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
may shed light of the
small
substances.
Acknowledgements. This investigation was supported in part by United States Public Health Service Research Grants No. AI-08899 and CA-17276 and by The excellent assistance of Mr. A.J. Trott, the John A. Hartford Foundation. Mr. R. Maturski, Mrs. J. Shaver and Miss P. Overturf is deeply appreciated.
429
Vol.70,No.2,
1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
References 1. :: 4. 2 7. 8. 1:: i:: 2 15. 16. 17. 18.
Nilsson, K., Evrin, P.E., Berggird, I. and Ponten, J., Nature New Biol. -244: 44 (1973). Berggird, I. and Bearn, A.G., J. Biol. Chem. 243: 4095 (1968). Peterson, P.A., Cunningham, B.A., Berggsrd, Ixnd Edelman, G.M., Proc. Nat. Acad. Sci. U.S.A. 69: 1697 (1972). Tanigaki, N. and Pressman, D., Transpl. Rev. 21: 15 (1974). Smithies, 0. and Poulik, M.D., Proc. Nat. Acaz Sci. U.S.A. 69: 2914 (1972). Cunningham, B.A. and Berggird, I., Science 183: 1079 (1975).Natori, T., Katagiri, M., Tanigaki, N. and Pressman, D., Transplantation 18: 550 (1974). Ktori, T., Tanigaki, N., Appella, E. and Pressman, D., Biochem. Biophys. Res. Comnun. 65: 611 (1975). Gray, W.R., Methods Enzymol. 25: 121 (1972). Summers, M.R., Smythers, G.W.and Oroszlan, S., Anal. Biochem. -53: 624 (1973). Zimmerman, C.L. and Pisano, J.J., unpublished. Smithies, O., Gibson, D., Fanning, E.M., Goodfliesh, R.M., Gilman, J.G. and Ballantyne, D.L., Biochemistry 10: 4912 (1971). Vitetta, E.S., Uhr, J.W. and Boyse,T.A., J. Imnunol. 114: 252 (1975). Anundi, H., Rask, L., Ostberg, L. and Peterson, P.A.,Biochemistry -14: 5046 (1975). Capra, J.D., Vitetta, E.S. and Klein, J., J. Exp. Med. 142: 664 (1975). Vitetta, E.S., Artzt, K., Bennett, D., Boyse, E.A. and Jacob, F., Proc. Nat. Acad. Sci. U.S.A. 72: 3215 (1975). Armerding, D., Kubo, R.'iT, Grey, H.M. and Katz, D.H., Proc. Nat. Acad. ;;;,,;.",A. 11: 4577 (1975). Immunol. II 2: 135 (1974). , ., Hunig, Th. and Wecker, E., Progr.
430
1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
PARTIAL AMINO ACID SEQUENCE OF MOUSE B,-MICROGLOBULIN Appella,
E.,
Tanigaki,
N.,
Natori,
T. and Pressman,
Laboratory of Cell Biology, National Cancer Institute, Maryland 20014 and Department of Immunology Research, Park Memorial Institute, Buffalo, New York 14263
Received
March
D. Bethesda, Roswell
17,1976
SUMMARY: A highly purified preparation of mouse B,-microglobulin has been obtained from sodium thiocyanate extracts of liver cell membranes of A/J strain mice and the amino acid sequence has been partially determined. The first 40 residues have been assigned except for position 34. In the sequence, the mouse protein differs only at 9 positions from human f+-microglobulin at 8 positions from the dog homologue and at 11 positions from the rabbit homologue. The sequence has also a homology to the constant regions of mouse yG most closely to the C 3 region. These data support the conclusion that t i i'mouse protein is indee L! the mouse homologue of human Bz-microglobulin. Human e2-microglobulin and is
present
types
in various
as well
(2).
homology Recent
work
body fluids
The amino
to the
constant
acid
has demonstrated
in other
that
amino
species
acid
sequences
A mouse protein globulin
in molecular
purified
from
this protein
cell
communication, and show it
appears
to be similar
kidney
determined
and the
has been pointed
out
is associated
The major to contain
cell
(3).
with
histocompatibility a human B,-microglobulin
have been isolated
and the partial
(5,6).
to be the mouse homologue acid
of spleen the
with
human b,-microglobulin
amino
we report
of patients
has been totally
(4).
11,800
of various
sequence
have been reported
membranes
urine
weight
surface
from
in dog and rabbit
weight,
cell
isolated
have been found
that
of a molecular
and on the
antigens
The homologues
homologue.
protein
of IgG heavy chain
regions
human HLA histocompatibility antigens
a simple
It was first
(1).
malfunction
is
composition and liver
partial
amino
of human 8,-micro-
and antigenicity of A/J
acid
strain
mice
sequence
to human 8,-microglobulin
has been (7,8).
of this and the
mouse homologues
in dog and rabbit. MATERIALS AND METHODS: cell
membranes
The mouse s2-microglobulin
of A/J strain
mice
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
by sodium 425
was extracted
thiocyanate
from
In
liver
(NaSCN) and purified
Vol.70,No.2,1976
by gel
BIOCHEMICAL
filtration,
described
ion-exchange
previously
chloride
method
sulphate.
Automatic by using
Sequencer
with
acids
polyamide graphy
chromatography
NH2-terminal
according
performed
amino
(5).
to Gray
sequence
program
(9)
identified
layers
(10).
on columns
and by hydrolysis
buffer
in nanomoles, degradation with
of the
data
obtained
permitted residue
the
in a nuclear
purified from
sequence
qualitative
assessment.
differences When aligned appears
there
of the amino
34.
(11)
with
rabbit
only
The extent
obtained
differences
and the homology
the corresponding regions
between regions
portions
the mouse protein
is
have No
at position
from
the
acids
(Fig. the from
1).
depended with
upon
the
Of the
human protein, the rabbit
39 eight
homologue.
of 8,-microglobulin any of the other
NH2-terminal
of mouse IgG is
IgG (Table
After
is compared
of human and rabbit
of human and rabbit
in conjunction
40 residues.
of IgG than
the CH3 region
of homology
by
chromatography
of PTH-amino
the mouse homologue
resemble
data
of cysteine
and dog homologue
nine
recovered,
human B,-microglobulin.
identification sequence
by
obtained
layer
The assignment
homology
The partial
are
and thin
maximum homology,
that
on
to be Ile
acids
These
for
and the homology the conclusion
was found
and 11 differences
regions.
between
residue
the dog homologue,
mouse BP-microglobulin found
gas chromatograph
from
to more closely
homology
PTH-
was made by gas chromato-
of 39 of the first
on the
and the
compared,
Ca.).
phenylthiohydantions
gas-chromatography
low and the
human 8,-microglobulin residues
Alto,
890 B
chromatography
mouse B,-microglobulin.
at position
were
mg) was
Chicago
amounts
of the
determination
was recovered
30, yields
terminal
1 shows the
HI hydrolysis
25 is at the moment based step
(1.3
HI (12).
Table
after
Palo
identification
RESULTS AND DISCUSSION: The amino method.
dodecyl
in the Beckman Model
by thin-layer
Quantitative
with
protein
as
by the dansyl-
of 2% sodium
native
Instruments,
qualitatively
electrophoresis
was determined
presence
of the
(Beckman
of 2% OV-275
acid
in the
analysis
111314
and column
amino
dimethylallylamine
were
the danysl
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2).
the mouse homologue
portion similar
of to that
8,-microglobulins The data
support
of human &-micro-
Vol. 70, No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Automated Degradation
1
Amino
of Mouse
Terminal 62-Microglobulin Cycle
Residue
57.1 53.2
26
Tw
4.0
27
Val
8.2
LYS Thr
43.2
28
Thr
5.8
45.9
29
Glu
4.4
Pro
44.5
30
Phe
3.0
Glu
40.8
7
Ile
36.6
8
Glu
33.0
9
Val
31.9
10
Tyr Ser
21.0
Arg His
C
14 15
Pro
27.2
Pro
26.0
16
Glu
17.3
17
Cycle
1 2 3 4 5 6
11
12 13
Residue
Yielda
Ile Glu
8.3
b
Asp
9.6
18
GUY
12.9
19
LYS Pro
6.7
20 21 22 23 24 25
Yielda
9.5 6.4
Asp Ile
8.0
Leu
6.5
Asp
5.0
CYS
aThe yields (nmoles) for the major amino acid phenylthiohydantoins identified at each cycle were determined by HI hydrolysis and were not corrected for either overlap or background. bThe phenylthiohydantoin Pauly reaction.
derivative
of histidine
'The phenylthiohydantoin phenanthrenequinone
derivative reaction.
of arginine
427
was identified was identified
by the by the
VoL70,No.
2, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5
6
7
8
9
10
15
16
17
18
19
20
Human WI
Vai
/Gin 1
His
Pro
Rabbit
Val
M
Arg
Ala
11
12
13
14
bbuse
SW
Arg
His
Pro
Hlmwl
SW
Arg
His
PI-0
wi
Ser
Arg
His
PI-0
Rabbit
Ser
Am
His
Pro
21
22
23
24
Pro
1 Ala
Ala
9
25
Glu
AM
GUY
LYS
PI-0
Glu
Asn
GUY
LYS
Ser
Glu
Asn
GUY
Lys
Pro
IGlu
Am
Gly
Lysl
Asp
27
28
29
30
26
kJUS.?
Human Dog Rabbit
MWSe
Human Dog Rabbit
Figure 1 - Comparison of the amino acid sequences of mouse, human, dog and rabbit B2-microglobulin. The data for the human protein are from Smithies and Poulik (21) and from Peterson et al. (3); for the dog protei! from Smithies and Poulik -7 (13); for the rabbit protein from Cunningham and Beggard (6).
Table Identical Mouse
Residues
2 in
the Sequences
BP-Microglobulin
f32m
and Mouse YG2a1
cH1
cH2
B2m
-
CHl
11
cH2
5
B
cH3
13
13
10
CL
7
8
10
1 Residues used for comparison mouse yG2a 173 (22).
of
have
428
been taken
BH3
DL
15 from
the
sequence
of
Vol.70,No.2,1976
globulin
BIOCHEMICAL
and indicate
mouse are closely Since domains
the
protein
work
was found
(15)
component
(16)
is
coded coded
for for
microglobulin
production
by the T/t
rabbit
and
histocompatibility have,been
B,-microglobulin
the major
complex,
appear
TL antigens
a possible
the
capacity
culture
is
that
Bn-microanti-human
of a T-cell
factor
Thus,
Bz-microglobulin
(18).
to have an important
equivalent Effect
to contain
a report
role
and Ss and with
embryonic Allogeneic
has been reported there
(13,14) complex
Furthermore,
In humans,
blocks
with
or a B,-micro-
histocompatibility
complex.
in in vitro
substances
that
by the major
(17).
antiserum
B2-
to stimulate or the
in the mechanism
of cell
interaction.
We have found serum raised
that
against
J2,000-dalton
our
preparation
of H-2 antigens,
the
subunits
small
identical
to mouse B,-microglobulin. to mouse 8,-microglobulin subunit
on the evolutional found
in various
reported
in function
for they
react
with
and biological
immunologically
these
substances
may perhaps
well
are not
still
be very
Isolation
of a
and T-cell
structure
significance important
binds
anti-
mouse 82-microglobulin
Fg antigen
of chemical
rabbit
that
and structure.
Ss protein,
by the analysis
relationship
i.e.
However,
from TL antigen,
when accompanied
do not
of mouse B,-microglobulin
component
Therefore,
dalton
and Fg antigen
Ss protein
(19,20).
subunits
dog,
to imnunoglobulin
of the major
associated
immune mediator,
determinants
analogous
present
histocompatibility
a soluble
globulin
factors
human,
significance
in mouse has revealed
both
of the major
12,000
from
to have a homology
and immunobiological
substance
Fg antigen
similar
proteins
related.
genetic
globulin-like
to the
four
to be elucidated.
Recent
to cell
the
and to be a fundamental
problem
antibody
all
8,-microglobulin
antigens,
Factor,
that
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
may shed light of the
small
substances.
Acknowledgements. This investigation was supported in part by United States Public Health Service Research Grants No. AI-08899 and CA-17276 and by The excellent assistance of Mr. A.J. Trott, the John A. Hartford Foundation. Mr. R. Maturski, Mrs. J. Shaver and Miss P. Overturf is deeply appreciated.
429
Vol.70,No.2,
1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
References 1. :: 4. 2 7. 8. 1:: i:: 2 15. 16. 17. 18.
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