Download Variable regions of a human anti-DNA antibody 0

Nucleic Acids Research, Vol. 20, No. 10 2601
© 7992 Oxford University Press
Variable regions of a human anti-DNA antibody 0-81
possessing lupus nephritis-associated idiotype
Yasuhiko Hirabayashi, Yasuhiko Munakata, Takeshi Sasaki* and Hiroshi Sano1
The Second Department of Internal Medicine, Tohoku University School of Medicine, Seiryo-cho 1-1,
Aoba-ku, Sendai 980 and biotechnology Institute, Akita Prefectural College of Agriculture, Minami
2-2, Ogata-mura, Akita 010-04, Japan
Submitted April 7, 1992
An anti-DNA antibody idiotype (Id), termed 0-81 Id, was in the
antigen-binding sites of human IgM monoclonal anti-single
stranded (ss) DNA antibodies secreted from the 0-81 clone,
derived from a patient with active lupus nephritis (1, 2). The
O-81 Id was specifically detected in circulating immune complex
IgG and renal immune deposits of patients with lupus nephritis
(3,4). The paratopes of O-81 were responsible for the idiotypic
expression of 0-81 (unpublished data). These findings suggested
that the sequence analysis of 0-81 may contribute to
understanding the origin of pathogenic autoantibodies in humans.
Poly(A+)RNA was prepared from cells of O-81 clone by
using Micro Fast Track (Invitrogen, San Diego, CA) and used
to prepare ss-cDNA which was primed with oligo-d(T)|2_i8
primer. For amplification of the variable regions by polymerase
chain reaction (PCR) the following primers were used:
VH3-leader 5' ATGGAGTTTGGGCTGAGC 3', Qi 5' TGGAAGAGGCACGTTCTTTTC 3', Vk2-leader 5' GAAGCTTATGAGGCTCCCTGCTC 3', Ck 5' TCTAGAACTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCAAG 3'. The
products were digested by EcoRI (heavy chain) and Hindm-Xbal
Gight chain), respectively, and were ligated into the vector
pUCl 18. Sequences were determined by the chain termination
method. To avoid misincorporation of TaqI polymerase, DNA
amplification was independently performed three times and at
least one clone was obtained at each time. The sequences of these
clones were identical.
The nucleotide sequences revealed V H 3 - D H Q 5 2 - J H 4 and
VK2-JK5. The VH segment of 0-81 showed the highest
homology with the V H m germline H l l gene (5), with 86.9%
matching (leader-FR3). It was also 86.9% homologous with the
FL2-2 gene obtained from cDNA library of fetal liver (6). The
VK segment was 98.1% homologous with the germline V6410
gene (7). It was suggested that the VK segment was encoded by
the V6410 gene or its relative genes but the VH segment might
be originated from an unreported VH3 germline gene.
EMBL accession nos X59134 and X59135
REFERENCES
1. Sasaki.T., Endo.F., Sekiguchi.Y., Mikami.M., Tada.K., Ishida.N. and
Yoshinaga.K. (1984) J. Immunol. Methods 72, 157.
2. Tamate.E., Sasaki.T., Muryoi.T., Takai,O., Otani,K., Tada,K. and
Yoshinaga.K. (1986) J. Immunol. 136, 1241.
3. Muryoi.T., Sasaki.T., Hatakeyama,A., Shibata.S., Seino.T. and
Yoshinaga.K. (1990) J. Immunol. 144, 3856.
4. Sasaki.T., Muryoi.T., Hatakeyama.A., Suzuki,M., Sato.H., Seino.J.,
Saito.T. and Yoshinaga.K. (1991) Am. J. Med. 91, 335.
5. Rechavi.G., Bienz.B., Ram.D., Ben-Neriah.Y., Cohen.J.B., Zakut.R. and
Givol.D. (1982) Proc. Nail. Acad. Sci. USA 79, 4405.
6. Nickerson.K.G., Berman.J., Glickman.E., Chess.L. and Alt.F.W. (1989)
J. Exp. Med. 169, 1391.
7. Klobeck.H.G., Meindl.A., Combriato.G., Solomon.A. and Zachau.H.G.
(1985) Nucleic Acids Res. 13, 6499.
Heavy chain
Leader
ATGGACTTTi
V
Q
t
V
GCGAAGACGCT)
E
S
G
G
C
L
V
Q
P
G
G
S
L
R
L
rra3CCAACGTGAACCAGGATGGAAGTGCCAGGTACTATGCG
CACTCTGTGAGGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTATATCTC
D S V R G R F T I 8 R D N A K N S L Y L
CAAATGCACAGCCTCACAGCCCAOyWACGGCCGTGTATTACTGTGCCACATCAACCGGC
Q H P S L R A D D T A V Y Y C A R S T G
TAATCCTCTGGGTCCCAGGATCCAGTCCC
ATCTCCTGCAGGTCTACTCAAACCCTCGTACACAGTGATGGAAACACCTACTTGAATTGG
I S C R S S O S L V H S D G N T Y L N W
rrTCACCAGAGGCCAGGCCAATCTCCAACGCGTCTAATTTATACCGTTTCTAACCGCGAC
F 0 0 R P C Q 8 P H B L I Y R V S H R 0
ACATTCAGCGCCAGTGGCTCAGGCACTGATTTCACACTGAAAATC
ACKNOWLEDGEMENTS
This work was supported by a grant-in-aid for Scientific Research
from the Ministry of Education, Science and Culture, and by
a research grant for autoimmune diseases from the Ministry of
Health and Welfare, Japan.
* To whom correspondence should be addressed
Figure 1. Nucleotide and deduced amino acid sequences encoding the VH and
VK regions of the O-81. Asterisks indicate complementary determing regions
(CDR).