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Sequence of i-e genes from autoimmune new zealand white mice.

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583
BRIEF REPORT
SEQUENCE OF I-E GENES FROM AUTOIMMUNE
NEW ZEALAND WHITE MICE
LAWRENCE R. SMITH and ARGYRIOS N. THEOFILOPOULOS
Autoimmune New Zealand white (NZW) mice
contribute to (New Zealand black X New Zealand
white)F, mice 1 or more major histocompatibility complex-linked genes that strongly correlate with susceptibility to murine lupus. The NZW class I1 major histocompatibility complex genes, I-Ea and I-EP, were
cloned and sequenced and Found to differ from normal
B1O.PL (H-2") mice by 3 amino acids in the first domain
of the I-EP subunit. Of these differences, the arginine at
position 72 of NZW mice could be an important disease
determinant since it lies in a predicted antigen-binding
cleft.
The genetic basis underlying the lupus-like autoimmune disease of (New Zealand black x New Zealand
white)F, ([NZB x NZWIF,; H-2d and H-2") mice is
hypothesized to occur by complementation of genes
derived from both parents (1). (NZB X NZW)F, mice
demonstrate high IgG autoantibody titers and immune
complex glomerulonephritis and develop an accelerated,
often fatal lupus disease that is uncommon to either
parental strain (2).
Although multiple factors probably contribute
to the severity of lupus in (NZB X NZW)F, mice,
Publication 5963IMM from the Department of Immunology,
Research Institute of Scripps Clinic.
From the Department of Immunology, Research Institute of
Scripps Clinic, La Jolla, California.
Supported in part by NIH grants AR-31203 and AR-39555.
Dr. Smith is an Irvington House Fellow.
Lawrence R. Smith, PhD; Argyrios N. Theofilopoulos,
MD.
Address reprint requests to Argyrios N. Theofilopoulos,
MD, Immunology Department/IMM3, Scripps Clinic and Research
Foundation, 10666 North Torrey Pines Road, La Jolla. CA 92037.
Submitted for publication September 6, 1989; accepted in
revised form November 6. 1989.
Arthritis and Rheumatism, Vol. 33, No. 4 (April 1990)
several studies have shown a strong correlation between disease and a single NZW gene linked to the
major histocompatibility complex (MHC) locus. The
evidence is as follows: 1) Ninety percent of (NZB X
NZW)F, mice backcrossed to NZB mice that carry the
NZW H-2" loci develop disease, compared with 12%
of those that carry the NZW H-2d'd loci (3). 2) When a
congenic strain of NZW mice carrying the H-2d loci
was crossed with NZB mice, the (NZB X NZW)F,
mice were found to have significantly lower levels of
IgG anti-DNA antibodies, gp70 immune complexes,
and proteinuria, as well as a lower mortality rate, than
the classic H-2d'" hybrids (4). 3) The MHC-linked
gene, tumor necrosis factor a (TNFa), has been reported to be an important contributor to disease in
(NZB x NZW)F, mice in that a restriction fragment
polymorphism in the NZW TNFa gene correlated with
reduced levels of TNFa (5).
Collectively, these results suggest that an NZW
MHC-linked gene(s) intensifies disease. Since the
highly polymorphic class I1 genes of the MHC, I-A and
I-E, exert profound positive and negative selection
effects over the T cell repertoire and are involved in
antigen binding and presentation (6), subtle differences
in these genes of NZW mice, in the presence of other
genes contributed by NZB mice, may confer autoimmune responsiveness to (NZB X NZW)F, mice.
Although NZW is the only strain of mice known
to possess the H-2" haplotype, serologic surveys have
suggested that the I-A and I-E subregions are closely
related to the H-2" haplotype (7). Both I-A and I-E
genes from an H-2" haplotype of a normal B1O.PL
mouse have been sequenced (8-1 l), and the I-Aa and
I-AP sequences of NZW mice have been found to be
identical with the I-A" haplotype of normal BIO.PL
584
mice (Acha-Orbea H: personal communication). However, to our knowledge, no studies have demonstrated
the sequence of NZW I-Err and I-EP genes. Therefore,
these genes were cloned and sequenced from NZW
mice and compared with other I-E haplotypes to define
any structural characteristics unique to NZW mice
that might, in the presence of complementing genes,
predispose (NZB x NZW)F, mice for autoreactivity .
Materials and methods. Total RNA was isolated
(12) from the spleens of female NZW mice (Jackson
Laboratories, Bar Harbor, ME). One microgram of
purified poly(A+) messenger RNA was reversetranscribed into complementary DNA (cDNA) (cDNA
Synthesis System; Promega Biotech, Madison, WI),
and a cDNA library was constructed in hgem4
(Promega Biotech). The library was screened with 1 kb
of genomic DNA and cDNA inserts corresponding to
Ead and EPb genes, respectively (the generous gift of
Dr. George Widera, Scripps Clinic and Research
Foundation). Five positive clones for each gene (aand
P) were rescreened, and 2 of each were selected for
sequencing. Clones were primed with T7 and SP6
oligonucleotides (Promega Biotech) and sequenced by
the dideoxy method (13) with Sequenase (United
States Biochemical, Cleveland, OH). Nested deletions
were created using exonuclease 3/mung bean nuclease
(Stragene, La Jolla, CA) to sequence the entire E d E P
cDNA. Sequence comparisons were made with those
listed in Microgenie (Beckman Instruments, Irvine,
CA) andor the literature.
Results and discussion. An NZW splenocyte
cDNA library was screened using Ead and EPb
probes. A comparison with the Ea" sequence of
BlO.PL mice (11) revealed no differences in the coding
sequences of the Err from B1O.PL mice and the NZW
mice (data not shown). Despite the Ea identity with
BIO.PL mice, the NZW EP sequence differs from the
I-EP sequence reported for BlO.PL mice (lO,ll), as
shown in Figure 1. The 5'-untranslated and leader
sequences of the NZW mouse share 97% and 96%
nucleotide sequence similarities, respectively, with
the corresponding sequences from E@ and EPb mice.
The first 2 amino acids of the NZW mouse EP sequence, valine and arginine, typical of mice with the
EPmhaplotype, are different from the arginine and
glycine reported to be the first 2 amino acids in BlO.PL
mice (10,ll). Although the BlO.PL mouse E P leader
sequence has not been previously reported, one can
speculate that a recombination event may have occurred between either the E@ or E@ leader sequence,
including the nucleotides encoding the first 2 amino
BRIEF REPORTS
NZW lEf3
ThlVl(iTTAUGTCTGMGCTTGCCT1CCCC~TGACTC~TGTCTCCTCTCCTGCAGC
-26
ATG ATG TGG CTC CCC AGA G l T CCC TGT GTG G U GCT GTG ATC Cn l T G Cn A U GTG CTG
p u PTO A r g V ~ FTO
I
c y s val A I ~ A I ~v a l 11e L ~ U
L ~ U
L ~ U~ h v
ra l m u
net net ~ r m
I0
-1
GAC TCC AGA C U CGG TTT TIG GGA TAT TCT ACA TCT
ASP ser A r q pro APhe IBU GIY ~ y r
ser mr ser
20
30
GAG TGT CAT T T C TAC M C GGC ACC CAC CCC CTG CGG TlT CTG GAC AGA TAC TIT T I C M C
Glu Cys Hi8 P h a Tyr A811 G l y T h r Gln A r q Val A r q P h e Leu Asp A r g Tyr Phe Tyr M n
40
50
CGG GAG GAG TGG GTG CGC T T C GAC AGC GAC GTG GGC GAG TAC CGC GCG GTG ACC GAG CE
A r g G I U Glu Trp Val A r q P h e Asp Ser A s p Val Gly Glu Tyyr A r g A l a Val T h r G l u Leu
60
70
GGG CGG CCA G M GCC GAG AAC TGG AAC AGC CAG CCG GAG ATC CE GAG C M
C l y Arg Pro Glu A I a GI" Asn Trp Asn Ser Gln P r o G l u I l e Leu G l u Gln
80
90
GCC GTG GAC ACG TAC TGC AGA CAC AAC TAT GAG ATC T C C GAT A M T T C C T T GTG CGG CGG
A l a Val A s p T h r T y r C y s A r g HIS A a n Tyr G I " Ile Ser A s p Ly6 Phe Leu 1111 A r q A r g
100
ACA GIT GAG CCT ACG GIG ACT GTG T I C
A r q Val Glu P r o T h r Val T h r Val Tyr
G
120
CTC CTG GTC TGC TCT GIG ACT GAC T I C
Leu Leu Val cys Ser Val Ser Asp P h e
140
I10
CCC ACA A X ACG CAG CCC CTC C M CAT CAC
P r o T h r Lys T h r Gln P r o 1Bu G I " H i s H i s
C
130
TAC CCT CCC M C A T T G M GTC AGA TGG T T C
T y r P r o Gly A m Ile Glu Val AT r p Phe
M C
A m
CGG
Arg
150
M T CCC M G GAG GAG AM ACA GGA A T 7 GTG TCC ACG GGC CTG GTC CGA AAT GGA GAC TGC
Asn Gly Lys G l u Glu Lys T h r Gly Ile Val Ser T h r Gly Leu Val A r q Assn Gly A s p Trp
160
170
ACC TX CAG ACA CI'G G I G ATG CTG GAG ACG GTT CCT CAG GGT GGA GAG GTT T I C ACC TGC
T h r P h e G l n T h r Leu Val net Leu GI" T h r Val Pro G l n G l y Gly Glu Val T y r T h r Cys
I80
190
CAG GTG GAG CAT CCC ACC CTG ACC GAC CCT GTC ACA GTC CAC TGG AM GCA C M TCC ACA
G l n Val G1U H i s Pro S e r Leu T h r Asp P r o Val T h r Val GI" T r p Lys A l a G l n Ser T h r
200
210
'ITT GCA CAC M C M G A X TTG ACT GGA GTT GGG GGC TTC GTG CTG GGC C T C C T C T T C CTC
ser A h Gln A s n Lys net Leu Ser Gly Val Cly Gly P h e V a l Lou Gly Leu Leu phe Leu
220
230
GGA GCC CCC CTG T I C ATC TAC T I C AGG M T CAG AM GWL CAG T C T GGA cI1' CAG CCA ACA
Gly A l a Cly Leu P h e Ile Tyr P h e A r g Asn Gln LYS G l Y Gln Ser Cly Leu Gln P T D T h r
CTC Cn AGC
G l y Leu Leu ser
GGA
Figure 1. Nucleotide and predicted amino acid sequence of I-Ep
complementary DNA from NZW mice. The H-2" EP amino acid
sequence of BIO.PL mice is identical to that of NZW mice, except
for residues 1, 2, and 72 (boxes). The only additional nucleotide
differences, at amino acids 96 and 112, do not result in different
amino acids.
acids, and the remaining nucleotides of the Efl" sequence. Only 1 other productive change exists between the B1O.PL mouse EP sequence and that of
NZW mice, as seen at amino acid 72; NZW mice have
an arginine at 72, while B 1O.PL mice have a threonine.
Two other nucleotide differences between NZW and
BlO.PL mice, occurring at amino acid positions 96 and
112, are silent. Thus, while B1O.PL and NZW mice are
100% identical for coding sequences of the Err locus,
they differ by 3 amino acids (98.9% identical) at the
EP locus.
Of these 3 differences, the arginine at position
72 could potentially have a profound effect on EP
function. Although the crystal structure of a class I1
molecule is unknown, a theoretical model (14) has
been described with reference to the crystallized
HLA-A2 class I molecule (15). According to this
model, the arginine at EP 72 (aligned with amino acid
156 of HLA-A2 or amino acid 71 of I-Ab) lies in one of
585
BRIEF REPORTS
the most polymorphic regions of the class I1 PI domain
in a predicted a-helical region, proposed to be an
antigenic peptide-binding site (14,15).
Close associations of class I1 MHC alleles with
a variety of human a s well as animal autoimmune
diseases have been documented (16). In several instances, disease susceptibility or resistance correlates
with either a particular amino acid at a single position
or within a short distance. For example, residues
70-74 in the third hypervariable region of DRPl exert
dominant effects in disease susceptibility to rheumatoid arthritis (16,17). In diabetes mellitus, amino acid
57 of DQP appears to confer susceptibility to disease if
it is alanine, serine, or valine, or resistance to disease
if it is aspartic acid. Furthermore, the unique I-AP
subunit of the nonobese diabetic mouse has a serine
instead of a n aspartic acid at position 57 (16,18). In
phemphigus vulgaris, the susceptibility of the DQP
allele DQwl.9 differs from other DQwl alleles by
only a valine-to-aspartic acid substitution at position
57 (16). These observations highlight the importance
of one, or a few, amino acids in determining the
3-dimensional structure of MHC and, presumably, its
ability to bind autoantigen and/or appropriate T cell
receptors. Thus, the Thr,,-Arg,,
substitution in
autoimmune-contributing NZW EP mice may affect
these functions and lead to autoimmune disease development in [NZB X NZW)F, mice.
Perhaps disease development in (NZB x
NZW)F, mice is dependent upon a transassociation of
NZB and NZW class I1 molecules. Further classic
genetic experiments with H-2' congenic lupus and
normal mice, as well as experiments with transgenic
mice, in which the NZW EP gene is introduced into
the NZB mice, might provide direct evidence for these
possibilities. It should also be noted that our studies do
not clarify whether the disease-predisposing effect is
specific for H-2" I-EP or is, in general, associated with
the I-MI-E molecules of the H-2" haplotype. We are
currently preparing NZB x PL/J crosses to answer
this question. It is clear, however, that the NZW
mouse is not the only strain contributing to accelerated
autoimmunity, since crosses of NZB (H-2d) mice with
SWR (H-2q)mice also produce a disease similar to that
in (NZB x NZW)F, mice (19). Finally, even if this
apparently unique I-EP sequence of the NZW mouse
is found to be necessary for disease expression, it
appears that it is, in itself, insufficient, and additional
genes contributed by both the NZW and the NZB
parent are required. This is clearly demonstrated by
the fact that NZW mice themselves are relatively free
of disease, and NZB mice crossed with BALB/c mice
rendered congenic for the H-2' haplotype remain free
of disease (Smith LR, Theofilopoulos AN: manuscript
in preparation).
Addendum. During review of this paper, Schiffenbauer et a1 also reported a unique sequence of the NZW I-EP
chain containing an arginine at position 72 (20). Our results
and conclusions are similar, and we have also included the
complete coding sequence of the NZW I-EP gene, which
was found to differ from the BIO.PL I-EB sequence by the
first 2 amino acids of the mature protein.
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1985
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to lupus-like disease in (NZBxNZW)F, mice. J Exp
Med 165:1237-1251, 1987
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Y, Sato H, Shirai T: Enhancing effect of H-2-linked
NZW gene(s) on the autoimmune traits of (NZB x
NZW)F, mice. J Exp Med 158:228-233, 1983
5 . Jacob CO, McDevitt HO: Tumor necrosis factor-a in
murine autoimmune 'lupus' nephritis. Nature 33 1:356358, 1988
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what does the T cell receptor see? Adv Immunol45:107193, 1989
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haplotypes of strains DBR7, BIO.NZW, NFS, BQZ,
STU, TO1 and T02. Immunogenetics 15:431436, 1982
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Watanabe Y, Tokunaga K, Matsuki K, Takeuchi F,
BRIEF REPORTS
Matsuta K , Maeda H. Omoto K, Juji T: Putative amino
acid sequence of HLA-DRB chain contributing to rheumatoid arthritis susceptibility. J Exp Med 169:226>
2268, 1989
18. Acha-Orbea H, McDevitt IiO: The first external domain
of the nonobese diabetic mouse class I1 I-AP chain is
unique. Proc Natl Acad Sci USA 84:2435-2439, 1987
19. Ghatak S, Sainis K. Owens I:L, Datta SK: T-cell receptor
Band IAP chain genes of normal SWR mice are linked with
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NZW I-EP chain and its possible contribution to autoimmunity in the (NZB x NZW)F, mouse. J Exp Med
170:971-984, 1989
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