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Genomic organization and characterization of mouse SAP, the gene that is

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Article in Immunogenetics · September 2000

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Immunogenetics (2000) 51 : 805±815
Digital Object Identifier (DOI) 10.1007/s002510000215
ORIGINAL PAPER
Chengbin Wu ´ Joan Sayos ´ Ninghai Wang
Duncan Howie ´ Anthony Coyle ´ Cox Terhorst
Genomic organization and characterization of mouse SAP, the gene
that is altered in X-linked lymphoproliferative disease
Received: 13 April 2000 / Revised: 15 May 2000 / Published online: 1 July 2000
 Springer-Verlag 2000
Abstract X-linked lymphoproliferative (XLP) disease
is a fatal immunological disorder that renders the
immune system unable to respond effectively to
Epstein-Barr virus (EBV) infection. The gene that
encodes a protein termed SAP or SH2D1A is either
deleted or mutated in XLP patients, resulting in
uncontrolled B- and T-cell proliferation upon EBV
infection. Here, we report the cloning and character-
ization of the mouse SAP gene. It is localized on the
mouse X chromosome and comprises four exons span-
ning approximately 25 kb. Its expression appears to be
restricted to T lymphocytes. Whereas a high level of
SAP expression is observed in Th1 cells, only small
amounts are detectable in Th2 cells. Moreover, SAP
expression is down-regulated upon in vitro activation
of T cells, including CD4+, CD8+ single-positive T
cells, and Th1 and Th2 cells. This study provides val-
uable information for in-depth genetic and biochemi-
cal analysis of the function of SAP in the immune sys-
tem.
Key words SAP ´ SLAM ´ SH2D1A ´ XLP ´ EBV
Introduction
Epstein-Barr virus (EBV) is a ubiquitous B-cell virus
that infects children at an early age without causing
any diseases in the majority of infected individuals.
C. Wu ()) ´ J. Sayos ´ N. Wang ´ D. Howie ´ C. Terhorst
Division of Immunology, RE-201,
Beth Israel Deaconess Medical Center, Harvard Medical School,
330 Brookline Avenue, Boston, MA 02215, USA
E-mail: cwu@caregroup.harvard.edu
Phone: +1-617-6677144
Fax: +1-617-6677140
A. Coyle
Department of Biology, Inflammation Division,
Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA
However, males with XLP genetic mutations develop
fatal immunological disorders following EBV infec-
tion. X-linked lymphoproliferative (XLP) disease is a
fatal immunological disorder which renders the
immune system unable to respond effectively to EBV
infection. Other typical clinical phenotypes of XLP
include malignant lymphoma, dysgammaglobulinemia,
nasopharyngeal carcinomas, aplastic anemia, and vas-
culitis and pulmonary lymphomatoid granulomatosis
(Seemayer et al. 1995).
We recently cloned the gene that is affected in
XLP patients (Sayos et al. 1998), through its associa-
tion with a cell surface structure termed SLAM
(Cocks et al. 1995). It was therefore termed SLAM-as-
sociated protein or SAP. Simultaneously, the XLP
gene was cloned by positional cloning and was sub-
sequently called SH2D1A (Coffey et al. 1998). The
gene that encodes a protein termed SAP or SH2D1A,
is either deleted or mutated in the XLP patients
analyzed, resulting in uncontrolled B- and T-cell pro-
liferation upon EBV infection. Human SAP, which is
primarily expressed in T cells comprises a single SH2
domain followed by a short tail of 26 residues. The
SH2 domain binds to a specific segment around the
residue Tyr281 of the cytoplasmic domain of T- and
B-lymphocyte surface receptor SLAM (Sayos et al.
1998). Since binding of SAP to SLAM prevents
recruitment of the tyrosine phosphatase SHP-2, SAP
is thought to act as a regulator of the SLAM signal
transduction pathway in T cells. Surprisingly, the SH2
domain of SAP binds to a motif adjacent to Tyr281 in
the nonphosporylated form of SLAM (Poy et al.
1999).
Here we report the cloning and characterization of
the mouse SAP gene. The overall genomic and pri-
mary structure of mouse SAP is similar to that of
human SAP. The gene was found to span about 25 kb
and produce a single transcript of 0.9 kb. Northern
blot analysis indicated that mouse SAP is expressed
primarily in lymphoid tissues, most abundantly in thy-
806
mus. Further analysis revealed that SAP is expressed
in T cells, including CD8+, CD4+ single-positive (SP)
T cells, mostly Th1, but also in Th2 cells. Its expres-
sion is down-regulated upon T-cell activation in vitro.
Like human SAP, mouse SAP binds to the cytoplas-
mic domain of the T- and B-lymphocyte surface recep-
tor SLAM, supporting the notion that SAP is a regula-
tor of SLAM signal transduction pathways.
Materials and methods
Cloning of mouse SAP
Using a human SAP cDNA sequence, we searched for similar
mouse sequences in the sequence database, and identified a
294-nt sequence (GenBank accession number AA255258). This
sequence originated from mouse lymph nodes, and had a high
homology with human SAP (83%). Therefore, it was likely a
fragment of the mouse SAP cDNA. Based on this sequence, a
probe was designed to screen a mouse (C57BL/6) thymus library
(a kind gift of Dr. Linda Clayton) by colony hybridization, and
four identical mouse SAP clones were obtained. To confirm that
they were full-length cDNA clones, two primers were designed
toward the 59 and 39 ends of the fragment, and rapid amplifica-
tion of cDNA ends (RACE) analysis was performed using a
mouse spleen cDNA library (Clontech). The primers used were:
for the 59-end PCR: 59-AGC TAT CTG CTG CGA GAC AGC
GAG-39; for the 39-end PCR: 59-ATT CAG GCA GAT ATC
AGA ATC TCT-39. Two PCR products, representing the full-
length 59- and 39-end sequences of mouse SAP cDNA, were sub-
cloned into the pCR2.1 vector and sequenced. The sequences
obtained collectively revealed the full-length mouse cDNA
sequence.
Isolation and characterization of mouse SAP genomic clones
A BAC mouse (strain 129/SVJ) genomic library (Genome Sys-
tems) was screened by PCR using the following two primers
(covering the first exon of the mouse SAP gene): forward,
59-GGA TGC AGC GAC TGT GTA CCA C-39; reverse,
59-CCT TGA TAC AAA ACA CAC AGG C-39. The "Down-
To-The-Well" pools of genomic clones were subjected to PCR
screening according to the manufacturer©s instructions. The
DNA of the four positive pBAC clones obtained was prepared
using a Qiagen Maxiprep kit and subjected to restriction enzyme
mapping analysis. Four probes, p1, p2, p3, and p4, corresponding
to the mouse SAP 59 untranslated region (59UTR), SH2 domain,
tail sequence, and 39UTR, respectively, were used to determine
the number and position of exons. Several genomic fragments,
including a HindIII-digested fragment containing exon 1, an
EcoRI-digested fragment containing exon 4, and a BamHI-di-
gested fragment containing exons 2±4, were subcloned into the
pBC SK vector (Stratagene) or pCR2.1 vector (Invitrogen), and
intron-exon boundaries were determined by DNA sequencing.
Chromosome localization of mouse SAP by fluorescence in situ
hybridization
The direct R-banding fluorescence in situ hybridization (FISH)
method was employed for chromosome assignment of the mSAP
gene on mouse chromosomes. Preparation of mouse chromo-
somes and FISH were performed as previously described (Mat-
suda and Chapman 1995). Briefly, DNA from the mouse SAP
genomic pBAC clone was labeled with digoxigenin (DIG) dUTP
by nick translation. The labeled probe was combined with
sheared mouse DNA and hybridized to normal metaphase chro-
mosomes derived from mouse embryo fibroblast cells in a solu-
tion containing 50% formamide, 10% dextran sulfate, and
2”standard sodium citrate. A second set of FISH experiments
was performed involving independent co-hybridization with both
centromere- and telomere-specific probes, 6856 and 6824,
respectively as internal controls (Shi et al. 1997). Specific hybrid-
ization signals were detected by incubating the hybridized slides
in fluoresceinated antidigoxigenin antibodies followed by
counter-staining with DAPI. Assignment of SAP in the mouse
genome was obtained from ten specifically labeled X chromo-
somes. A total of 80 metaphase cells were analyzed with 70
exhibiting specific labeling.
Northern and Western blots, and immunoprecipitation analysis
Northern blots were performed according to standard pro-
cedures. Total RNA was prepared from mouse tissues or cell
suspensions using Trizol reagent (Life Technologies) and sub-
jected to electrophoresis. Immunoprecipitations and Western
blot were carried out as previously described (Sayos et al. 1998).
DIG RT-PCR
SAP and SLAM mRNAs were quantified with RT-PCR ELISA
according to the manufacturer©s instructions (Roche). Briefly,
equal amounts of total RNA (0.1±1 g) from different samples
were used in RT-PCR. The RT-PCR product was labeled with
DIG during the reaction, and immobilized onto a streptavidin-
coated 96-well plate using a biotinylated oligonucleotide probe
complementary to an internal sequence of the RT-PCR product.
horseradish peroxidase-conjugated anti-DIG was then added to
the plate. After incubation and washing, the samples were devel-
oped with the manufacturer©s developing solution (Roche), and
detected with a microplate reader at 415 nm. A standard curve
was constructed for each mRNA species using different RT-
PCR and assay conditions to ensure that the sample readings
were within the proper range. Each sample reading was also
normalized with CD3e (for T cells), and b-actin or GAPDH as a
housekeeping gene internal control for all assays. Samples were
assayed in triplicate.
T-cell preparation and activation
Single-cell suspensions were prepared from spleen and lymph
nodes of BALB/c mice (Jackson Laboratories), and then labeled
with biotinylated anti-CD11b, anti-Ly-6G, and anti-CD45R/B220
monoclonal antibodies (mAbs) (Pharmingen). The cells were
washed and resuspended in PBS containing streptavidin-con-
jugated metal beads (Miltenyi Biotec), followed by another
wash with PBS. T cells were purified by applying labeled cells to
an iron column (Miltenyi Biotec) in a magnetic field. According
to fluorescence-activated cell sorting analysis, 96% of the eluted
cells were CD4+/CD8+ T cells (data not shown). For certain
experiments, SP, DP, and DN T cells were isolated by a cell
sorter. The purified T cells were then transferred to 24-well
plates, which had been coated with anti-CD3e mAb (2C11)
(Pharmingen), and maintained in a 37C, 5% CO2 incubator for
different time periods. The cells were then collected and total
RNA extracted using Trizol reagent, and Northern blot or DIG
RT-PCT analysis was performed as described above.
SAP expression in Th1 and Th2 effector cells
Mice expressing the transgene for the DO11.10 ab-T-cell recep-
tor (TCR), which recognizes residues 323±339 of chicken ovalbu-
min (OVA) in association with I-Ad, were provided by Dr. D.
Loh (Washington University, St. Louis, Mo.). DO11.10 TCR-
transgenic CD4+ T cells were cultured in complete RPMI 1640

with OVA323±339- (1 m) and mitomycin C-treated splenocytes.
For Th1 phenotype development, recombinant mouse interleu-
kin (IL)-12 (10 ng/ml) and neutralizing anti-IL-4 mAb (11B11,
40 g/ml: R & D Systems) were added, and for Th2 devel-
opment, recombinant mouse IL-4 (10 ng/ml) and neutralizing
polyclonal anti-mouse IL-12 (TOSH-2, 3 g/ml; Endogen, Cam-
bridge, Mass.) were used. Cells were cultured for three rounds
of antigenic stimulations under polarizing conditions and then
stimulated on plate-bound anti-CD3 in the presence of hIL-2
(Endogen) for 6 h. Poly(A)+ RNA was isolated from activated
cells using the FastTrack mRNA Kit (Invitrogen), and 1.0 mg of
RNA was loaded per lane for Northern blot analysis. The mem-
brane was probed sequentially with probes for SAP, SLAM,
IFN-g, IL-4 (Clontech), and b-actin. The IFN-g probe comprised
a 344-nt fragment of mouse IFN-g covering the region from
position 532±876 (GenBank accession number M28621).
Results
Isolation of the mouse SAP cDNA
To pursue the importance of SAP in immune physi-
ology, we determined the structure of mouse SAP, to
allow in-depth genetic and physiological studies in this
species. A full-length mouse SAP cDNA was isolated
from a BALB/c mouse spleen cDNA library and a
C57BL/6 mouse thymus cDNA library, and its nucleo-
tide sequence determined. The mouse SAP cDNA
sequence is 80% identical with that of human SAP,
including 59UTR, coding, and 39UTR regions (Fig.
1A).
The deduced amino acid sequences of mouse and
human SAP share 87% identity and 89% similarity
overall, with the highest similarity in the SH2-domain
(Figure 1B). By comparing the SAP amino acid
sequence with the EST database (translated in all pos-
sible reading frames), a partial rat SAP sequence was
found to be very similar to the mouse sequence (Fig.
1B). Not surprisingly, many other proteins with signifi-
cant similarities in their SH2 domains were detected
(Coffey et al. 1998; Sayos et al. 1998). Of those, the
sequences of mouse and human EAT-2 are shown, as
the EAT-2 protein is the only other known protein
that consists of a mere SH2 domain and a short C-ter-
minal tail (Thompson et al. 1996). This sequence sim-
ilarity is of significance, because EAT-2 and SAP bind
to the same tyrosine motifs in SLAM (Poy et al. 1999;
M. Morra, unpublished data). Moreover, structural
similarities between SAP and EAT-2 are suggested by
analysis of the three-dimensional structure of SAP
(Poy et al. 1999).
Characterization of the mouse SAP gene
Three positive pBAC clones were obtained from a
mouse 129SV genomic library in pBAC by colony
hybridization. Their DNA was digested with restric-
tion enzymes and probed with four probes cor-
responding to four regions of mSAP cDNA, namely
the 59UTR, a segment encoding the SH2 domain, the
807
tail, and the 39UTR. All three positive clones con-
tained the complete transcribed sequence of mouse
SAP mRNA. Using extensive restriction analysis and
Southern hybridization with the four probes, the
mouse SAP gene was shown to consist of four exons
and three introns spanning approximately 25 kb (Fig.
2A). A restriction map of a representative positive
clone (no. 16594) is illustrated in Fig. 2A with the
positions of restriction enzymes EcoRI, HindIII, and
BamHI marked. Detailed sequencing analysis revealed
the size of each exon and intron/exon boundaries as
shown in Fig. 1 and Table 1 (all genomic sequences
are available under GenBank accession numbers AF
154503, AF 154504, and AF 154505). Each of the
mouse SAP splice junction sequences conforms to the
eukaryotic splice consensus sequence (AG/GT). A
partial exon-skipping SAP polymorphism represented
by a 55-nt deletion in exon 3 which is observed in
human was not detected in mice based on our RT-
PCR analysis (data not shown). The first exon of 299
bp contains all of the 59UTR and part of the SH2
domain. The SH2 domain of mSAP spans the first
three exons, whereas exon 4 encodes part of the tail
sequence and all of the 39UTR. Taken together, these
results suggest that SAP exists as a single gene in the
haploid mouse genome and that it encodes an SH2
domain and a 24-amino acid tail.
While there are two transcripts identified for
human SAP (0.9 kb and 2.5 kb; Sayos et al. 1998), we
only detected a single 0.9-kb transcript for mouse SAP
using both colony hybridization and Northern blot
techniques. However, comparison of the genomic
sequences beyond mouse exon 4 revealed that this
exon might extend and that a larger transcript is the-
oretically possible. Whereas the 39UTR of mouse SAP
mRNA contains three AUUUA motifs that are sig-
nals for rapid mRNA degradation, the 0.9-kb human
RNA contains two and the 2.5-kb human SAP mRNA
contains eight of these degradation signals [Fig. 1A
and accession number AF072903 (mouse) and
AF073019 and AF072930 (human)]. All three mRNAs
undergo rapid degradation upon in vitro T-cell activa-
tion (see below).
Analysis of the transcription start sites and the
sequence of the mouse SAP promoter region
We sequenced the 59 region flanking the first exon of
the mouse SAP gene, and analyzed the potential tran-
scription factor-binding sites in this region. The
sequence of the 59-flanking region (approximately 1.2
kb) of the mouse SAP gene is shown in Fig. 2B. The
consensus transcription factor-binding sites are indi-
cated in boxes, based on the information from the
Transcription Element Search System (Schug and
Overton 1997). There are multiple sites for transcrip-
tion initiation (Fig. 2B). Ubiquitous sites include TBP,
TFIID, GATA-1, OCT, delta-factor, CAC-binding
808

Fig. 1A,B Primary structure

of mouse SAP. A Alignment
of mouse and human SAP
mRNA sequences. Exon
boundaries are indicated as
horizontal arrows; boxes indi-
cate start and stop codons; a
vertical arrow indicates the
end of the shorter transcript of
human SAP; bold letters rep-
resent mRNA degradation sig-
nals. B Alignment of SAP and
EAT sequences from different
species. Secondary structural
elements (a helices and b
sheets) are indicated based on
the crystal structure of human
SAP (Poy et al. 1999)

protein, CP2, and AP-2. Factors that are important for
T-lymphocyte development and function include mul-
tiple glucocorticoid receptor (GR)-binding sites,
c-Ets-1, and IRF-1. In conclusion, the SAP gene con-
tains a number of cis-elements that are likely to be
involved in up- and down-regulation of its expression.
Chromosomal localization of the mouse SAP gene
Chromosomal localization of the mSAP gene was
determined by direct R-banding FISH using one of
the SAP genomic BAC clones as a probe (number
16594). Measurements of ten specifically labeled X
chromosomes demonstrated that mouse SAP was
located at a position which is 25% of the distance
from the heterochromatic-euchromatic boundary to
 809

Fig. 2A,B Genomic organiza-

tion of mouse SAP gene. A
Intron-exon organization of
mouse SAP (H HindIII, B
BamHI, E EcoRI). B Putative
transcription factor-binding
sites in the region 59 flanking
the first exon of mouse SAP.
GR glucocorticoid receptor;
c-Ets-1 a transcription factor
involved in hematopoiesis and
leukemogenesis, and in the
transactivation of the TCR
b-chain enhancer; TBP
TATA-binding protein; TFIID
transcription factor IID;
GATA-1 GATA-binding tran-
scription factors 1; OCT
octamer-binding transcription
factor; CP2 a-globin transcrip-
tion factor; HES-1 helix-loop-
helix factor 1; IRF-1 interferon
regulatory factor 1; CAC-bind-
ing protein an erythroid-spe-
cific transcription factor; Delta
factor a zinc finger transcrip-
tion factor that binds to down-
stream elements in several
polymerase II promoters

the telomere of the X chromosome, an area that cor-
responds to band XA5 (Fig. 3A,B). Because part of
band XA5 is syntenous with human Xq25, we exam-
ined whether an altered SAP gene could be involved
in the XLP disease and indeed we showed that a
series of patients with XLP had a mutation in SAP
(Sayos et al. 1998; and data not shown). Interestingly,
few other genes have been detected in the immediate
proximity of the human SAP gene (Coffey et al.
1998).
Expression of SAP mRNA
RNA blotting was performed to characterize the tis-
sue distribution of the mouse SAP mRNA. When nor-
810

Table 1 Intron-exon junction sequences of the mouse SAP gene

Exon Exon size 59 flanking sequence Exon and amino acid 39 flanking
(bp) sequences sequences

1 299 TTGTGAGGGG 111 GTTCTCTCTC . . . TGTGTGTTTT 299 GTGAGTATAT

. . . LeuCys ValLeu
2 64 TCTTTCACAG 300 GTATCAAGGT . . . GAGTGCCGAG 363 GTACAGCTCT
147 TyrGlnGly . . . SerAlaGlu 167
3 142 ATTTTTCCAG 364 ACAGCACCTG . . . GCTCCCACAG 505 GTGTGCTGCC
168 ThrAlaPro . . . AlaProThr 113
4 382 TTTTCTTTAG 506 GGAGAAGAGA . . . TTTTGGAAA 887 GCGGCATTTT
114 GlyArgArgAsp . . .

malized on total RNA, the highest steady-state levels
were detected in the thymus, with less in lymph nodes
and spleen (Fig. 4A). To further analyze SAP mRNA
expression in lymphoid organs, several mutant mice,
i.e., CD3gd±/±, which are deficient in T cells, and SCID
and RAG±/±, which are deficient in T and B lympho-
cytes, were used for Northern blot analysis (Fig. 4B).
In all three mutant mice, T-cell development was
arrested at the transition from double-negative (DN)
CD44±CD25+ to the double-positive (DP) stage (Chen
et al. 1994; Pla and Mahouy 1991; Wang et al. 1998).
SAP is expressed moderately in the DN thymocyte
population of the CD3gd±/± mouse, but very low levels
of SAP expression were detected in the equivalent
cells of the SCID or RAG±/± mouse. This result sug-
gests that the block in thymocyte development of the
CD3gd±/± mouse is different from that in SCID or
RAG±/±mice. To study SAP expression in different
developmental stages of thymic T cells, we analyzed
SAP mRNA levels in SP CD4+ and CD8+ cells, DP
(CD4+CD8+) cells, and DN (CD4±CD8±) cells (Fig.
4C). Our data show that SAP is expressed at a low
level in DN cells and a high level in DP cells. It is
expressed at moderately high levels in SP cells, with a
slightly higher level in CD8+ cells than in CD4+ cells
(Fig. 4C). Thus, SAP up-regulation could signify a dis-
tinct step in transition from DN to DP cells, and upon
positive selection, SAP is down-regulated.

Fig. 3A,B Chromosome local-

ization of mouse SAP. A
FISH analysis shows that SAP
is located at a position which
is 25% of the distance from
the heterochromatic-euchro-
matic boundary to the telom-
ere of the X chromosome, an
area that corresponds to band
XA5. The red arrows indicate
the position of SAP in the X
chromosome, and the white
arrows indicate the position of
an internal control probe. B
Schematic representation of
the mouse SAP gene in the X
chromosome

Fig. 4A±C Tissue distribution of mouse SAP. A Multiple
mouse tissue Northern blot shows SAP expression in different
tissues in comparison to SLAM expression. B SAP and SLAM
expression in tissues of immunodificient mice. C SAP and
SLAM expression in different populations of thymic T cells.
Cells were isolated by cell sorter, and total RNA was prepared
and assayed for SAP and SLAM using the DIG RT-PCR
method
Co-expression of mouse SAP and SLAM
Since human SAP had been cloned using a two-hybrid
system in which the cytoplasmic tail of SLAM had
been used as a bait, and because these interactions
were shown to occur in human T lymphocytes, expres-
sion of SLAM was compared with that of mouse SAP.
RNA blotting demonstrated that mouse SLAM tran-
scripts are found at high levels in the thymus and at
lower levels in lymph nodes and spleen. Low levels of
mouse SLAM mRNA were detected in other tissues
as well (Fig. 4A). Whereas in the thymus, expression
of SLAM mRNA followed that of SAP, there were
differences in the lymph nodes and spleen (Fig. 4B).
These can easily be explained by the fact that SLAM,
811
but not SAP, is expressed in B cells. This is based
upon studies with monoclonal anti-human SLAM
(Cocks et al. 1995). Indeed, mouse SLAM cannot be
detected in SCID or RAG±/± mice which have neither
T nor B lymphocytes (Fig. 4B). Although human
SLAM has been found on the surface of dendritic
cells, expression is low on resting dendritic cells (Mik-
halap et al. 1999). This would explain why no signifi-
cant amounts of SLAM mRNA were detected in
lymph nodes of SCID and RAG±/± mice.
Western blot analysis demonstrated that mouse
SAP mRNA indeed encoded a protein of Mr 15,000 in
thymus, spleen, and lymph node cells. (Fig. 5A).
Moreover, upon precipitation of SLAM from a deter-
gent lysate made from either a C57BL/6 (Fig. 5B) or a
Balb/c thymus (data not shown), SAP protein was
shown to be associated as demonstrated by Western
blotting with an anti-SAP reagent. We conclude that
in thymocytes, SAP and SLAM are co-expressed and
form a complex, but that co-expression of SAP and
SLAM only occurs in cells of T lineage. In contrast, B
cells express only SLAM.
812
Fig. 5A,B SAP binds to SLAM. A Western blot shows that
mouse SAP is expressed in thymus, spleen, and lymph nodes. B
SAP co-precipitates with SLAM. In C57BL/6 mice, SLAM
immunoprecipitation followed by SAP Western blot indicates
that SAP binds to SLAM. In the control experiment, a rat Ig
was used in the immunoprecipitation
Regulation of SAP expression during T-cell activation
Based on its three-dimensional structure (Poy et al.
1999), we proposed that there are two principal mech-
anisms by which SAP could regulate the function of
SLAM. Either (1) the tight interaction between SAP
and SLAM could block phosphorylation of Tyr281 or
Tyr326, or (2) SAP could block SH2 domain-dependent
recruitment of an adapter or enzyme molecule.
Whereas evidence for the latter mechanism was pro-
vided by demonstrating that SAP can block recruit-
ment of the tyrosine phosphatase SHP-2 to the cyto-
plasmic tail of SLAM (Sayos et al. 1998), no data are
available to support the notion that SAP can block
phosphorylation. However, in either model, regulation
of expression of the two proteins could influence the
signaling function induced by engagement of SLAM.
First, Northern blot analysis was used to follow
expression of mouse SAP and SLAM upon activation
of purified T cells with the mitogen anti-CD3e. While
SAP mRNA expression was down-regulated upon
activation of T cells, SLAM mRNA expression was
initially up-regulated and reached a maximal level at
24 h post-stimulation, and then decreased gradually to
a level similar to that of unstimulated cells at 7 days
post-stimulation (Fig. 6A). Disappearance of the SAP
protein upon triggering of the TCR for antigen was
also easily detected by Western blot (Fig. 6B). SAP
expression is down-regulated in both CD4+ and CD8+
SP T cells upon anti-CD3e stimulation (Fig. 6C).
To examine the changes in expression in special-
ized T cells upon antigenic stimulation, activated Th1
and Th2 cells were used (Fig. 6D). In the inactivated
state, SAP expression is much greater in Th1 cells
than in Th2 cells, while SLAM displays a comparable
distribution in these cells. When Th1 and Th2 cells
were stimulated for 6 h with plate-bound anti-CD3e in
the presence of hIL-2, SAP expression decreased in
both Th1 and Th2 cells. In contrast, the expression of
SLAM was markedly augmented in activated Th1 cells
while no considerable up-regulation of SLAM was
observed in activated Th2 cells. Thus, expression of
the SAP and SLAM transcripts are regulated in an
entirely different fashion, allowing for different rel-
ative amounts of SAP or SLAM within the cell,
depending upon its activation status.
Discussion
The mouse SAP cDNA encodes a protein of 126
amino acids whose sequence is highly homologous
with human SAP (Sayos et al. 1998) and its SH2
domain shares approximately 45% and 35% sequence
identity with that of SHIP and Abl, respectively. SAP
also shares about 40% sequence homology with
EAT-2, a protein expressed in response to cellular
transformation (Thompson et al. 1996). Similar to
SAP, EAT-2 contains an SH2 domain followed by a
short C-terminal tail. But it is expressed in different
mouse tissues (Thompson et al. 1996). The structural
similarity between SAP and EAT-2 suggests a compa-
rable mechanism of action and thus the existence of a
SAP-like protein family, which defines natural inhibi-
tors of docking sites of SH2 domains.
The X chromosome is a major repository of genes
that must be crucial to immune function given that
many immunodeficiencies are X-linked. Our FISH
result showed that mouse SAP maps to chromosome
XA5, a region that is syntenic to human chromosome
Xq25 to which the XLP gene has been assigned (Har-
ris et al. 1988; Lamartine et al. 1996; Lanyi et al. 1997;
 813

Fig. 6 Regulation of SAP in

T cells. A,B Down-regulation
of SAP induced by in vitro
activation of T cells by plate-
bound anti-CD3 is dem-
onstrated by both Northern
(A) and Western (B) blot
analyses. C SAP is also down-
regulated in CD4+ and CD8+
single-positive (SP) T cells.
Splenic T cells or SP cells
were purified with MACS col-
umns and activated by plate-
bound anti-CD3 (2C11) at 10
g/ml. At the different time
points indicated, cells were
collected and total RNA pre-
pared. SAP mRNA levels
were assayed by the DIG RT-
PCR method. D Northern blot
shows that, in Th1 and Th2
cells, SAP was down-regulated
upon T-cell activation in vitro.
The phenotypes of Th1 and
Th2 cells were confirmed by
IFN-g and IL-4 Northern blot
analyses

Monier et al. 1998; Sanger et al. 1990; Sylla et al. 1989;
Wang et al. 1993). Indeed, that information prompted
us to examine XLP patients for mutations in the SAP
gene (Sayos et al. 1998). Not surprisingly, the organi-
zation of the mouse SAP gene is identical to that pub-
lished for the human gene (Coffey et al. 1998).
In XLP patients, mutations have been identified in
all four exons of the SAP gene, as well as in intronic
regions (Brandau et al. 1999; Coffey et al. 1998; Nich-
ols et al. 1998; Sayos et al. 1998). We have recently
identified a point mutation in the 59 promoter/regula-
tory region (M. Morra and C. Terhorst, unpublished
data). The sequence analysis of the 59-flanking region
of the mouse SAP gene revealed the heterogeneous
transcription factor-binding sites, among which or
those important for T-cell development and function
is a c-Ets-1 site 11 bp upstream of the transcription
start site, which is also found in the similar region of
the human SAP gene. Ets-1 is preferentially expressed
at high levels in B and T cells of adult mice and is
regulated during both thymocyte development and
T-cell activation, being required for the normal sur-
vival and activation of mouse T cells (Ho et al. 1990).
In addition, resting T cells express high levels of
c-Ets-1 mRNA and protein. However, upon T-cell
activation, c-Ets-1 gene expression decreases to very
low levels (Bhat et al. 1990). Since SAP is also rapidly
down-regulated in activated T cells, Ets-1 is possibly
involved in the regulation of SAP transcription. There
are also multiple GR-binding sites found throughout
814
the 1.2 kb sequence in the 59 regulatory region of the
mouse SAP gene. GR involvment in T-cell devel-
opment (Sacedon et al. 1999a, 1999b; Vacchio and
Ashwell 1997), T-cell apoptosis (Ramdas et al. 1999),
and cytokine production (Faunce et al. 1998; Mozo et
al. 1998; Visser et al. 1998) is well documented. In
addition, an IRF-1 site was also found, which regulates
transcription of many cytokines and thus lymphocyte
development (Matsuyama et al. 1993). Our data show-
ing that SAP is expressed constitutively in resting T
cells suggests a T cell-specific transcription regulation,
and more detailed analysis will be required to address
which transcription factor is specifically responsible
for promoting SAP transcription.
Our results showed that SAP expression is rapidly
down-regulated in in vitro-activated T cells, including
the Th1 and Th2 subsets. This indicates that in mature
T cells, SAP expression may be modulated at least in
part by certain T-cell activation molecules. SLAM, by
contrast, is up-regulated early in T-cell activation.
Typical mRNA degradation signals (AUUUA) are
found in the 39UTR of both mouse and human SAP
(Fig. 2), but not in SLAM sequences. The AUUUA
motif is a highly conserved sequence in the 39UTR of
short-lived RNAs encoded by many cytokine genes
and oncogenes, and has been demonstrated to induce
rapid mRNA degradation (Chen and Shyu 1995). In
certain cases, such as IFN-b, the AUUUA motif can
bind to other intracellular protein(s) to form a mRNA
degradation complex, which is up-regulated in
response to viral induction (Raj and Pitha 1993).
Therefore, even though SAP is not transiently
expressed, it is rapidly degraded, possibly by up-regu-
lated mRNA degradation protein(s) upon T-cell acti-
vation. Thus, SAP is likely regulated through multiple
mechanisms, at the transcriptional level by transcrip-
tion factors such as Ets-1, and at the post-transcrip-
tional level by mRNA degradation molecules.
SAP is expressed in all major T-cell subsets, with
highest expression in the DP, and lowest in the DN
population. SAP is expressed at much higher levels in
the thymus of CD3gd±/± mice than in that of RAG±/±
or SCID mice. This suggests that the developmental
arrest from the DN to DP stage in the CD3gd±/± mice
may be partial and incomplete, when compared to
RAG±/± and SCID mice. In humans, SAP is not
expressed in B cells isolated from healthy donors, but
detectable by RT-PCR in EBV-transformed B cells
(Coffey et al. 1998; Nichols et al. 1998; Sayos et al.
1998). Our results show that SAP expression was also
detected in spleen and lymph nodes of all three
mutant mice. This suggests that SAP is expressed in
natural killer (NK) cells, and possibly other cell types
in the periphery except B cells. In fact, we have found
that SAP is expressed in NK cells, and that it binds to
the NK cell surface receptor 2B4 (J. Sayos, K.B.
Nguyen, C. Wu, S.E. Stepp, D. Howie, I.D. Schatzle,
V. Kumar, C.A. Biron, C. Terhorst, unpublished data).
Moreover, our preliminary data indicate that SAP is
not expressed in activated mouse dendritic cells,
whereas SLAM can be induced in dendritic cells (data
not shown).
The level of SAP expression in Th1 cells is much
greater than in Th2 cells. Thus, Th1 cells may be
involved more substantially in response to EBV infec-
tion. The diminished inhibition of EBV-infected B-cell
growth was previously shown to be correlated with the
decreased IFN-g production observed in T cells, sug-
gesting that suppressor cells may be activated in the
patients with XLP, but the frequent development of
B-cell lymphoma indicates that immunoglobulin sup-
pression may not exert enough pressure on the in vivo
growth of EBV-infected B cells. Perhaps defective
T-cell responses, in the absence of SAP, cause
impaired regulation of B-cell function, through cyto-
kines and other mediators. The decreased secretion of
IFN-g may be, at least partially, responsible for the
abnormal T-cell activity found in patients with XLP
(Yasuda et al. 1991). Since engagement of SLAM
induces IFN-g production and redirects the Th2 phe-
notype to a Th1/Th0 phenotype (Carballido et al.
1997), an impaired SLAM/SAP signal transduction
pathway in XLP patients would likely cause an unbal-
anced differentiation of Th cells and cytokine produc-
tion profile, which in turn would lead to uncontrolled
B-cell proliferation.
Acknowledgements We thank Dr. Linda Clayton for providing
the mouse (C57BL/6) thymus library. This work is supported by
grant AI-35714 from the NIH. Chengbin Wu is supported by a
grant from the Cancer Research Institute. Joan Sayos is sup-
ported by a fellowship of the Ministerio de Education y Cultura
de Espana. Duncan Howie is supported by a fellowship grant
from the Leukemia Society.
References
Bhat NK, Thompson CB, Lindsten T, June CH, Fujiwara S, Koi-
zumi S, Fisher RJ, Papas TS (1990) Reciprocal expression of
human ETS1 and ETS2 genes during T-cell activation: regu-
latory role for the protooncogene ETS1. Proc Natl Acad Sci
USA 87:3723±3727
Brandau O, Schuster V, Weiss M, Hellebrand H, Fink FM,
Kreczy A, Friedrich W, Strahm B, Niemeyer C, Belohradsky
BH, Meindl A (1999) Epstein-Barr virus-negative boys with
non-Hodgkin lymphoma are mutated in the SH2D1A gene,
as are patients with X-linked lymphoproliferative disease
(XLP). Hum Mol Genet 8:2407±2413
Carballido JM, Aversa G, Kaltoft K, Cocks BG, Punnonen J,
Yssel H, Thestrup-Pedersen K, Vries JE de (1997) Reversal
of human allergic T helper 2 responses by engagement of sig-
naling lymphocytic activation molecule. J Immunol
159:4316±4321
Chen CY, Shyu AB. (1995) AU-rich elements: characterization
and importance in mRNA degradation. Trends Biochem Sci
20:465±470
Chen J, Shinkai Y, Young F, Alt FW (1994) Probing immune
functions in RAG-deficient mice. Curr Opin Immunol
6:313±319
Cocks BG, Chang CC, Carballido JM, Yssel H, Vries JE de,
Aversa G (1995) A novel receptor involved in T-cell activa-
tion. Nature 376:260±263

Coffey AJ, Brooksbank RA, Brandau O, Oohashi T, Howell
GR, Bye JM, Cahn AP, Durham J, Heath P, Wray P, Pavitt
R, Wilkinson J, Leversha M, Huckle E, Shaw-Smith CJ,
Dunham A, Rhodes S, Schuster V, Porta G, Yin L, et al.
(1998) Host response to EBV infection in X-linked lymphop-
roliferative disease results from mutations in an SH2-domain
encoding gene. Nat Genet 20:129±135
Faunce DE, Gregory MS, Kovacs EJ (1998) Glucocorticoids
protect against suppression of T cell responses in a murine
model of acute ethanol exposure and thermal injury by regu-
lating IL-6. J Leukoc Biol 64:724±732
Harris A, Lenoir GM, Lankester SA (1988) X-linked lymphop-
roliferative disease: linkage studies using DNA probes. Clin
Genet 33:162±168
Ho IC, Bhat NK, Gottschalk LR, Lindsten T, Thompson CB,
Papas TS, Leiden JM (1990) Sequence-specific binding of
human Ets-1 to the T cell receptor alpha gene enhancer.
Science 250:814±818
Lamartine J, Nichols KE, Yin L, Krainer M, Heitzmann F, Ber-
nard A, Gaudi S, Lenoir GM, Sullivan JL, Ikeda JE, Porta
G, Schlessinger D, Romeo G, Haber DA, Sylla BS, Harkin
DP (1996) Physical map and cosmid contig encompassing a
new interstitial deletion of the X-linked lymphoproliferative
syndrome region. Eur J Hum Genet 4:342±351
Lanyi A, Li B, Li S, Talmadge CB, Brichacek B, Davis JR,
Kozel BA, Trask B, Engh G van den, Uzvolgyi E, Stan-
bridge EJ, Nelson DL, Chinault C, Heslop H, Gross TG,
Seemayer TA, Klein G, Purtilo DT, Sumegi J (1997) A yeast
artificial chromosome (YAC) contig encompassing the criti-
cal region of the X-linked lymphoproliferative disease (XLP)
locus. Genomics 39:55±65
Matsuda Y, Chapman VM (1995) Application of fluorescence in
situ hybridization in genome analysis of the mouse. Electro-
phoresis 16:261±272
Matsuyama T, Kimura T, Kitagawa M, Pfeffer K, Kawakami T,
Watanabe N, Kundig TM, Amakawa R, Kishihara K, Wake-
ham A (1993) Targeted disruption of IRF-1 or IRF-2 results
in abnormal type I IFN gene induction and aberrant lympho-
cyte development. Cell 75:83±97
Mikhalap SV, Shlapatska LM, Berdova AG, Law CL, Clark
EA, Sidorenko SP (1999) CDw150 associates with src-ho-
mology 2-containing inositol phosphatase and modulates
CD95-mediated apoptosis. J Immunol 162:5719±5727
Monier K, Michalet X, Lamartine J, Schurra C, Heitzmann F,
Yin L, Cinti R, Sylla BS, Creaven M, Porta G, Vourc©h C,
Robert-Nicoud M, Bensimon A, Romeo G (1998) High-res-
olution mapping of the X-linked lymphoproliferative syn-
drome region by FISH on combed DNA. Cytogenet Cell
Genet 81:259±264
Mozo L, Gayo A, Suarez A, Rivas D, Zamorano J, Gutierrez C
(1998) Glucocorticoids inhibit IL-4 and mitogen-induced
IL-4R alpha chain expression by different posttranscriptional
mechanisms. J Allergy Clin Immunol 102:968±976
Nichols KE, Harkin DP, Levitz S, Krainer M, Kolquist KA,
Genovese C, Bernard A, Ferguson M, Zuo L, Snyder E,
Buckler AJ, Wise C, Ashley J, Lovett M, Valentine MB,
Look AT, Gerald W, Housman DE, Haber DA (1998) Inac-
tivating mutations in an SH2 domain-encoding gene in
X-linked lymphoproliferative syndrome. Proc Natl Acad Sci
USA 95:13765±13770
Pla M, Mahouy G (1991) The SCID mouse. Nouv Rev Fr
Hematol 33:489±491
Poy F, Yaffe MB, Sayos J, Saxena K, Morra M, Sumegi J, Can-
tley LC, Terhorst C, Eck MJ (1999) Crystal structures of the
XLP protein SAP reveal a class of SH2 domains with
extended, phosphotyrosine-independent sequence recogni-
tion. Mol Cell 4:555±561
View publication stats
815
Raj NB, Pitha PM (1993) 65-kDa protein binds to destabilizing
sequences in the IFN-beta mRNA coding and 39 UTR.
FASEB J 7:702±710
Ramdas J, Liu W, Harmon JM (1999) Glucocorticoid-induced
cell death requires autoinduction of glucocorticoid receptor
expression in human leukemic T cells. Cancer Res
59:1378±1385
Sacedon R, Vicente A, Varas A, Jimenez E, Munoz JJ, Zapata
AG (1999a) Early maturation of T-cell progenitors in the
absence of glucocorticoids. Blood 94:2819±2826
Sacedon R, Vicente A, Varas A, Morale MC, Barden N, Mar-
chetti B, Zapata AG (1999b) Partial blockade of T-cell dif-
ferentiation during ontogeny and marked alterations of the
thymic microenvironment in transgenic mice with impaired
glucocorticoid receptor function. J Neuroimmunol
98:157±167
Sanger WG, Grierson HL, Skare J, Wyandt H, Pirruccello S,
Fordyce R, Purtilo DT (1990) Partial Xq25 deletion in a
family with the X-linked lymphoproliferative disease (XLP).
Cancer Genet Cytogenet 47:163±169
Sayos J, Wu C, Morra M, Wang N, Zhang X, Allen D, Schaik S
van, Notarangelo L, Geha R, Roncarolo MG, Oettgen H,
Vries JE de, Aversa G, Terhorst C (1998) The X-linked lym-
phoproliferative-disease gene product SAP regulates signals
induced through the co-receptor SLAM. Nature 395:462±469
Schug J, Overton GC (1997) TESS: transcription element search
software on the WWW. Technical Report CBIL-
TR-1997-1001-v0.0. Computational Biology and Informatics
Laboratory, School of Medicine, University of Pennsylvania
Seemayer TA, Gross TG, Egeler RM, Pirruccello SJ, Davis JR,
Kelly CM, Okano M, Lanyi A, Sumegi J (1995) X-linked
lymphoproliferative disease: twenty-five years after the dis-
covery. Pediatr Res 38:471±478
Shi YP, Mohapatra G, Miller J, Hanahan D, Lander E, Gold P,
Pinkel D, Gray J (1997) FISH probes for mouse chromo-
some identification. Genomics 45:42±47
Sylla BS, Wang Q, Hayoz D, Lathrop GM, Lenoir GM (1989)
Multipoint linkage mapping of the Xq25±q26 region in a
family affected by the X-linked lymphoproliferative syn-
drome. Clin Genet 36:459±462
Thompson AD, Braun BS, Arvand A, Stewart SD, May WA,
Chen E, Korenberg J, Denny C (1996) EAT-2 is a novel
SH2 domain containing protein that is up regulated by
Ewing©s sarcoma EWS/FLI1 fusion gene. Oncogene
13:2649±2658
Vacchio MS, Ashwell JD (1997) Thymus-derived glucocorticoids
regulate antigen-specific positive selection. J Exp Med
185:2033±2038
Visser J, Boxel-Dezaire A van, Methorst D, Brunt T, Kloet ER
de, Nagelkerken L (1998) Differential regulation of interleu-
kin-10 (IL-10) and IL-12 by glucocorticoids in vitro. Blood
91:4255±4264
Wang B, Wang N, Salio M, Sharpe A, Allen D, She J, Terhorst
C (1998) Essential and partially overlapping role of
CD3gamma and CD3delta for development of alphabeta and
gammadelta T lymphocytes. J Exp Med 188:1375±1380
Wang Q, Ishikawa-Brush Y, Monaco AP, Nelson DL, Caskey
CT, Pauly SP, Lenoir GM, Sylla BS (1993) Physical mapping
of Xq24±25 around loci closely linked to the X-linked lym-
phoproliferative syndrome locus: an overlapping YAC map
and linkage between DXS12, DXS42, and DXS37. Eur J
Hum Genet 1:64±71
Yasuda N, Lai PK, Rogers J, Purtlo DT (1991) Defective con-
trol of Epstein-Barr virus-infected B cell growth in patients
with X-linked lymphoproliferative disease. Clin Exp Immu-
nol 83:10±16