CD40 Ligand Mutants Responsible for X-linked Hyper-IgM
Syndrome Associate with Wild Type CD40 Ligand*
Kuniaki
Seyama
§,
William R. A.
Osborne
, and
Hans D.
Ochs
¶
From the
Department of Pediatrics, University of
Washington School of Medicine, Seattle, Washington 98195 and the
§ Department of Respiratory Medicine, Juntendo University
School of Medicine, 2-1-1, Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
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ABSTRACT |
CD40 ligand (CD40L) is a 33-kDa type II membrane
glycoprotein mainly expressed on activated CD4+ T
cells in trimeric form. When it is mutated, the clinical consequences are X-linked hyper-IgM syndrome (XHIM), a primary immunodeficiency disorder characterized by low levels of IgG, IgA, and elevated or
normal levels of IgM. Mutated CD40L can no longer bind CD40 nor provide
signals for B cells to proliferate and to switch from IgM to other
immunoglobulin isotypes. When considering gene therapy for XHIM, it is
important to address the possibility that the mutated CD40L associates
with transduced wild type CD40L, and as a consequence, immune
reconstitution is not attained. In this study, we demonstrate that the
various mutated CD40L species we have identified in patients with XHIM,
including both full-length and truncated mutants, associate with wild
type CD40L on the cell surface of co-transfected COS cells. The
association between wild type and mutated CD40L was also observed in
CD4+ T cell lines established from XHIM patients with leaky
splice site mutations. The clinical phenotype of these patients
suggests that this association between wild type and mutated CD40L
species may result in less efficient cross-linking of CD40.
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INTRODUCTION |
CD40 ligand (CD40L)1
(CD154, gp39, or TRAP) is a type II membrane glycoprotein, consisting
of 261 amino acid residues, and is expressed mainly on activated
CD4+ T cells (1, 2). The natural receptor for CD40L is
CD40, a member of the TNF receptor superfamily, expressed on a variety of cells including B cells, macrophages/monocytes, dendritic cells, vascular endothelial cells, and epithelial cells (3). The interaction between CD40L and CD40 therefore plays a crucial role in the immune system (3, 4). The cross-linking of CD40 by CD40L induces a signal for
B cells to undergo proliferation and immunoglobulin isotype switching
and to escape apoptosis (3, 4). In addition, CD40L-CD40 interaction
influences many aspects of T cell-mediated inflammatory responses, such
as up-regulation of adhesion molecules, cell extravasation, production
of inflammatory cytokines and chemokines, as well as activation of
macrophage effector function (3).
The physiologic significance of the CD40L-CD40 interaction has been
underscored by the observation that mutations of the CD40L gene cause X-linked hyper-IgM syndrome (XHIM) (5-9), a primary immunodeficiency disorder characterized by low or absence of IgG, IgA,
and IgE and normal or elevated IgM. Mutations of the CD40L gene identified in XHIM patients are highly heterogeneous. They include
missense, nonsense, and splice site mutations, and insertions or
deletions (10, 11), and are distributed throughout the CD40L
gene which consists of 5 exons and 4 introns and spreads over 12 kilobase pairs in genomic DNA (12, 13). More than 75 unique mutations
have been reported to date (10, 11). In most instances, the mutated
CD40L on the cell surface of activated T cells is undetectable if
anti-CD40L monoclonal antibodies (mAbs) or CD40-Ig, a fusion protein
consisting of the extracellular domain of CD40 and the Fc portion of
human immunoglobulin G, are used. However, if a polyclonal anti-CD40L
antiserum is used, the expression of mutated CD40L by activated T cells
is detected in the majority of XHIM patients (11).
CD40L is a member of the TNF superfamily that includes TNF-
,
TNF-
, Fas ligand (FasL), and CD30 ligand (CD30L) (14). Based on
x-ray crystallographic structures available for TNF-
(15, 16),
TNF-
(17, 18), and CD40L (19), it appears that members of the TNF
superfamily characteristically form homotrimers consisting of three
monomers, each folding with a "jelly roll" topology (15, 16, 19).
Most of the TNF superfamily members exist in both membrane-anchored and
soluble forms due to proteolytic cleavage (20-22), except for the
TNF-
homotrimer which is expressed only as soluble form.
Heterotrimer formation is known only among lymphotoxin
and TNF-
(23, 24). Each trimer, e.g. TNF-
and CD40L, can bind
three molecules of the counter-receptor along the surface groove
between two adjacent subunits (18, 25, 26), leading to receptor
clustering or aggregation required for activation signaling into the
target cells (18, 27, 28).
When considering gene therapy for XHIM patients, the possibility of an
association between the transduced wild type CD40L and the patient's
mutated CD40L resulting in heterotrimer formation has to be addressed.
Since two CD40L monomers contribute to form one functional CD40-binding
site (25, 26), the association of wild type CD40L monomer with mutated
CD40L monomer is expected to generate decreased numbers of CD40-binding
sites and, consequently, render the heterotrimer less efficient in
clustering CD40. In this study, the association of wild type CD40L with
various mutated CD40L species isolated from XHIM patients is
demonstrated in transfected COS cells and in activated T cell lines
established from XHIM patients with different splice site mutations.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Antibodies--
COS cells, murine
IgG1 anti-human CD40L monoclonal antibody 106 (mAb 106),
and biotinylated mAb 106 (bio-106), rabbit anti-human CD40L antiserum,
and the CD40-Ig construct consisting of the extracellular domain of
CD40 fused with the Fc region of human IgG1 were provided by Dr. Diane Hollenbaugh (Bristol-Myers Squibb). COS cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated fetal calf serum (FCS) (HyClone, Logan, UT),
2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml of streptomycin. Murine anti-CD40L mAb 5c8 (IgG2a) and Rb784,
a rabbit antiserum (27) recognizing the N-terminal 15 amino acids of CD40L, were provided by Biogen Inc. (Cambridge, MA). Murine anti-Flag mAbs, M5 (IgG1), and biotinylated M5 (bio-M5), were
purchased from Eastman Kodak Co.). Murine anti-human FasL mAb NOK1
(IgG1) (28) was a generous gift from Dr. Hideo Yagita
(Juntendo University, Tokyo, Japan) and murine anti-human CD30L mAbs,
M80 (IgG2b), M81 (IgG2b), and M82
(IgG2a) were provided by Immunex Corp. (Seattle, WA).
Interleukin-2-dependent CD4+ T cell lines
(>90% CD4+) were prepared from T cell lines established
from XHIM patients using a magnetic cell sorting system (Miltenyi
Biotec Inc., Auburn, CA) and maintained in Yssel's medium (Gemini
Biological Products, Calabasas, CA) supplemented with 8%
heat-inactivated FCS, 2% heat-inactivated human AB serum (Gemini
Biological Products), 100 units/ml penicillin, and 100 µg/ml
streptomycin according to standard methods.
Construction of Expression Plasmid--
A schematic
representation of the protein structures we expressed in COS cells and
the arbitrary designation of each plasmid and corresponding protein are
shown in Fig. 1 and Table
I, respectively. All of the expression
plasmids were constructed by reverse transcription-polymerase chain
reaction (RT-PCR) with Pfu DNA polymerase (Stratagene, La Jolla, CA) using cDNA isolated from activated peripheral blood mononuclear cells derived from a healthy volunteer or from selected XHIM patients (11). The human CD40L cDNA consisting of nt 1-807 (nucleotide numbering is based on the sequence data of Diane
Hollenbaugh et al. (1)) was amplified by RT-PCR using sense
primer (SP) 1 (5'CGCGGATCCATTTCAACTTTAACACAGC3',
recognition sequence underlined) and antisense primer (AP)
1 (5'GCGCTCGAGTCAGAGTTTGAGTAAGCCAAAGG3'), and
subsequently cloned into BamHI and
XhoI sites of pcDNA3.1/Zeo(+) (Invitrogen, Carlsbad,
CA). Naturally occurring mutant cDNAs, exon 2-skipped cDNA, 19 nucleotides of intron 2-inserted cDNA, and exon 3-skipped cDNA,
were similarly cloned into pcDNA3.1/Zeo(+) using cDNA generated
from the appropriate XHIM patients. The expression vector of the CD40L
lacking the cytoplasmic domain (CytDel,
Met21-Leu261) was generated using SP2
(5'CGCGGATCCATTTCAACTTTAACACAGCATGAAAATTTTTATGTATTTAC3') and AP1 and cloned into BamHI and XhoI sites of
pcDNA3.1/Zeo(+). The expression vectors of Flag-tagged wild type
and the naturally occurring mutant CD40L were generated by RT-PCR using
primers SP3
(5'CGCGGATCCATTTCAACTTTAACACAGCATGGATTACAAGGACGATGACGACAAGATCGAAACATACAACCAAACTTC3') and AP1, cloned into the same vector; Flag peptides consisting of
DKYDDDDL were inserted between Met1 and Ile2 of
wild type or mutant CD40L. In constructing pF-L258S, AP2
(5'GCGCTCGAGTCAGAGTTTGAGTGAGCCAAAGG3') was designed to have
a missense mutation within the antisense primer sequence and was used
for amplification instead of AP1. The Flag-tagged expression vector,
pF-Stalk, expressing the remainder of the CD40L molecule after the
extracellular domain of CD40L (Gln114-Leu261)
had been cleaved off (20, 21) was constructed using SP3 and AP3
(5'GCGCTCGAGTCACTACATTTCAAAGCTGTTTTCTTTC3'). The control expression vectors, pF-HybFasL and pF-HybCD30L, expressing a
Flag-tagged fusion protein consisting of the cytoplasmic and
transmembrane domains of CD40L and the extracellular domain of FasL
(Gln103-Leu281) (29) and CD30L
(Gln63-Asp234) (30), respectively, were
constructed as follows. The coding sequence of cytoplasmic and
transmembrane domains of CD40L was amplified by RT-PCR using SP4
(5'ATAAGAATGCGGCCGCATTTCAACTTTAACACAGCATGGATTACAAGGACGATGACGACAAGATCGAAACATACAACCAAACTTC3') and AP4 (5'CGCGGATCCCGAAGATACACAGCAAAAAGTGCTG3') and
subsequently cloned into NotI and BamHI sites of
pcDNA3.1/Zeo(+). The coding sequences of the extracellular domain
of the FasL and CD30L were amplified by PCR using SP5
(5'CGCGGATCCACAGCTCTTCCACCTACAGAAGGAG3') and AP5
(5'GCGCTCGAGTTAGAGCTTATATAAGCCGAAAAACGTCTG3') for FasL and
SP6 (5'CGCGGATCCACAGAGGACGGACTCCATTCC3') and AP6
(5'GCGCTCGAGTCAGTCTGAATTACTGTATAAG3') for CD30L,
respectively, and then fused into BamHI and XhoI
sites of the pcDNA3.1/Zeo(+) into which the coding sequence of the
cytoplasmic and transmembrane domains of CD40L had already been cloned.
All expression vectors were sequenced to verify the correct nucleotide sequences.

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Fig. 1.
Schematic presentation of the constructs
including wild type CD40L, naturally occurring mutant CD40Ls, and
control constructs used in this study. Shown are the contribution
of each exon of the CD40L gene to the domain structure: IC,
intracellular tail; TM, transmembrane domain;
ECU, extracellular unique region; and TNFH, TNF
homology domain. The number on the right side of
the protein structure represents the amino acid number where each
domain starts or the one where each protein or truncated ECU domain
ends. In order to discriminate wild type CD40L both by protein size and
by antigenicity from various mutant CD40Ls, a Flag peptide (DKYDDDDL)
was inserted at the N terminus between the first amino acid methionine
and the second amino acid isoleucine of wild type CD40L. Flag-tagged
proteins are represented by the "F-" preceding each protein name.
The constructs designed for this study included the following: cartoon
1, wild type CD40L; 2, CD40L lacking the
intracellular tail and starting at Met21 (CytDel);
3, Flag-tagged wild type CD40L (F-Wild); 4, Flag-tagged CD40L with the extracellular part of CD40L consisting of
Gln114-Leu261 (soluble CD40L) cleaved off
(F-Stalk); 5, Flag-tagged mutant CD40Ls with one or two
amino acid substitutions in TNFH selected from patients with XHIM,
including F-DM (double mutations resulting in S128R/E129G), F-T147N,
F-Y170C, F-G227V, F-A235P, F-T254M, and F-L258S; 6 and
7, Flag-tagged mutant CD40Ls with premature termination
selected from patients with XHIM, representing F-W140X and
F-Q186X, respectively; 8, mutant CD40L with
in-frame deletion of 44 amino acids encoded by exon 2 and identified in
XHIM patients with intron 2 splice donor site mutation (E2skip);
9 and 10, a truncated mutant CD40L without or
with the N terminus Flag peptide (E2ins and F-E2ins, respectively),
resulting from the translation of the mRNA transcripts carrying the
19 nucleotides' insertion of intron 2 and identified in XHIM patients
with intron 2 splice donor site mutation; 11 and
12, a truncated mutant CD40L without or with the N terminus
Flag peptide (E3skip and F-E3skip, respectively), resulting from the
translation of exon 3-skipped mRNA transcripts identified in XHIM
patients with intron 3 splice donor site mutation; 13, Flag-tagged control protein construct (F-HybFasL or F-HybCD30L),
representing the hybrid protein in which the extracellular region of
the human FasL (from Gln103 to Leu281) or of
human CD30L (from Gln63 to Asp234) was fused to
the IC and TM of CD40L. Both F-HybFasL and F-HybCD30L contain three
extra amino acids, RDP, at the fusion site. See also Table I; the
number of each cartoon shown here corresponds to that in Table I.
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Table I
Expression plasmids and structural feature of proteins expressed by
plasmids
Expression plasmids and proteins expressed by them when transfected to
COS cells were named arbitrarily for convenience on explaining the
experimental results. See also Fig. 1 since the numbers in the
left column correspond to those of Fig. 1 in which structure of protein
is shown schematically.
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Transfection of COS Cells--
COS cells were electroporated
with supercoiled expression plasmid under the following conditions: 10 µg of plasmid and 4 × 106 cells were mixed in 0.4 ml of serum- and antibiotic-free DMEM and electroporated at 210 V and
960 microfarads using GenePulser (Bio-Rad). Forty eight hours after
electroporation, cells were harvested after incubation in
phosphate-buffered saline (PBS), 0.5% bovine serum albumin, 5 mM EDTA for 10 min and used either to examine surface
expression of the transduced gene product by flow cytometry or to
biochemically characterize the expressed proteins.
Surface Biotinylation and Immunoprecipitation of Proteins
Transiently Expressed by COS Cells--
Forty eight hours after
electroporation, cells were harvested as described above. When
co-transfection with an expression plasmid of wild type CD40L (pWild)
and of a CD40L mutant was intended, 5 µg of each plasmid was mixed
and electroporated under the same conditions. Cells were resuspended at
a concentration of 5 × 106/ml and kept for 30 min on
ice in lysis buffer containing 1% Triton X-100, 10 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 0.025% NaN3,
freshly supplemented with 200 µg/ml phenylmethylsulfonyl fluoride
(Sigma), 10 µg/ml aprotinin (Roche Molecular Biochemicals), 10 µg/ml leupeptin (Roche Molecular Biochemicals), and 10 mM
iodoacetamide (Sigma). For surface biotinylation, cells were washed
with PBS twice, suspended at 5 × 106/ml in PBS, and
Sulfo-NHS-biotin (Pierce) added to a final concentration of 500 µg/ml. Cells were incubated for 30 min with rotation at room
temperature and then lysed. The lysate was cleared by centrifugation at
14,000 × g for 10 min at 4 °C. Protein
concentration was measured using Bio-Rad DC Protein Assay (Bio-Rad) and
bovine
-globulin as standard. The lysate was adjusted to 200 µg of
protein in 200 µl of lysis buffer and precleared overnight with 20 µl of protein G-Sepharose or protein A-Sepharose (50% slurry)
(Pierce) at 4 °C, followed by immunoprecipitation with 3 µg of
specific antibody or 5 µg of CD40-Ig for 1.5 h at 4 °C. The
immune complexes were collected using 10 µl of protein G-Sepharose or
protein A-Sepharose. The Sepharose beads were washed three times with
0.1% Triton X-100, 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.025% NaN3, followed by a single
wash with 10 mM Tris-HCl (pH 7.5), 150 mM NaCl,
0.025% NaN3, and with 50 mM Tris-HCl (pH 6.8).
The Sepharose beads-absorbed immune complexes were treated with 20 µl
of 2× Laemmli's sample buffer containing
-mercaptoethanol and were
incubated for 5 min in boiling water. The eluted proteins were resolved
by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
N-Glycosidase F Treatment of the Immunoprecipitates--
The
Sepharose beads-absorbed immune complexes were suspended in 20 µl of
0.5% SDS, 0.1 M
-mercaptoethanol and incubated for 5 min in boiling water. After centrifugation, the supernatant was mixed
with or without 5 units of protease-free N-glycosidase F
(Roche Molecular Biochemicals) in a total volume of 60 µl containing 50 mM sodium phosphate buffer (pH 7.2), 1%
n-octyl glucoside (Roche Molecular Biochemicals) and
incubated for 18 h at 37 °C. The reaction mixture was then
treated with 12 µl of 6× Laemmli's sample buffer containing
-mercaptoethanol for 5 min in boiling water and resolved by
SDS-PAGE.
Detection of Expressed Proteins by Western
Blotting--
Following immunoprecipitation from the lysate of
transfected COS cells, proteins were resolved on SDS-PAGE and
transferred to an ImmobilonTM-P membrane (Millipore Corp., Bedford,
MA). The blotted membrane was incubated in a blocking solution
containing 5% blocking non-fat milk (Bio-Rad) in 20 mM
Tris-HCl (pH 7.6), 137 mM NaCl, 0.1% Tween 20 (TBS-T), for
2 h at room temperature or overnight at 4 °C and then probed
with a specific antibody at 2 µg/ml in blocking solution for 1 h
at room temperature. Membranes were washed three times with TBS-T and
then incubated with TBS-T containing 1:2000-diluted
streptavidin-horseradish peroxidase conjugate (Life Technologies, Inc.)
or 1:5000-diluted goat anti-rabbit immunoglobulin-horseradish
peroxidase conjugate (BioSource International) for
1 h at room temperature. After washing three times with TBS-T, proteins recognized by the specific antibody were visualized by the ECL
system (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Metabolic Labeling, Immunoprecipitation, and SDS-PAGE
Analysis--
Metabolic radiolabeling of activated
interleukin-2-dependent CD4+ T cell lines was
performed in Met/Cys-free RPMI 1640 medium (Sigma) supplemented with
10% dialyzed heat-inactivated FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin. CD4+ T cells were suspended at 5 × 106 cells/ml in labeling medium containing 0.2 mCi/ml
EXPRE35S35S (NEN Life Science Products) and
activated with 1 µg/ml ionomycin and 10 ng/ml phorbol 12-myristate
13-acetate (Sigma) for 4 h at 37 °C. Radiolabeled cells were
lysed at 5 × 107 cells/ml in lysis buffer. The
resultant lysate was precleared and immunoprecipitated as described
above. Proteins were resolved by 13% SDS-PAGE, and fluorography was
performed using ENTENSIFYTM (NEN Life Science Products) according to
the manufacturer's instruction. For metabolic radiolabeling of
transiently transfected COS cells, the cells were washed 36 h
after transfection with PBS and incubated in Met/Cys-free DMEM (Sigma)
supplemented with 5% dialyzed heat-inactivated FCS, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 0.2 mCi/ml
EXPRE35S35S for 4 h and processed similarly.
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RESULTS |
Surface Expression of Transduced Protein--
Surface expression
of wild type CD40L, mutant CD40L species, and control hybrid proteins
(F-HybFasL and F-HybCD30L) by transfected COS cells was confirmed by
flow cytometry (data not shown). Wild type CD40L and Flag-tagged wild
type CD40L (F-Wild) were expressed abundantly by transfected COS cells
and detected by mAb 5c8 as well as CD40-Ig construct, suggesting that
the Flag peptide at the N terminus does not affect the structure and
function of CD40L. Two unique truncated mutants, CytDel and E2skip,
both found in XHIM patients (11), were detected in transfected COS
cells using mAb 5c8 and the CD40-Ig construct, respectively. The
expression of other mutated CD40L species, including mutants with amino
acid substitutions and truncated mutants, was confirmed by the binding of a polyclonal anti-CD40L antiserum. Similarly, both F-HybFasL and
F-HybCD30L expressed by transfected COS cells were detected well by mAb
NOK1 and three different anti-CD30L mAb preparations (M80, M81, and
M82), respectively. Since NOK1 recognizes the antigenic epitope
consisting of two FasL monomers, it seems likely that F-HybFasL retains
the inherent structural integrity of FasL and forms a trimer. The
finding that F-HybCD30L expression was detectable by three different
anti-CD30L mAbs suggests that this hybrid construct also retains the
inherent structural integrities of CD30L and forms a trimer.
Biochemical Characterization of Wild Type CD40L Transiently
Expressed by COS Cells--
The biochemical characteristics of
proteins expressed by COS cells transfected with pWild, pCytDel, and
pF-Wild were analyzed by immunoprecipitation followed by Western
blotting. From COS cells transfected with pWild, CD40-Ig
immunoprecipitated two CD40L species, one with an apparent molecular
mass of 33 kDa (p33) and the other of 31 kDa (p31) when probed with mAb
bio-106. CD40Ls obtained from pCytDel-transfected cells were found to
have an apparent molecular mass of 29 (p29) and 27 kDa (p27), and those obtained from pF-Wild-transfected COS cells had a molecular mass of 34 (p34), 32 kDa (p32), and p29, respectively (Fig.
2A). Because CD40L has two
potential N-glycosylation sites, one in the cytoplasmic domain and the other in the C terminus of the TNF homology (TNFH) domain, N-glycosidase F treatment was performed to determine
whether glycosylation contributes to the existence of multiple species of CD40L. After deglycosylation, p31 and p27 were detected in pWild-transfected cells, a single band, p27, in pCytDel-transfected cells, and p32 and p27 in pF-Wild-transfected cells (Fig.
2A). These results suggest that COS cells express
full-length CD40L as well as CD40L lacking the cytoplasmic domain and
that both species exist in glycosylated (p33, p29, respectively) and
unglycosylated forms (p31, p27, respectively) when transfected with
pWild. This interpretation was further supported by the results of
probing the same preparation with Rb784 (Fig. 2B), an
antiserum recognizing only the cytoplasmic domain of CD40L; p29 and p27
were no longer detectable, and immunoprecipitation of
pCytDel-transfected cells failed to identify a band. The production of
F-Wild and CytDel both in glycosylated and in unglycosylated forms was
also noted in pF-Wild-transfected cells. As reported by others (27),
the CytDel form of CD40L occurs and associates with full-length CD40L in Jurkat cells that constitutively express CD40L and in COS cells transfected with the expression plasmid carrying a full-length CD40L
cDNA. Interestingly, Rb784 identified a band of 16 kDa (p16) in the
immunoprecipitates of pWild-transfected cells and an 18-kDa (p18) band
in pF-Wild-transfected cells (Fig. 2B). These small proteins
appear to be the N-terminal fragment of CD40L consisting of
Met1-Met113 generated after cleavage of the
extracellular domain known as soluble CD40L (20, 21); p18, observed in
the immunoprecipitate of pF-Wild-transfected COS cells, was detected by
anti-Flag mAb bio-M5 (Fig. 2C). As shown later (Fig. 5), a
protein derived from COS cells to which pF-Stalk, a plasmid expressing
Flag-tagged Met1-Met113, was transfected
migrated to the same position as the p18 band. Since the mobility of
p16 of pWild- and p18 of pF-Wild-transfected cells on SDS-PAGE did not
change following N-glycosidase F treatment (Fig. 2,
B and C), the postulated
N-glycosylation site of the intracellular domain of CD40L
does not seem to be glycosylated. These finding demonstrate that CD40L
forms a multiheteromeric complex consisting of full-length CD40L (wild
type CD40L or F-Wild) with or without glycosylation, a CD40L lacking
the cytoplasmic domain (CytDel) with or without glycosylation, and the
N terminus fragment of CD40L (Stalk or F-Stalk) and that CD40-Ig can
co-precipitate even Stalk and F-Stalk fragments that lack the
extracellular TNFH domain required for the binding of CD40 (5, 10, 19,
31).

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Fig. 2.
Detection of proteins in transfected COS
cells by immunoprecipitation and subsequent Western blotting. COS
cells were transfected with either pWild, pCytDel, or pF-Wild, and cell
lysates prepared 48 h after electroporation were subjected to
immunoprecipitation with CD40-Ig. Subsequently, each immunoprecipitate
was incubated with or without protease-free N-glycosidase F
(see "Experimental Procedures"); represents untreated and + treated immunoprecipitate. Samples were resolved by 13% SDS-PAGE,
transferred onto ImmobilonTM P membrane, and detected by either mAb
bio-106 (A), Rb784 antisera (B), or mAb bio-M5
(C). The sizes of the molecular mass standard proteins are
indicated as kDa on the left of each panel.
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Naturally Occurring Mutants of CD40L Co-precipitate with Wild Type
CD40L--
To study whether wild type CD40L and Flag-tagged naturally
occurring mutant CD40L species can associate with each other,
immunoprecipitation with CD40-Ig or with the anti-Flag mAb M5 was
performed using lysates from COS cells to which pWild and various
plasmids expressing different Flag-tagged mutant CD40L species were
co-transfected. To test the system, we co-transfected COS cells with
pWild and either pF-Wild, pF-HybFasL, or pF-HybCD30L and examined their association. As shown in Fig.
3A, F-Wild was detected by
bio-M5 in CD40-Ig-generated immunoprecipitates of lysates from pWild + pF-Wild co-transfected cells, but neither F-HybFasL nor F-HybCD30L was
detected in lysate from pWild + pF-HybFasL or pF-HybCD30L co-transfected cells, respectively. When a membrane with similarly treated samples was probed with mAb bio-106, we found wild type CD40L
in all lysates (Fig. 3B), demonstrating that neither
F-HybFasL nor F-HybCD30L associate with wild type CD40L. Similarly,
wild type CD40L was detected by mAb bio-106 in the mAb M5-generated immunoprecipitates only if pWild + pF-Wild were co-transfected but not if pWild + pF-HybFasL or pF-HybCD30L were co-transfected (Fig. 3C), whereas F-Wild, F-HybFasL, and F-HybCD30L were
successfully detected by mAb bio-M5 in the appropriate co-transfectant
(Fig. 3D). These results demonstrate that F-Wild associates
with wild type CD40L and that Flag peptide at the N terminus of CD40L
does not affect trimer formation of CD40L monomers.

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Fig. 3.
Immunoprecipitation and Western blotting of
the lysates from COS cells co-transfected with pWild and either
pF-Wild, pF-HybFasL, or pF-HybCD30L. The lysates of COS cells
co-transfected with pWild and either pF-Wild, pF-HybFasL, or
pF-HybCD30L were subjected to immunoprecipitation (IP)
with either CD40-Ig (A and B) or mAb M5
(C and D). After the immunoprecipitates were
resolved by 12% SDS-PAGE and transferred onto ImmobilonTM P membrane,
either mAb bio-M5 (A and D) or mAb bio-106
(B and C) was used to detect expressed proteins.
The sizes of the molecular mass standard proteins are indicated as kDa
on the left of each panel. Note that only F-Wild can
co-immunoprecipitate with wild type CD40L and that neither F-HybFasL
nor F-HybCD30L can.
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By using the same technique, naturally occurring mutants of CD40L were
co-transfected with wild type CD40L to test whether mutants can
associate with wild type CD40L. Seven mutants with a single or two
amino acid substitutions were studied (Table I). F-DM which has two
amino acid substitutions (S1128R/E129G) and F-T147N are Flag-tagged
mutants with amino acid substitutions at positions that directly
compromise the CD40-binding site (31). F-Y170C and F-G227V are
Flag-tagged mutants of CD40L with amino acid substitutions at positions
that are important for trimer formation (31). F-A235P, F-T254M, and
F-L258S are Flag-tagged mutants whose amino acid substitutions
interfere with monomer packaging and folding (31). As shown in Fig.
4A, all seven Flag-tagged missense mutants were expressed by singly transfected COS cells and
immunoprecipitated with anti-Flag mAb M5 from the lysate. In contrast,
six of seven Flag-tagged mutants failed to be immunoprecipitated with
CD40-Ig (Fig. 4B) as expected since these mutants were
isolated from patients with XHIM. F-T147N, which has a larger molecular mass than F-Wild and other Flag-tagged missense mutants due to the
generation of a cryptic N-glycosylation site by the amino acid substitution, was the only mutant detected in the
CD40-Ig-generated immunoprecipitate, although only as an extremely
faint band (Fig. 4B). In contrast, all mutants with amino
acid substitutions studied were detected (Fig. 4C) in
immunoprecipitates obtained with CD40-Ig if COS cells were
co-transfected with pWild and expression plasmids of Flag-tagged
missense mutants. Similarly, wild type CD40L was readily demonstrated
in immunoprecipitates obtained with mAb M5 from the lysate of each
co-transfectant (data not shown). These findings clearly demonstrate
that all mutants with amino acid substitutions studied associate with
wild type CD40L and that the resultant complexes, although they include
mutants that cannot bind CD40-Ig by themselves, can bind CD40-Ig. The
inability of these mutants to bind CD40-Ig appears not to be due to
their inability to form complexes by themselves; when plasmids carrying
Flag-tagged mutants and those carrying corresponding plain mutants were
co-transfected to COS cells, both Flag-tagged and plain mutants were
detected in the immunoprecipitates obtained with mAb M5 using an
anti-CD40L polyclonal antiserum (data not shown). This observation
implies that these mutants can form complexes themselves but fail to
generate binding sites for CD40.

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Fig. 4.
Immunoprecipitation and Western blotting of
the lysates from COS cells transfected with plasmids carrying missense
mutations or from COS cells co-transfected with pWild and plasmids
carrying missense mutations. A and B, COS cells
transfected with a plasmid expressing either F-Wild or Flag-tagged
mutant CD40L with an amino acid substitution in TNFH domain. The
lysates were immunoprecipitated (IP) with either mAb M5
(A) or CD40-Ig (B), resolved by 12% SDS-PAGE,
and transferred onto ImmobilonTM P membrane. mAb bio-M5 was used for
detection. The sizes of the molecular mass standard proteins are
indicated as kDa on the left of each panel. Note that all
Flag-tagged mutant CD40Ls with amino acid substitutions tested could be
detected in the immunoprecipitates obtained with mAb M5 (A)
but not in those obtained with CD40-Ig (B). However, F-T147N
mutant, which is slightly larger than other Flag-tagged proteins due to
the generation of a cryptic glycosylation site by the missense
mutation, showed minimal binding with CD40-Ig. C, lysates
from COS cells co-transfected with pWild and either pF-Wild or a
plasmid carrying Flag-tagged mutant CD40L with an amino acid
substitution were subjected to immunoprecipitation with CD40-Ig. The
immunoprecipitates were resolved by 12% SDS-PAGE, transferred onto
ImmobilonTM P membrane, and probed with mAb bio-M5. The sizes of the
molecular mass standard proteins are indicated as kDa on the
left of each panel. Note that all mutant CD40Ls with amino
acid substitutions tested could be detected in the immunoprecipitates
obtained with CD40-Ig when co-expressed with wild type CD40L
(C), although they could not be immunoprecipitated with
CD40-Ig when expressed alone in COS cells (see B).
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To investigate if truncated CD40L can associate with wild type CD40L,
we transfected COS cells with the following Flag-tagged truncated
mutants (Table I): F-Stalk; F-W140X, the most commonly identified nonsense mutant in XHIM patients (10); F-Q186X, a truncated mutant carrying a part of TNFH domain, found in XHIM patients
(11); F-E2ins, the dominant CD40L mutant found in XHIM patients with
intron 2 splice donor site mutations (11); and F-E3skip, the dominant
CD40L mutant found in XHIM patients with intron 3 splice donor site
mutations (11). Although all truncated CD40L mutants tested were
expressed by singly transfected COS cells and detected in the
immunoprecipitates obtained with mAb M5 (Fig.
5A), none was
immunoprecipitated with CD40-Ig, as expected (Fig. 5B).
However, all truncated mutants tested could be demonstrated in the
CD40-Ig-generated immunoprecipitates (Fig. 5C) if each expression plasmid of truncated mutant CD40L was co-transfected with pWild. These results suggest that the truncated CD40L species tested are able to associate with wild type CD40L. This was further confirmed by the observation that wild type CD40L was detected in the
immunoprecipitates obtained with mAb M5 from the lysates of
co-transfectants (data not shown).

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Fig. 5.
Immunoprecipitation and Western blotting of
the lysates from COS cells expressing a truncated CD40L or from COS
cells co-expressing wild type CD40L and a truncated CD40L.
A and B, COS cells transfected with a plasmid
expressing either F-Wild or Flag-tagged truncated mutant CD40L. The
lysates were immunoprecipitated (IP) with either M5
(A) or CD40-Ig (B), resolved by 12% SDS-PAGE,
and transferred onto ImmobilonTM P membrane. mAb bio-M5 was used for
detection. The sizes of the molecular mass standard proteins are
indicated as kDa on the left of each panel. Note that all
Flag-tagged truncated mutant CD40Ls tested could be detected in the
immunoprecipitates obtained with mAb M5 but not in those obtained with
CD40-Ig. C, the lysates of COS cells co-transfected with
pWild and either pF-Wild or a plasmid expressing a truncated mutant
CD40L were subjected to immunoprecipitation with CD40-Ig. The
immunoprecipitates were resolved by 12% SDS-PAGE, transferred onto
ImmobilonTM P membrane, and probed with mAb bio-M5. The sizes of the
molecular mass standard proteins are indicated as kDa on the
left of each panel. Note that all truncated mutant CD40Ls
tested could be detected in the immunoprecipitates obtained with
CD40-Ig when co-expressed with wild type CD40L (C), although
they could not be immunoprecipitated with CD40-Ig when expressed alone
in COS cells (see B).
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The Mutant CD40L Lacking the Exon 2-encoded Stalk Is Less Efficient
in forming a Complex with Wild Type CD40L--
The mutant E2skip,
generated by exon 2-skipping, is a unique truncated CD40L that can
still bind CD40-Ig since the entire TNFH domain is preserved. When
pE2skip and pF-E2ins (both products generated from patients with intron
2 splice donor site mutations) were co-transfected into COS cells, we
failed to demonstrate an association between these two mutants. F-E2ins
could not be identified by mAb bio-M5 in the CD40-Ig-generated
immunoprecipitates from lysates of pE2skip + pF-E2ins co-transfected
cells (Fig. 6, left panel),
whereas the association of wild type CD40L with F-E2ins was observed
(see Fig. 5C). Similarly, if COS cells were co-transfected with pE2skip and pF-E2ins, followed by the immunoprecipitate with mAb
M5, E2skip could not be detected by mAb bio-106 (data not shown). These
observations suggest that the stalk region, which is mainly encoded by
exon 2 of the CD40L gene, as well as the TNFH domain, play
an important role in the association of CD40L monomers. This conclusion
is further supported by the observation that the amount of E2skip found
in the mAb M5-generated immunoprecipitate from a pF-Wild + pE2skip
co-transfectant was very low (Fig. 6, right lane in
right panel) when compared with the amount of E2skip found
in the immunoprecipitate obtained with CD40-Ig by which wild type CD40L
and E2skip are independently immunoprecipitated regardless of
association (Fig. 6, left lane in right
panel).

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Fig. 6.
Co-expression of Flag-tagged wild type CD40L
and mutated CD40L (E2skip or F-E2ins) identified in a patient with
intron 2 splice donor site mutation. COS cells were singly
transfected or co-transfected with the plasmids indicated and the cell
lysates immunoprecipitated (IP) with either CD40-Ig or mAb
M5. The immunoprecipitates were resolved by 13% SDS-PAGE, transferred
onto ImmobilonTM P membrane, and detected by mAb bio-106 or mAb
bio-M5. Note that E2skip, a truncated mutant CD40L that lacks the exon
2-encoded extracellular stalk preserving the entire TNFH domain, could
be immunoprecipitated with CD40-Ig (middle panel and
left lane in right panel) but did not associate
with the truncated F-E2ins mutant (right lane in left
panel) (E2ins is co-expressed with E2skip in XHIM patients with
intron 2 splice donor site mutation), whereas wild type CD40L could
associate with the F-E2ins mutant (see Fig. 5C). In
addition, Flag-tagged wild type CD40L (F-Wild) and E2skip mutant
associate poorly with each other (right lane in right
panel) despite the fact that both have the entire TNFH
domain.
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Another interesting feature of the E2skip mutant is the fact that the
efficiency of E2skip to transduce a signal through CD40 appears to be
less than that of wild type CD40L if E2skip is anchored on cell
surface, although the binding of E2skip and CD40-Ig in the cell lysate
is normal. Although E2skip is efficiently recovered in
CD40-Ig-generated immunoprecipitates from transfected COS cells (Fig.
6, middle panel and left lane in right
panel), the staining intensity of pE2skip-transfected COS cells
with CD40-Ig (as shown by flow cytometry) is much lower than that of
pWild-transfected cells: whereas the mean fluorescence intensity (MFI)
of E2skip-expressing COS cells was 60.4, the MFI of wild type
CD40L-expressing COS cells was 159.6 when detected by CD40-Ig
(immunostained histograms obtained by flow cytometry not shown). On the
other hand, similar MFI were obtained for E2skip- and wild type
CD40L-expressing COS cells when mAb 5c8 was used for
detection (421.7 and 406.8, respectively). We hypothesize that the
weaker intensity of CD40-Ig binding by membrane-expressed E2skip, when
compared with the bright staining with mAb 5c8, is due to steric
hindrance resulting from the loss of the extracellular stalk which is
encoded mainly by exon 2.
Association Occurs When Mutant and Wild Type CD40L Are Co-expressed
and the Resultant Complex Is Present on the Cell Surface--
To
demonstrate that the association of mutated CD40L with wild type CD40L
occurs exclusively when they are co-expressed, we compared the
immunoprecipitation of lysates from co-transfectants with that of
lysate mixtures prepared from corresponding single transfectants. We
selected F-T147N and F-W140X, representing a CD40L with an
amino acid substitution and a CD40L that is truncated, respectively,
since both are expressed well when transfected into COS cells.
Following preclearance of lysate mixture with protein G-Sepharose beads
overnight at 4 °C and further incubation for 1 h at either 4 or
37 °C, immunoprecipitation with mAb M5 was performed. Since wild
type, F-Wild, and F-T147N are equally well recognized by mAb 106 (11)
and expressed by COS cells in glycosylated and unglycosylated form, the
discrimination between wild type, F-Wild, and the mutant is difficult.
We therefore treated the immunoprecipitates with
N-glycosidase F before resolving with SDS-PAGE. When
compared with the immunoprecipitate of COS cells co-transfected with
pWild and pF-Wild, only a trace (at 4 °C) or a very low amount (at
37 °C) of wild type CD40L was recovered from the immunoprecipitate
of the mixture of the lysates of each single transfectant (Fig.
7A), suggesting that
association of wild type CD40L and F-Wild occurs during co-expression
by COS cells but not during the preclearing or immunoprecipitation
process. However, a very small amount of wild type CD40L did associate with F-Wild at 37 °C (3rd lane from the left
in Fig. 7A). To test the association of mutants with wild
type CD40L, similar experiments were performed using F-T147N and
F-W140X. Similar to the observation made with wild type
CD40L and F-Wild, a strong association between wild type CD40L and
F-T147N occurred only in co-transfected cells but not if lysates of
singly transfected COS cells were mixed in vitro at 4 °C,
and only at very small quantities when mixed at 37 °C (Fig.
7B). No association between wild type CD40L and a truncated
mutant F-W140X was observed if mixed in vitro at
4 and at 37 °C, respectively (Fig. 7C).

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Fig. 7.
Lysates from co-transfected COS cells but not
mixtures of lysates from singly transfected COS cells show an
association between wild type CD40L and mutants. A,
comparing immunoprecipitates using expression plasmid pWild and
pF-Wild. COS cells were either co-transfected with pWild and pF-Wild,
singly transfected with pWild, or singly transfected with pF-Wild, and
lysates from each transfectant prepared (see "Experimental
Procedures"). The lysates from co-transfected COS cells were
precleared overnight with protein G-Sepharose at 4 °C and
subsequently immunoprecipitated (IP) with mAb M5. The
lysates from COS cells transfected with either pWild or pF-Wild were
mixed, precleared overnight at 4 °C, and the supernatant further
incubated either at 4 °C for 1 h (2nd lane from the
left) or at 37 °C for 1 h (3rd lane), and
immunoprecipitated with mAb M5. To indicate the position of wild type
CD40L and F-Wild, respectively, on SDS-PAGE, lysates of COS cells
transfected with pWild and with pF-Wild were immunoprecipitated with
mAb 106 and M5, respectively. All immunoprecipitates were subsequently
treated with N-glycosidase F to simplify the identification
of wild type CD40L and F-Wild, resolved by 12% SDS-PAGE, and detected
by mAb bio-106. Note that wild type CD40L could barely be detected in
the mixture of lysates from each corresponding single transfectant when
incubated at 4 °C, whereas a small amount of wild type CD40L could
be identified when incubated at 37 °C; in contrast a significant
amount of wild type CD40L was immunoprecipitated together with F-Wild
in lysates from the co-transfectant. B, comparing
immunoprecipitates using expression plasmid pWild and pF-T147N. This
experiment is similar to that shown in A. We used pF-T147N
instead of F-Wild to represent a CD40L mutant with an amino acid
substitution. The preparation and treatment of cell lysates was the
same as those described in A. Note that wild type CD40L
could barely be detected in the mixture of lysates from each
corresponding single transfectant when incubated at 4 °C, although a
small amount of wild type CD40L was found when incubated at 37 °C.
In contrast, larger amount of wild type CD40L was immunoprecipitated
together with F-T147N in lysates from the co-transfectant.
C, comparing immunoprecipitates using expression plasmid
pWild and pF-W140X. This experiment is similar to those
shown in A and B. We used pF-W140X as
the representative for a truncated CD40L caused by a nonsense mutation.
The preparation and treatment of cell lysates were the same as those in
A and B except that N-glycosidase F
treatment was not performed. Although wild type CD40L could be detected
in lysates from the co-transfectant, there was no wild type CD40L
demonstrable in the mixture of lysates from each corresponding single
transfectant even when incubated at 37 °C.
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To demonstrate that the complexes formed between wild type and mutated
CD40L are anchored in the cell membrane, surface biotinylation of the
transfectant was performed using membrane-impermeable Sulfo-NHS-biotin, followed by immunoprecipitation and Western blotting (Fig.
8). An association between wild type
CD40L and F-T147N or F-W140X, respectively, was observed on
the cell surface in both CD40-Ig- and mAb M5-generated
immunoprecipitates (Fig. 8, A and B,
respectively) from surface-biotinylated co-transfected COS cells.

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Fig. 8.
Surface biotinylation of transfected COS
cells followed by immunoprecipitation and Western blotting demonstrates
association on the cell surface. COS cells were transfected with
either pWild (1st lane from the left), pF-T147N
(2nd lane), pWild + pF-T147N (3rd lane),
pF-W140X (4th lane), or pWild + pF-W140X (5th lane) and biotinylated using
Sulfo-NHS-biotin which is membrane-impermeable (see "Experimental
Procedures"). After cells were lysed, expressed proteins were
immunoprecipitated (IP) with either CD40-Ig (A)
or mAb M5 (B) and resolved by 13% SDS-PAGE. Following
transfer, the membrane was probed with streptavidin-horseradish
peroxidase conjugates. Note that F-T147N and F-W140X are
immunoprecipitated with CD40-Ig only when co-transfected with pWild
(A) and that wild type CD40L is immunoprecipitated with mAb
M5 only when co-transfected with pF-T147N or pF-W140X
(B), suggesting that the association occurs on the cell
surface of transfected COS cells.
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Association of Mutant and Wild Type CD40L Occurs in
CD4+ T Cell Lines Established from XHIM Patients with Leaky
Splice Site Mutations--
The association of wild type CD40L with
E3skip were further confirmed in CD4+ T cell lines
established from two XHIM patients with different intron 3 splice donor
site mutations, nt 367G
A in one patient (Fig.
9A, lane 3) and nt 367 + 5g
a in the other (Fig. 9A, lane 4). CD4+ T
cells from both patients are able to generate normally spliced and exon
3-skipped mRNA (11). When metabolically labeled activated cultured
CD4+ T cells were lysed and immunoprecipitated with
CD40-Ig, E3skip was clearly detected in lysates from both patients
(Fig. 9A, lanes 3 and 4) and in a lysate of pWild + pE3skip co-transfected COS cells (Fig. 9A, lane 7). On the
other hand, no association of E2skip with E2ins was observed in a
CD4+ T cell line from an XHIM patient with the intron 2 splice donor site mutation nt 309 + 2t
a (11) (Fig. 9B),
as expected from the finding in co-transfected COS cells (Fig. 8).
E2ins could not be detected in the immunoprecipitate obtained with
CD40-Ig from the metabolically labeled lysate of the patient's
CD4+ T cells (Fig. 9B, lane 2) nor in the lysate
of pE2skip + pE2ins co-transfected COS cells (Fig. 9B, lane
6). In contrast, the immunoprecipitates obtained with Rb784 from
the patient's CD4+ T cell line (Fig. 9B, lane
4) and from pE2skip + pE2ins co-transfected COS cells (Fig.
9B, lane 8) demonstrated that E2ins was expressed but did
not co-immunoprecipitate with E2skip; the short protein band in Fig.
9B, lanes 4 and 8, has the same position as the
single band shown in the immunoprecipitate obtained with Rb784 from
pE2ins-transfected COS cells (Fig. 9B, lane 10).

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Fig. 9.
Immunoprecipitation of metabolically labeled
CD4+ T cell lines carrying intron 2 or intron 3 splice site
mutations. A, association of a truncated E3skip mutant
with wild type CD40L, both being co-expressed in CD4+ T
cell lines from XHIM patients with intron 3 splice site mutations.
Activated CD4+ T cell lines were metabolically labeled and
lysed (see "Experimental Procedures"). Similarly, COS cells were
transfected with either pWild, pE3skip, or pWild + pE3skip and
metabolically labeled. Cell lysates are as follows: lanes 1 and 2, CD4+ T cell line from a normal control;
lane 3, CD4+ T cell line from a patient with the
mutation nt 367G A; lane 4, CD4+ T cell line
from a patient with the mutation nt 367 + 5g a; lane 5, COS cells transfected with pWild; lane 6, COS cells
transfected with pE3skip; and lane 7, COS cells
co-transfected with pWild and pE3skip. CD40-Ig was used for the
immunoprecipitation in lanes 2-7, whereas human
IgG1 was used in lane 1 as a control reagent.
The sizes of the molecular mass standard proteins are indicated as kDa
on the left. B, a truncated E2skip mutant does
not associate with a truncated E2ins mutant in a CD4+ T
cell line from an XHIM patient with intron 2 splice site mutation (nt
309 + 2t a). An activated CD4+ T cell line derived from
the patient with the mutation nt 309 + 2t a was metabolically
labeled and then lysed. Similarly, COS cells were either co-transfected
with pE2skip and pE2ins or transfected with pE2ins, metabolically
labeled, and then lysed as a control experiment. Cell lysates are as
follows: lanes 1, 2, 3, and 4, CD4+ T
cell line from the XHIM patient with nt 309 + 2t a; lanes 5, 6, 7, and 8, COS cells co-transfected with pE2skip and
pE2ins; lanes 9 and 10, COS cells transfected
with pE2ins. As indicated, control human IgG1 was used for
the immunoprecipitation in lanes 1 and 5, CD40-Ig
in lanes 2 and 6, control rabbit sera in
lanes 3, 7, and 9, and Rb784 in lanes 4, 8, and 10. The sizes of the molecular mass standard
proteins are indicated as kDa on the left. In contrast to
A which shows that wild type CD40L and E3skip can associate
with each other and be immunoprecipitated with CD40-Ig, E2skip, a
truncated mutant which can still bind CD40-Ig, does not associate with
E2ins; CD40-Ig was able to immunoprecipitate only E2skip although E2ins
was co-expressed by CD4+ T cells effectively as
demonstrated by the immunoprecipitation with Rb784, an antiserum
recognizing the intracellular domain of CD40L.
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DISCUSSION |
CD40L, a member of the TNF superfamily, is expressed on the cell
surface as a trimer (5, 19) similar to most other members of this
family, including TNF, CD30L, and FasL (14). In addition to the
full-length wild type CD40L, other derivatives of CD40L participate in
forming a heteromultimeric complex. In this study of activated
CD4+ T cells and CD40L-transfected COS cells, we have
demonstrated that this heterocomplex contains not only the full-length
wild type CD40L but also a CD40L derivative lacking the intracellular tail (CytDel) and a truncated CD40L ("Stalk") generated by cleaving off the extracellular TNFH region to form soluble CD40L (20, 21). The
formation of a heteromultimeric complex consisting of full-length
CD40L, CytDel, and soluble CD40L, the latter being a minor fraction of
the complex, has previously been reported (27). It has been proposed
that CytDel and soluble CD40L are generated by proteolytic cleavage on
the cell surface after the full-length CD40L has been transported from
the intracellular compartment containing CD40L to the surface (32).
Although we have not clearly detected soluble CD40L as one of the
constituents in the immunoprecipitates obtained with CD40-Ig or with
anti-Flag mAb, we have seen a very faint band, approximately 18 kDa in
size (the 1st lane from the left in Fig.
8A) in an immunoprecipitate obtained with CD40-Ig from COS
cells that were transfected with pWild and followed by surface
biotinylation before lysis. This band may represent soluble CD40L,
although we have no direct evidence. The proteolytic cleavage leading
to a CD40L trimer containing significant molecules of "Stalk" may
play a role in attenuating the CD40L-CD40 interaction in
vivo by decreasing the binding site for CD40 and, as a
consequence, make CD40 clustering less efficient. This may be an
important strategy to limit CD40L activity under physiologic conditions.
Several lines of evidence suggest that the extracellular TNFH domain is
of importance for trimer formation. A soluble form of CD40L exists as a
trimer (19, 21) and was found to be biologically active in stimulating
B cell proliferation and immunoglobulin class switch (21, 33). Based on
x-ray crystallography (19) and computer-based structural analysis (31),
several amino acid residues in the TNFH domain have been shown to be of
importance for trimer formation. Mutations occurring at those residues
have been identified in patients with XHIM (10, 11). It is of interest that CD40L mutants that have amino acid substitutions at positions Y170C and G227V, known to be important for trimer formation, were still
able to associate with wild type CD40L if transfected together with
wild type CD40L into COS cells. Furthermore, our data suggest that the
Stalk region (His47-Met113), which is mainly
encoded by exon 2 of the CD40L gene, must also be important
for complex formation since all mutants with intact extracellular Stalk
region tested were able to associate with wild type CD40L, even if a
mutant lacks the entire TNFH domain (Stalk), has a truncated TNFH
domain (F-W140X and F-Q186X), or has cryptic
amino acid residues after Lys96 (F-E2ins and F-E3skip). The
mutated CD40L lacking the exon 2-encoded Stalk due to an in-frame
deletion from Ile53 to Lys96 (E2skip) was able
to associate with wild type CD40L, although much less efficiently
compared with the complex formation between wild type CD40L and either
Flag-tagged wild type CD40L or Flag-tagged mutants carrying amino acid
substitutions. Thus, the integrity of the entire extracellular part of
CD40L, including the extracellular unique region as well as the TNFH
domain, is of importance for efficient complex formation of CD40L.
Similar to the mechanism involved in TNF receptor activation by TNF-
and TNF-
, ligand-induced receptor clustering or aggregation has been
associated with CD40 signaling. Several observations support this
hypothesis as follows: (i) both agonistic and non-agonistic anti-CD40
mAbs have been identified (34-36); (ii) non-agonistic mAb can be
rendered bioactive when cross-linked with a secondary antibody (34,
35); (iii) agonistic activity of a mAb is not dependent on the epitope
to which it binds, and mAbs bound to different epitopes of CD40 can
elicit strong CD40 signaling (35); (iv) anti-CD40 mAb BL-C4, a
pentameric IgM mAb, has been reported to generate a strong signal that
induces monocytes to produce interleukin-1 (37), whereas the anti-CD40
mAb G28-5, known to be agonistic to stimulating B cells (36), did not.
The generation of expression plasmids from cDNA derived from XHIM
patients with diverse, informative mutations of CD40L provided us with
the opportunity to co-transfect COS cells with wild type and mutated
CD40L to investigate heterotrimer formation. We found that various
naturally occurring CD40L mutants can physically associate with wild
type CD40L and form a complex on the cell surface if simultaneously
translated. It is intriguing to consider the possible physiologic
function of these complexes, although the stoichiometry of these
heterotrimers has not been determined in this study. Binding of CD40
takes place in three grooves formed by the three monomers (25, 26), and
each monomer contributes to form the functional groove for CD40
binding. A homotrimer consisting of wild type CD40L is expected to bind
three CD40 molecules resulting in the clustering of CD40 and signal
transduction. In contrast, a heterotrimer consisting of two wild type
CD40L molecules and one mutated CD40L molecule is expected to bind only
one CD40 molecule. A heterotrimer consisting of one wild type and two
mutated CD40L molecules can no longer bind CD40. Thus, heterotrimer
formation is expected to decrease the number of binding sites for CD40
and may no longer be able to ligate CD40 sufficiently to initiate signal transduction in CD40-expressing cells such as B lymphocytes, monocytes, and follicular dendritic cells. As previously reported (11),
activated T cells from most patients with XHIM have mutated CD40L
mRNA transcript levels that are comparable to those found in normal
activated T cells and, following activation, express mutated CD40L on
the cell surface. If the association between wild type and mutated
CD40L species, as we have observed in this study, leads to heterotrimer
formation and to a less efficient CD40 engagement on CD40-expressing
cells, gene therapy for patients with XHIM may face additional difficulties.
A dominant negative effect due to heterotrimer formation may be
responsible for the immunodeficiency in some XHIM patients with splice
site mutations. As previously reported (11) some splice site mutations
of CD40L are "leaky" and allow T cells to generate both normally
and abnormally spliced mRNA transcripts. This is a naturally
occurring in vivo situation where the association of wild
type CD40L and mutant CD40L is likely to take place with physiologic
consequences. By using an RNase protection assay, we have determined
the ratio of mis-spliced to normally spliced mRNA transcripts and
found in most instances that the dominant mRNA species among
multiple splicing products are the mis-spliced mRNAs (11). In a
patient with intron 3 splice donor site mutation (nt 367G
A)
(patient AM in Ref. 11), the amount of exon 3-skipped transcript was
approximately twice that of the normally spliced transcript. A similar
ratio of the transcript carrying a 19 nucleotide insertion of intron 2 (producing E2ins) to the exon 2-skipped transcript, which generates a
mutated CD40L (E2skip) that can still bind CD40-Ig, was found in a
patient with the intron 2 splice donor site mutation nt 309 + 2t
a
(patient PS in Ref. 11). In both cases, the amount of normally spliced
and the amount of exon 2-skipped transcripts, respectively, were
quantitated as being 20-30% of normal control, which by itself may be
too high to be entirely responsible for the clinical phenotype of XHIM. Interestingly, the clinical phenotype of patient PS, whose mutation generates two mutated CD40L species (E2skip and E2ins) that do not
associate with each other, is milder than that of patient AM (nt 367G
A) and patient JE (11) (nt 367 + 5g
a) whose intron 3 splice
site mutations generate wild type CD40L and E3skip that complex with
each other. Whereas patient PS was completely healthy until he
developed parvovirus B19-induced anemia at 17 years of age, patient AM
presented at 5 years of age with recurrent infections, and patient JE
developed Pneumocystis carinii pneumonia at 10 months of age
(11). Based on these clinical observations that suggest a correlation
between the physiologic co-expression of functional CD40L and
non-functioning mutated CD40L in XHIM patients with splice site
mutations, it is likely that the association of wild type CD40L with
mutated CD40L has a disadvantage in the in vivo signal
transduction through CD40. To carry out functional assays to assess
signal transduction via CD40 by heterotrimers consisting of wild type
CD40L and mutant CD40L, it is necessary to establish stably transfected
cell lines. If such experiments prove a dominant negative effect on
signaling, gene therapy for XHIM will be difficult to achieve.
Nevertheless, in considering gene therapy for XHIM, it is important to
determine the type of mutation responsible for XHIM in a given family,
the amount of expressed mRNA transcript, the quantity of mutant
CD40L generated, and the ability of a given mutant CD40L to form
complexes with wild type CD40L.
 |
ACKNOWLEDGEMENT |
We thank Dr. Jiangchen Xu for helping us
constructing Flag-tagged expression plasmids and transfecting COS cells
by electroporation.
 |
FOOTNOTES |
*
This study was supported by National Institutes of Health
Grants HD17427 and AI40102, the March of Dimes Birth Defects Foundation Grant 6-FY96-0330, and the Immune Deficiency Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Pediatrics, University of Washington School of Medicine, Box 356320, Seattle, WA 98195-6320. Tel.: 206-543-3207; Fax: 206-543-3184; E-mail:
allgau{at}u.washington.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
CD40L, CD40 ligand;
AP, antisense primer;
CD30L, CD30 ligand;
DMEM, Dulbecco's modified
Eagle's medium;
FasL, Fas ligand;
FCS, fetal calf serum;
mAb, monoclonal antibody;
MFI, mean fluorescence intensity;
nt, nucleotide
number;
PBS, phosphate-buffered saline;
PMSF, phenylmethylsulfonyl
fluoride;
RT-PCR, reverse transcription-polymerase chain reaction;
PAGE, polyacrylamide gel electrophoresis;
SP, sense primer;
TNF, tumor
necrosis factor;
XHIM, X-linked hyper-IgM syndrome.
 |
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