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INTRODUCTION |
Interaction of CD40L (CD154) with its receptor, CD40, provides
essential signals for the development of a protective humoral immune
response and is a key mechanism in the regulation of defense against
pathogenic assault (for review see Ref. 1). CD40, a 50-kDa
transmembrane glycoprotein, is a member of the tumor necrosis factor
(TNF)1 receptor superfamily
(for review see Refs. 2 and 3) and is expressed on antigen-presenting
cells including dendritic cells, B cells, macrophages, follicular
dendritic cells, and fibroblasts. CD40 is also expressed on a number of
other cell types including endothelial cells and a significant
proportion of carcinomas.
CD40L is predominantly expressed on activated CD4+ T cells,
but variable expression has also been reported on CD8+ T
cells, mast cells, basophils, B cells, monocytes, NK cells, and
activated platelets (1-4). Generally, CD40L does not exist naturally
in a soluble form, although exceptions have been reported (5, 6).
Initial studies focusing on the interaction of CD40L with its receptor
on B cells show that CD40L is largely responsible for the helper T cell
function that drives B cell proliferation and Ig class switching and
protects B cells from apoptotic cell death. More recent studies on
other cell types have indicated an important role for CD40L in the
induction of T cell-mediated effector functions, antigen presentation,
and costimulatory activity of antigen-presenting cells and in the
production of many cytokines (for review see Ref. 1).
CD40L is a type II membrane glycoprotein and is a member of the TNF
superfamily. CD40L has an extracellular domain consisting of a 75-amino
acid spacer region immediately adjacent to the membrane spanning region
that is not shared by other family members and a receptor-binding
domain that consists of two stacked
-sheets. The receptor-binding
domain has low amino acid sequence homology with TNF family members
(<30%) but has high structural homology (7). Soluble forms of CD40L
have been expressed that are able to induce B cell proliferation,
costimulate Ig class switching, and suppress the induction of apoptosis
(8-10). As with cell surface expressed CD40L, maximal activity of
soluble forms of the molecule is dependent on the presence of
costimulatory cytokines. Interestingly, in contrast to its generally
stimulatory effects on normal B cells, interaction of soluble
recombinant CD40L with CD40 on neoplastic B cells can lead to growth
inhibition in vitro and in vivo. The in
vivo effects appear to be mediated via both direct and indirect mechanisms. These findings suggest that soluble CD40L may have therapeutic potential for the treatment of some B cell lymphomas (3,
11, 12).
Earlier studies with CD40 monoclonal antibodies (mAbs), both whole
antibody and F(ab)'2 fragments, and various forms of
soluble CD40L have demonstrated that each reagent has a unique ability to signal through the receptor. In general, the more multimeric the
signaling agent, the more sustained is the subsequent signaling. Thus,
bivalent mAb and dimeric or trimeric soluble forms of CD40L have more
potent biological activity than either F(ab)'2 antibody fragments or soluble CD40L lacking multimerization domains (8). Moreover, although monovalent F(ab) fragments of CD40 mAbs can bind to
CD40, they are not stimulatory and in fact antagonize signaling
mediated via soluble trimeric CD40L (8). These data indicate not only
that CD40 cross-linking is necessary for signaling but also that the
degree of cross-linking determines the magnitude of the response.
Recent investigations have shown that soluble forms of recombinant
CD40L consisting only of the TNF homologous region spontaneously
associate as trimers (13). It has not been clear, therefore, why the
addition of a multimerization domain would increase the activity of
soluble CD40L.
In the current report we have explored further the role of the
trimerization domain in enhancing the activity of soluble human CD40L.
We have expressed soluble forms of both the TNF homologous region and
the entire extracellular domain of human CD40L and compared the
activity of these molecules with that of a hybrid molecule consisting
of the CD40L TNF homologous region fused to an isoleucine zipper (IZ)
trimerization domain at its N terminus. We have found that the soluble
CD40L molecules containing either the spacer region or the
trimerization domain have significantly higher biological activity than
constructs containing just the TNF homologous region. Furthermore, the
CD40L construct consisting of only the TNF homologous region is
considerably less stable than the form containing the trimerization
domain. Taken together, the findings presented here suggest that the
superior biological activity seen with the IZ-containing form of
soluble CD40L is likely to be due to the ability of this trimerization
motif to stabilize soluble CD40L into an orientation that is more
favorable for receptor signaling.
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MATERIALS AND METHODS |
Expression of Soluble CD40L Variants in Chinese Hamster Ovary
Cells--
The TNF homologous region of human CD40L (amino acids
113-261) (14) was polymerase chain reaction-amplified, cloned 3' to the human growth hormone leader sequence (15) in Bluescript (Stratagene, La Jolla, CA) and sequence verified. The final protein sequence included a Thr-Ser dipeptide that is retained in the N
terminus after processing of the growth hormone leader. The CD40L
molecule encoded by this plasmid is termed CD40L receptor-binding domain (RBD). Similarly the entire extracellular region of CD40L (amino
acids 52-261) was polymerase chain reaction-amplified and cloned 3' to
the human growth hormone leader sequence, and this CD40L molecule was
designated CD40L FL (full length). CD40L FL also contained a Thr-Ser at
the N terminus after processing of the growth hormone leader sequence.
Soluble human CD40L was also expressed as a hybrid protein containing a
trimerization domain fused to the N terminus of the TNF homologous
region (amino acids 112-261). The trimerization domain is based upon a
leucine zipper motif (16) modified to form a trimer (17). This soluble
CD40L molecule, designated CD40L IZ, includes a Thr-Ser-Ser-Asp
sequence at its N terminus and Leu-Leu at the junction between the IZ
and RBD regions of the molecule. For expression in CHO cells, the various CD40L constructs were cloned into the expression vector 2a5I
(18).
The final CD40L constructs were transfected into DXB11
DHFR
CHO cells (19) using LipofectAMINETM
(Life Technologies, Inc.). After 48 h, the cells were
subcultivated into DHFR selection medium lacking glycine, hypoxanthine,
and thymidine (
GHT) and grown to confluence. The cells were then selected in
GHT medium containing 50 µM methotrexate.
Cell clones from each transfection were pooled and used in production
cultures to generate milligram quantities of each soluble CD40L molecule.
Purification--
One molar Tris, pH 8.5, was added to the
harvested cell culture supernatant to a final molarity of 80 mM and adjusted to pH 8.5 with 2 N NaOH. The
adjusted cell culture supernatant was then diluted 1:5 with water
before loading onto a Macro-Prep High Q column (4.4cmd × 4.2cmh)
at a flow rate of 70 ml/min that was previously equilibrated in 25 mM Tris, pH 8.5. The resulting unbound fraction was then
directly loaded at a flow rate of 70 ml/min onto a Fractogel
SO3
650 M column (4.4-cm
diameter × 1.8-cm height) that was previously equilibrated in 25 mM Tris, pH 8. The column was then washed in sequence with
equilibration buffer followed by 50 mM NaCl, 25 mM Tris, pH 8.5. The recombinant CD40L was then eluted from
the column with 0.3 M NaCl, 25 mM Tris, pH
8.5.
The eluted CD40L was then dialyzed into 25 mM Tris, 4%
mannitol, 1% sucrose, pH 7.4 buffer and subsequently 0.2µ filtered. The final concentration of CD40L in the purified preparations was
determined by amino acid analysis (20).
Polyacrylamide Gel Electrophoresis and Western
Blotting--
Western blots were performed using polyacrylamide gel
electrophoresis (4-20% Tris-Glycine gels, Novex, San Diego, CA).
After electrophoresis, the gels were either stained with Coomassie Blue or blotted onto nitrocellulose paper (Novex, San Diego, CA.) for Western blotting to visualize the protein bands. Western blots were
performed using the mouse anti-human CD40L mAb M90 (21), which was
generated at Immunex using recombinant human CD40L as immunogen.
Horseradish peroxidase-conjugated anti-mouse antibody (Bio-Rad) was
used as the secondary reagent, and Western blots were developed using a
chemiluminecent reagent (LumiGlo, Kirkegaard, and Perry Labs,
Gaithersburg, MA).
Bioassays--
Human peripheral blood mononuclear cells were
isolated from heparinized blood of healthy donors by centrifugation
over Isolymph (Gallard-Schlesinger Industries Inc., Carle Place, NY).
Purification of B cells was achieved by removal of cells rosetting with
2-aminoethylisothiouronium bromide-treated sheep red blood cells
followed by positive enrichment by magnetic cell separation technology
(Miltenyi Biotec, Auburn, CA) using CD19-coated magnetic beads. The
resultant B cell population was >98% CD20+ with no
detectable CD3+ T cells as determined by flow cytometric
analysis on a FACScan (Becton Dickinson, Mountain View, CA).
B cell proliferation assays were conducted in RPMI medium + 10%
heat-inactivated fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO2. 50,000 cells/well were cultured
in triplicate in round-bottomed 96-well microtiter plates (Corning,
Corning, NY) for 90 h in the presence of the appropriate additives
as described under "Results." Cells were pulsed with 1 µCi/well
tritiated thymidine (25 Ci/mmol, Amersham Pharmacia Biotech) for the
final 18 h of culture. Cells were harvested, and the number of cpm
incorporated was determined by tritium-sensitive avalanche gas
ionization detection on a Matrix 96 Direct Beta Counter (Packard,
Meriden, CT). For IgE secretion, cells (5 × 104/well)
were cultured in Iscove's modified Dulbecco's medium + 10%
heat-inactivated fetal bovine serum, 50 µg/ml transferrin (Sigma), 5 µg/ml bovine insulin (Sigma), 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere of 10%
CO2. Quantitation of IgE levels in supernatants after 10 days of culture was performed by enzyme-linked immunosorbent assay as
described (22).
Three-detector Light Scattering Assessment of Molecular
Mass--
The nonglycosylated polypeptide molecular masses of the
CD40L IZ and CD40L RBD constructs were determined using the
SEC-UV/LS/RI method (on-line SEC followed by detection using
ultraviolet absorbance at 280 nm, light scattering at 90 °, and
refractive index detectors in series). Light scattering and refractive
index measurements were collected using a DAWN DSP and Optilab
differential refractometer (products of Wyatt Technology Corporation,
Santa Barbara, CA). The experiments were carried out using an Integral
high pressure liquid chromatography system (PerSeptive Biosystems,
Inc., Farmingham, MA) with a Superdex 200 HR 10/30 column (Amersham
Pharmacia Biotech). Column loads for the two cases studied ranged from
150 to 200 µg. The flow rate was 1 ml/min with a mobile phase of 200 mM sodium phosphate at pH 8. All detector systems were
calibrated as described (23-25) using ovalbumin (Sigma), bovine serum
albumin monomer (Sigma), and bovine serum albumin dimer (Sigma) at a
nominal concentration of 1 mg/ml in each case. The resulting plot of
(LS × UV)/[
× (RI)2] as a function of molecular mass was
linear, passing through the origin and having an
r2 = 0.994. LS is the light
scattering intensity, RI is the refractive index, and
UV is the absorbance. The slope of this plot provided the
combined instrumental calibration constant k" used in the determination of the molecular mass of the CD40L IZ and CD40L RBD.
Theoretical molar extinction coefficients (
) were determined from
the amino acid composition (26) for the CD40L RBD and CD40L IZ versions
of soluble CD40L and were found to be 0.76 and 0.65, respectively. Data
were acquired using ASTRA (accompanying software from Wyatt Technology Corporation).
Differential Scanning Calorimetry--
Calorimetric measurements
were carried out using a MicroCal MC-2 DSC. Samples were buffer
exchanged into 25 mM Tris, pH 7.6, 400 mM NaCl,
and 10% glycerol. Protein concentrations under these conditions were
nominally between 2 and 3 mg/ml. The scan rate in each case tested was
68 °C/h. The thermograms were background corrected and rescans were
employed to evaluate the reversibility of the unfolding transitions. In
all cases examined the second upscan yielded no reversible enthalpy,
suggesting that the sample was irreversibly denatured as indicated by
the presence of aggregate when withdrawing the sample from the DSC. In
this study, 90 °C was set as the upper temperature limit, which was
beyond the completion of the major melting transition of the soluble
CD40L molecules. All data manipulation was performed using the Origin
software provided with the instrument.
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RESULTS |
Expression and Purification of the Soluble CD40L Variants--
The
three CD40L molecules compared in this study are diagrammed in Fig.
1A. After purification as
described under "Materials and Methods," each soluble CD40L
preparation was analyzed for purity using Western blots (Fig.
1B, lanes 1-3) and Coomassie gels (Fig.
1B, lanes 4-6). Each molecule had one main band
that migrated slightly higher than the predicted molecular mass and at
least one faint lower molecular mass band that migrated closer to the
expected monomeric molecular mass. The calculated peptide molecular
masses for CD40L FL, CD40L RBD, and CD40L IZ are 24.0, 16.4, and 20.9 kDa, respectively. The preparations of CD40L IZ and CD40L RBD showed no
bands on Coomassie gels besides the CD40L bands. The Coomassie
preparation of CD40L FL (Fig. 1B, lane 4) had a
large number of background bands that were not detected by Western
blot. This molecule did not express as well as the other two, and thus
it was more difficult to obtain a highly purified final preparation.
Previous analysis using N-glycanase of CD40L molecules
expressed in CHO cells have indicated that the higher molecular mass
bands detected on the Western blot are glycoforms of CD40L.

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Fig. 1.
Expression of recombinant human soluble forms
of CD40L. A, schematic representation of soluble CD40L
molecules. CD40L RBD consists of just the TNF homologous region of the
extracellular domain (gray box). CD40L FL consists of the
extracellular spacer between the cell membrane and the TNF homologous
region (hatched box) and the TNF homologous region. CD40L IZ
consists of the receptor-binding domain and an IZ motif at the amino
end of the molecule (black box). B, Western blots
and Coomassie stain of various soluble human CD40L constructs.
Lanes 1-3, Western blot. Lane 1, CD40L FL;
lane 2, CD40L RBD; lane 3, CD40L IZ. Lanes
4-6, Coomassie stained gel. Lane 4, CD40L FL;
lane 5, CD40L RBD; lane 6, CD40L IZ.
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Bioactivity of the CD40L Variants--
The biological activities
of CD40L RBD and CD40L IZ were compared in a human B cell proliferation
assay performed in the presence of IL-4 as a costimulus (Fig.
2A). Although both forms of
CD40L induce B cell proliferation, the IZ construct showed
significantly higher biological activity with effects seen at ligand
concentrations as low as 20 ng/ml. Proliferation induced by these forms
of soluble CD40L could be inhibited by addition of anti-CD40L
antibodies to the cultures (data not shown). A similar distinction in
the activities of the two CD40L constructs was seen in the induction of
IgE secretion from IL-4-costimulated B cells (Fig. 2B).

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Fig. 2.
Biological activity of CD40L RBD and CD40L
IZ. A, purified peripheral blood B cells were cultured
for 90 h in the presence of IL-4 (5 ng/ml) and a titration of
CD40L RBD ( ) or CD40L IZ ( ). Tritiated thymidine was added to
cells for the final 18 h of culture. Results are expressed as the
mean cpm of triplicate cultures and are representative of three
experiments performed. B, purified peripheral blood B cells
were cultured for 10 days in the presence of IL-4 (5 ng/ml) and a
titration of CD40L RBD ( ) or CD40L IZ ( ). IgE levels in day 10 culture supernatants were determined by enzyme-linked immunosorbent
assay. Results are expressed as the mean values from triplicate
cultures and are representative of three experiments performed.
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The CD40L RBD and CD40L FL were compared also for their ability to
induce proliferation of peripheral blood B cells (Fig. 3). An effect of the CD40L RBD was seen
at concentrations higher than 1 µg/ml, whereas the full-length form
of the ligand had greater biological activity, measurable down to 300 ng/ml.

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Fig. 3.
Biological activity of CD40L RBD and CD40L
FL. Purified peripheral blood B cells were cultured for 90 h
in the presence of IL-4 (5 ng/ml) and a titration of CD40L RBD ( ) or
CD40L FL ( ). Tritiated thymidine was added to cells for the final
18 h of culture. Results are expressed as the mean cpm of
triplicate cultures and are representative of three experiments
performed.
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Three-detector Analysis of CD40L--
One possible explanation for
the increased activity of CD40L IZ compared with that of the CD40L RBD
construct could be attributed to it existing in a higher order
multimeric form in solution. To evaluate the multimeric state of these
two recombinant CD40L variants, SEC followed by three-detector analysis
was employed. Evaluation of the molecular mass of glycosylated proteins
using the SEC-UV/LS/RI method offers the advantage of being able to assess the molecular mass of the molecule as if it were
nonglycosylated; glycosylation is essentially transparent at 280 nm
(23). Fig. 4A shows the
elution profile of the CD40L RBD with a peak elution volume of 13.6 ml.
The SEC-UV/LS/RI method yielded a measured molecular mass of 49.47 ± 0.81 kDa. Utilizing the monomer polypeptide molecular mass of this
molecule (16.4 kDa), it was determined that CD40L RBD is indeed a
trimer. This confirms and extends the findings from sucrose gradient
sedimentation experiments published earlier (10). Similarly, the
polypeptide molecular mass of the CD40L IZ as measured by SEC-UV/LS/RI
was 62.2 ± 0.14 kDa (Fig. 4B). This is approximately
three times the monomer molecular mass (20.9 kDa). These results
provide evidence that both soluble forms of CD40L are trimeric.

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Fig. 4.
The SEC elution profiles for CD40L RBD
(A) and CD40L IZ (B). Labeled are the
corresponding eluting components as measured by the UV absorbance at
280 nm, light scattering at 90 ° (LS), and refractometer
(RI). The vertical dashed lines in each case
display the bracketed region designated for molecular mass
characterization (after computing the molecular mass per slice)
surrounding the peak maximum.
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An alternative explanation for the increased activity of CD40L IZ
compared with CD40L RBD is that the latter may disassociate into
monomers at lower concentrations. Disassociation of trimeric CD40L RBD
at low concentrations could greatly impair its ability to cross-link
and thus signal through CD40. To address this issue, CD40L RBD was
diluted to 2 µg/ml, and disassociation was monitored using the SEC-UV
method. No disassociation of the CD40L RBD was detected (Fig.
5), suggesting that this was not the
reason for the differences observed in the biological activity of the
CD40L IZ and CD40L RBD constructs. At 2 µg/ml the CD40L IZ molecule showed greater activity than CD40L RBD (Fig. 2A); thus,
disassociation of CD40L RBD into monomers does not explain the
differences in activity.

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Fig. 5.
SEC showing (from top to
bottom) the buffer blank, lysozyme standard, CD40L RBD
(1.95 mg/ml), and CD40L RBD (1.95 µg/ml). The effect of dilution
does not show discernible evidence of further disassociation of the
trimer.
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DSC--
It is possible that the distinct activities of the CD40L
IZ and CD40L RBD could be due to a difference in stability. To address this issue, differential scanning calorimetry (DSC) experiments were
performed. The DSC data distinctly showed a 10 °C difference in the
melting transition, with the CD40L RBD having the lower melting
transition (Fig. 6). Collectively, these
data suggest that in the CD40L IZ, the IZ confers appreciable
conformational stability compared with that seen in CD40L RBD, despite
both existing in solution as trimers. That is to say, it is more
difficult thermodynamically for the IZ version of the molecule to
unfold under the given solution conditions. This might be explained in
terms of the local restraint offered by the IZ in proximity to the
receptor-binding domain.

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Fig. 6.
Concentration-normalized DSC scans depicting
the endothermic melting transitions of CD40L RBD and CD40L IZ.
Both samples are in a common buffer system consisting of 25 mM Tris, pH 7.6, 400 mM NaCl, and 10%
glycerol. The exotherm at 80 °C (lower trace) is due to
the formation of aggregate.
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DISCUSSION |
The results presented here demonstrate that the three forms of
soluble CD40L examined, CD40L RBD, CD40L FL, and CD40L IZ, are
biologically active. The presence of the IZ trimerization domain or the
native extracellular spacer region significantly increased the activity
of soluble CD40L in B cell proliferation assays. The presence of the IZ
trimerization domain on CD40L RBD also increased IgE secretion.
Significantly enhanced activity of CD40L IZ constructs, compared with
that of CD40L RBD, was seen also in the induction of B cell activation
antigens, CD54, CD86, and HLA-DR (data not shown). Furthermore, the
SEC-UV/LS/RI method demonstrated that CD40L RBD and CD40L IZ exist as
trimers in solution. Finally, the DSC study indicated that addition of
the IZ serves to conformationally stabilize CD40L RBD.
Molecular modeling studies have shown (7) and crystallographic studies
at 2 Å resolution have confirmed (13) that the trimerization of CD40L
RBD involves Tyr170 and His224 to form an
unusual cluster of two triads along the 3-fold axis. The trimerized
assemblages are stabilized by hydrogen bonds and presumably salt bridge
interactions. The DSC data indicate that significant differences exist
in overall conformational stability when the IZ motif is incorporated
at the N terminus of the RBD. Without the IZ, the CD40L RBD molecule
would be more prone to unfold at the N terminus rather than the C
terminus, because the latter is buried in the interior of the
trimerized molecule (13). With the IZ one can begin to imagine how the
N terminus would be constrained, making it more difficult to unfold
from that end. The broad features in the unfolding profile of the IZ
may suggest an unraveling of the molecule that begins with an overall
loosening of the protein interior and ends with the sheet regions of
the molecule exposed. The trimerized assemblages may thus form
aggregates in the unfolded state (observations after removal of samples
from the DSC have shown visible signs of aggregation). Such structural restraint of the N terminus may also affect the way in which the ligand
interacts with the receptor and explain the enhanced bioactivity. Site-directed mutagenesis has shown that Lys143 and
Tyr145 are critical for CD40L interaction with CD40 (27).
These residues are in close proximity to the N terminus. Restraint at
the N terminus conferred by the IZ may stabilize the configuration in
this region and thus augment signaling.
In addition to improved conformational stability, the IZ may enhance
signaling through other mechanisms. Based on previous studies using
mAbs to mediate receptor ligation, it is predicted that binding of
soluble CD40L to CD40 is followed by rapid cell surface localization
and internalization of the receptor-ligand complex. The presence of the
IZ motif in the CD40L IZ molecule may result in binding to CD40 in such
a way that these processes are retarded, resulting in prolonged
signaling through CD40.
It is not clear why we were able to detect activity for the CD40L FL
and others were not (10). It could be that in the bacterial system it
was not possible to achieve the correct folding or that important
differences exist in the glycosylation achieved in bacteria compared
with CHO cells. Either could affect the biological activity of CD40L
FL. Furthermore, our studies with CHO cell-derived CD40L FL indicate
that this molecule has a tendency to aggregate, which could explain why
it has relatively good biological activity compared with CD40L RBD,
because in a partially aggregated form it could more effectively
mediate receptor cross-linking.
Modeling studies have pointed to structural similarities between CD40L
and TNF, a prototype member of the TNF/nerve growth factor superfamily
of ligands. However, it is important to note that although biologically
active TNF exists naturally as a soluble homotrimer, CD40L is
predominantly expressed as a membrane-anchored molecule. Results
presented in this study and those of Mazzei and colleagues (10) have
demonstrated that the receptor-binding domain of CD40L can associate to
form homotrimers in solution. However, in contrast to TNF, such a
configuration does not impart optimal biological activity to the
soluble CD40L molecule, because its stability appears to be enhanced
considerably by the presence of the 75-amino acid spacer region or the
IZ trimerization domain. This suggests that this region between the RBD
and the transmembrane domain of the CD40L molecule may be important in
facilitating formation of stable CD40L complexes on the cell surface.
Supporting evidence for the importance of N-terminal multimerization in
the structural integrity of CD40L comes from studies on a hyper IgM patient whose CD40L gene contained a deletion of the coding region for
the extracellular spacer domain (28). In this study the mutant CD40L
was expressed in COS cells and found to have a reduced ability to bind
to CD40 compared with a wild type CD40L.
Regardless of the mechanism responsible for the enhanced biological
activity observed, the results presented here shed new light on
signaling mediated by soluble forms of CD40L. In two examples, the
CD40L IZ and the CD40L FL, the presence of an N-terminal multimerization domain resulted in soluble forms of the ligand with
improved specific activities. This result is not predicted by protein
modeling studies, which suggest that the critical region for functional
association of the CD40L molecule is just contained within the TNF
homologous region. In fact, within the TNF ligand superfamily enhanced
biological activity of IZ-containing constructs is not confined to
CD40L. The activities of soluble Fas ligand and TRAIL, the
extracellular domains of which can occur naturally in soluble form, are
significantly enhanced by the addition of the IZ motif at the N
terminus.2 Further
investigations into the mechanism of action of the IZ motif should lead
to greater insight into regulation of receptor signaling and
development of therapeutic constructs for this important family of molecules.