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*
DNAX Research Institute, Palo Alto, CA 94304;
Laboratoire de Chimie Bioorganique, Unité Mixte de Recherche 7514 Centre National de la Recherche Scientifique-Université Louis Pasteur, Faculté de Pharmacie, Illkirch, France; and
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Like the Aplysia cyclase enzyme, the extracellular domain of CD38 possesses ADP-ribosyl cyclase activity 13, 14 . However, unlike the Aplysia cyclase enzyme, CD38 is also able to mediate the catalysis of both NAD+ and cADPR into ADP-ribose (ADPR) through its NAD+ and cADPR hydrolase activities 13 . In fact, CD38 is a more efficient NAD+ glycohydrolase enzyme than ADP-ribosyl cyclase enzyme, as >97% of the total product produced by CD38 is ADPR 13 . Nevertheless, it has been hypothesized that the cADPR produced via the cyclase activity of CD38 may be an important regulator of calcium signaling 15 .
In the hemopoietic system, CD38 has been shown to regulate lymphocyte activation and effector functions 1, 16 . We and others have shown that engagement of CD38 using anti-CD38 Abs can induce a proliferative response in B and T lymphocytes 17, 18, 19 , and that coligation of CD38 and the Ag receptor on B cells (BCR) can augment BCR-mediated responses 20 . Additionally, anti-CD38 stimulation has been shown to be a potent inducer of cytokine production in T and B cells 20, 21, 22 . Finally, CD38 clearly plays an important role in the immune system in vivo, as animals deficient in CD38 have marked deficiencies in their ability to mount humoral immune responses 23 .
To examine the molecular requirements for anti-CD38-mediated activation and costimulation in murine B cells, we recently established an in vitro CD38 signaling model system using the A20 murine B cell lymphoma 20 . A20 cells are CD38 negative but express a functional BCR complex, which, when cross-linked, will initiate the synthesis of IL-2 24, 25, 26 . When CD38 was stably expressed in A20 cells, the resultant CD38+ clones inducibly produced IL-2 after CD38 was cross-linked with a polyclonal Ab (polyanti-CD38) 20 . Furthermore, coligation of CD38 and the BCR induced a potent synergistic IL-2 response in these cells, suggesting that CD38 might function as a BCR coreceptor. Interestingly, CD38-mediated signaling and coreceptor activity in this system was found to be completely dependent upon expression of a functional BCR complex 20 . Surprisingly, a large truncation of the cytoplasmic tail of CD38 did not noticeably depress CD38-mediated signal transduction, suggesting that the cytoplasmic tail of CD38 might be unnecessary for anti-CD38-mediated signaling 20 .
In this report we have attempted to reconcile the signaling capacity of murine CD38 with its enzymatic properties. We have performed site-directed mutagenesis to produce mutated forms of CD38 and have examined the consequences of these mutations on CD38-mediated signal transduction and enzyme activity in our A20 B lymphoma model system. The results are presented and discussed in the context of the predicted structure for CD38.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The A20/2J cell line was obtained from American Type Culture Collection (Manassas, VA), and the cytokine-dependent cell line HT-2 was a gift from Dr. David Woodland (St. Jude Children’s Research Hospital, Memphis, TN). The cells were maintained as previously described 20 . Polyclonal rabbit anti-mouse CD38 antiserum was prepared by immunizing rabbits with purified recombinant soluble CD38 20 . The IgG fraction of the antiserum was subsequently concentrated by ammonium sulfate purification, absorbed on the CD38-negative A20 B cell line, and then protein A purified. Approximately 13% of the protein A-purified rabbit anti-CD38 was specific for CD38 as measured using soluble CD38 in an ELISA. The same procedure was used to purify normal rabbit IgGs from normal rabbit serum (Colorado Serum Co., Denver, CO). Affinity-purified rabbit anti-mouse IgG and F(ab')2 goat anti-mouse IgG were purchased from Cappel (West Chester, PA).
Construction of CD38 mutant cDNAs
The wild-type CD38 expression vector (CD38-pME18S/neo) contains
the full-length coding region of the murine CD38 cDNA cloned downstream
of the SR
promoter in the pME18S/neo expression vector, as
previously described 3 . All the different CD38 mutant constructs were
generated by PCR using the primers listed below (cloning sites are
underlined, and the altered nucleotides that correspond to the
replacement amino acids codons are indicated in lower case italics):
5'-µATG, 5'-(CCC GAA TTC ATG aag gtg aag ATC
GGT CTC GGA GTG GGT CTC CTG G)-3'; 5'-µTFR-I, 5'-(tg gtc
atc ttc ttc ctc ata ggc ttc tcc gga AGG CCG CGC TCA CTC CTG GTG
TGG)-3'; 5'-µTFR-II, 5'-(CCC GAA TTC atg aag gtc
aag cta tgc ttc gca gcc ata gcc ttg gtc atc ttc ttc ctc ata)-3';
5'-E150L/D151V, 5'-(T ACT TGG ATC CAG GGA AAG AT G TTC ACC
CT G ctg gtc ACC CTG C)-3'; 5'- E150Q/D151N, 5'-(T ACT
TGG ATC CAG GGA AAG AT G TTC ACC CT G cag aac
ACC CTG C)-3'; 5'-CD38wt, 5'-(GGG GAA TTC ATG GCT AAC TAT
GAA TTT AGC CAG G)-3'; 3'-XbaI, 5'-(CCC TCT AGA
CCA GAT CCT TCA CGT ATT AAG TCT ACA CG)-3'; 3'-cys, 5'-(CC CTG
GAT CCA AGT ATA TTG ATG GGC CAG GTG TTT GGA TTT GCT CCA AAA GAG
AGT CTT GTT ctt TGG TAT GG)-3'; and 3'-hyaluronate (3'-HA),
5'-(C CTG GAT CCAAGT ATA TTG ATG GGC CAG GTG tgt
GGA gct GCT CC)-3'. The appropriate PCR reaction products
were then isolated, purified, and cloned into the PME18S/neo expression
vector. The cloned products were sequenced in both directions to
ascertain that the appropriate mutation was introduced and that no
polymerase or cloning errors were present in the rest of the molecule.
Generation of CD38 mutant A20 stable transfectants
CD38-negative A20 B lymphoma cells were transfected with the different CD38 expression constructs and then selected in G418 (Geneticin, Life Technologies, Grand Island, NY) as previously described 20 . The G418-resistant clones were grown out and analyzed for the expression of CD38 and surface Ig by immunostaining (described below) using polyclonal rabbit anti-mouse CD38 and an R-phycoerythrin-conjugated goat F(ab')2 anti-mouse Ig (Southern Biotechnology Associates, Birmingham, AL). Ten CD38+, BCR+ clones for each of the different CD38 mutant or wild-type transfectants were chosen and single cell subcloned. After subcloning, all the clones were further selected for inducible IL-2 production after stimulation with both PMA and Ca2+ ionophore or with F(ab')2 anti-Ig (Cappel).
Mixed clones were then generated for cells expressing the recombinant wild-type CD38 (WILD-CD38) and for each of the different mutant CD38 clones. For example, 10 independently selected WILD-CD38 clones were expanded in log phase for 1 wk. Equal numbers of these 10 clones were then mixed together, and 30 identical aliquots of the mixed WILD-CD38 cells were frozen (-80°C) in 10% DMSO/90% FBS.
FACS staining of mixed mutant clones
An aliquot of all the mutant mixed clones was thawed, grown in tissue culture, and then analyzed for CD38 expression on a FACS Calibur (Becton Dickinson, Mountain View, CA) after staining. Cells were first blocked with 1 µg of purified Fc block (24G2, PharMingen, San Diego, CA) and then stained on ice with either protein A-purified rabbit anti-mouse CD38 or purified normal rabbit IgGs. The cells were washed, stained with rat anti-rabbit FITC (Zymed, South San Francisco, CA), and analyzed.
Preparation of cross-linked Ab conjugates
Cross-linked Abs were prepared as previously described 20 . Briefly, 2 mg of the purified rabbit anti-CD38 Ab and 8 mg of purified normal rabbit IgGs were mixed per 1.0 ml of recombinant protein A beads (Zymed). To prepare the substimulatory anti-CD38 cross-linked beads, 200 µg of anti-CD38 Ab plus 10 mg of normal rabbit IgGs were incubated per 1.0 ml of protein A beads. To prepare the substimulatory anti-Ig beads, 4 µg of rabbit anti-mouse IgG (Cappel) plus 10 mg of normal rabbit IgGs were incubated per 1.0 ml of protein A beads. To prepare the cocross-linked Abs, 200 µg of anti-CD38, 4 µg of anti-IgG, and 10 mg of normal rabbit IgG were incubated per 1.0 ml of protein A beads. All the Ab/bead combinations were incubated, with rocking, at room temperature for at least 1 h and then washed four times in medium to remove unbound Ab. Freshly prepared batches of cross-linked Ab were used in each experiment and were tested on the control CD38-negative A20 clone (NeoR). In no case did the cross-linked anti-CD38 beads induce the CD38-negative cells to produce IL-2.
Stimulation of CD38 mutant transfectants and IL-2 quantification
An aliquot of each of the mutant mixed clones was thawed on day 0 and put into tissue culture at 2 x 105 cell/ml. The cells were maintained in log phase growth for the next 2 days and were expanded at 5 x 105 cells/ml in fresh medium on day 3. On day 4 the cells were counted, assessed for viability, washed, and used in experiments on that day. Triplicate cultures of 2 x 105 mixed clone cells/well were plated in a 200-µl volume in complete medium with the appropriate stimulus. The cells were stimulated with soluble affinity-purified F(ab')2 goat anti-mouse IgG (25–50 µg/ml; Cappel), PMA (5 ng/ml), and A23187 (1/80,000 dilution of 1 µM) or with a 2.5-µl packed bead volume (25 µl of 10% bead/medium slurry) of the various Ab/protein A conjugates described above. Cells were stimulated for 8 h, and supernatants were removed and frozen at -80°C. To test for the presence of IL-2, supernatants were thawed and used in bioassays with the IL-2-dependent cell line, HT-2. One unit of IL-2 is defined as the amount of IL-2 required to induce half-maximal growth (as measured by Alomar Blue, Accumed, Cleveland, OH) of 5 x 103 HT-2 cells. The Alomar Blue assay used here could reproducibly detect IL-2 levels as low as 0.2 U/ml 20 . The concentration of IL-2 in the supernatants was calculated using purified recombinant murine IL-2 (20 U/ml; DNAX, Palo Alto, CA) as the standard on all plates, and the results were expressed as the mean ± SD.
Preparation of cell fractions
Mixed clone aliquots were thawed, expanded in tissue culture to 2 x 108 cells over a period of 7 days, washed twice by centrifugation at 1000 x g in PBS, and then frozen as a pellet at -80°C until use. To prepare the cell fractions, the pellets were diluted in 1 ml of a 50-mM potassium phosphate buffer, pH 6.8, and homogenized with a glass-glass Dounce potter. The homogenates were then centrifuged at 100,000 x g for 60 min, and the pellets were carefully resuspended (potter) in the same volume of buffer for enzyme activity testing. The protein concentration was determined with the bicinchoninic acid assay reagent (Pierce, Rockford, IL) using BSA as a standard.
Enzyme assays
The catalytic activity of CD38 was determined in the various cell fractions by two different radiometric methods. The first one determines, under saturating conditions, the release of [14C]nicotinamide from [14C-nicotinamide] NAD+ and yields the total NAD+ glycohydrolase and ADP-ribosyl cyclase activities. The second, by use of [14C-adenosine]NAD+, measures the formation of [14C]ADPR and [14C]cADPR and yields the two activities separately. Alternatively, when increased sensitivity was needed, [3H-adenine]NAD+ was used to generate [3H]ADPR and [3H]cADPR. [Carbonyl-14C]nicotinamide adenine dinucleotide (35 mCi/mmol) was purchased from Amersham (Aylesbury, U.K.). [Adenosine-U-14C]NAD+ (604 mCi/mmol) and [adenine-2,8-3H]NAD+ (4 Ci/mmol) were purchased from New England Nuclear (Boston, MA).
Nicotinamide-releasing activity
Cell fractions were added to an assay mixture containing [carbonyl-14C]nicotinamide adenine dinucleotide (2.5 x 105 dpm) and NAD+ (500 µM) in a 50-mM potassium phosphate buffer, pH 6.8 (250-µl final volume), and incubated at 37°C. At given times, 100-µl aliquots were withdrawn, and after acidification with 4 µl of 50% TCA, the reaction products were analyzed as described below. Each run (two time points; reaction progress, <30%) was repeated at least three times with different time points and amounts of cell fractions.
NAD+ glycohydrolase and ADP-ribosyl cyclase activity
Cell fractions were added to an assay mixture containing either [adenosine-U-14C]NAD+ (5.0 x 105 dpm) and NAD+ (final concentration, 25 µM) or [adenine-2,8-3H]NAD+ (5 x 106 dpm) and NAD+ (final concentration, 5 µM) in a 50-mM potassium phosphate buffer, pH 6.8 (500 µl final volume), and incubated at 37°C. At given time points, 100-µl aliquots were withdrawn, and the reaction product was analyzed as described below.
Analysis of the reaction products by HPLC
Product analysis was performed on TCA-precipitated aliquots of the reaction medium (centrifuged at 10,000 x g for 10 min at 4°C on Microcon-10 microconcentraters (Amicon, Danvers, MA)) using a Waters HPLC system (Waters Associates, Milford, MA). Chromatography was conducted on a 300 x 3.9-mm µBondapak C18 column (Waters) operated at ambient temperature at a flow rate of 1 ml/min. The compounds were eluted isocratically with a 10-mM ammonium phosphate buffer, pH 5.5, containing 1.2% (v/v) acetonitrile and were detected by their UV absorbance at 260 nm and by radiodetection (Flo-one, Packard-Radiometric Instruments, Meriden, CT). The reaction products were identified by coelution with standards, the radioactivity associated with the different peaks was integrated, and the cADPR/ADPR ratio was calculated 27 .
Calculation of adjusted enzyme activity
To normalize the enzyme activity of the cell homogenates, the enzyme activities were multiplied by a correction factor that compensated for the total protein per cell and the amount of CD38 expressed per cell. The amount of protein per cell was determined by a Bradford assay (Bio-Rad, Hercules CA), and the mean concentration was calculated from three independent experiments. The amount of CD38 protein expressed by each cell was quantitated by FACS. The mean fluorescence intensity was determined for individual mutant cells and was then normalized to WILD-CD38, which was set at a value of 1.0 arbitrary CD38 unit/cell. The FACS staining experiments were performed three times, and the average arbitrary CD38 units per cell for each of the mutants were calculated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using A20 B lymphoma cells that had been stably transfected with
murine CD38, we previously demonstrated that signaling through CD38 was
unimpaired by truncation of the cytoplasmic tail to four amino acids,
suggesting that the cytoplasmic tail of CD38 may not be necessary for
signal transduction 20 . Three other domains in the CD38 molecule have
been hypothesized to play a role in signaling; these include the
transmembrane domain, the putative HA binding sites (HA sites) 28 ,
and the extracellular enzymatic active site(s) 1, 13, 16 . To test
which of these domains is required for anti-CD38-mediated signal
transduction in B cells, we generated A20 clones expressing CD38
molecules that had been specifically altered by site-directed
mutagenesis in each of the regions described above. As diagrammed in
Fig. 1
, we generated two chimeric forms
of CD38 in which the cytoplasmic tail (µATG-CD38) or the cytoplasmic
tail and transmembrane domain (µTFR-CD38) of CD38 were completely
replaced. The CD38 cytoplasmic tail in both chimeric molecules was
replaced with the three amino acid sequence of the IgM BCR cytoplasmic
tail (KVK), as this sequence has been demonstrated to be inert in B
cell signaling assays 29, 30 . Furthermore, in µTFR-CD38, the
transmembrane domain of CD38 was replaced with the transmembrane domain
from another type II protein, the transferrin receptor 31 (Fig. 1).
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cDNAs encoding each of the different forms of CD38 as well as a cDNA encoding the wild-type CD38 molecule (WILD-CD38) were generated by PCR, cloned into the PME18S/neo mammalian expression vector 3 , and then stably expressed in the parental CD38-negative A20 lymphoma cells 20 . Multiple independent CD38-expressing clones were generated from cells transfected with WILD-CD38 or with each of the different CD38 mutant cDNAs and were tested to ensure that they inducibly produced IL-2 after treatment with PMA and Ca2+ ionophore treatment as well as after stimulation with anti-Ig reagents. At least 10 independent clones of cells expressing a particular transfected form of CD38 were then equally mixed (as described in Materials and Methods) to eliminate any clone biasing that might arise in the selection process. Thus, the effect of each of the different CD38 mutations could be assessed on a known, reproducible, mixed population of transfected cells.
In initial experiments using the mixed clones, the CD38 expression
levels were quantitated by FACS using purified polyanti-CD38 Ab or
normal rabbit IgG as a control. The resultant histograms are shown in
Fig. 2. The CD38 expression levels on the
mixed clones were evaluated with the polyclonal anti-CD38 and were
found to be nearly equivalent, with no more than a twofold difference
in the mean fluorescence intensity (in upper right corner of
each histogram) between WILD-CD38 and any of the mutant clones.
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CD38 is a multifunctional enzyme that is able to catalyze the
conversion of NAD+ into nicotinamide
(nicotinamide-releasing activity), ADPR (NAD+
glycohydrolase activity), and cADPR (ADP-ribosyl cyclase activity; Fig. 3). To assess the role of CD38’s
enzymatic activities on signal transduction, we first evaluated the
impact of the different structural mutations described above on CD38’s
various enzyme functions. To do this, the mixed clones expressing the
various mutant CD38 molecules were homogenized in the absence of
detergent to generate cell homogenates that preserved the integrity of
the cellular membranes. The protein concentration and enzyme activity
of the homogenates were then determined for each of the mutants. The
nicotinamide-releasing activity (Fig. 3), which is basically a
determination of the total catalytic activity of CD38 (combined
NAD+ glycohydrolase, ADP-ribosyl cyclase,
transglycosidation, and cADPR hydrolase activities), was assayed under
saturating condition by measuring the release of
[14C]nicotinamide from
[carbonyl-14C]nicotinamide adenine dinucleotide. The
reaction products were then analyzed by radiometric HPLC. The steady
state enzyme activity for the homogenates generated from each of the
CD38 mutant cell lines was expressed as nanomoles of product produced
per minute per milligrams of total protein, and the relative enzyme
activities of each of the homogenates was calculated as a percentage of
the activity of WILD-CD38 (Table I).
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). The elimination of the two putative HA
binding sites (HA-CD38) had a small, but measurable, effect and reduced
the catalytic activity of CD38 by approximately 50% (Table I). In
contrast, and in agreement with the findings of Grimaldi et al. 32 ,
selected point mutations at positions 150 and 151 greatly reduced the
nicotinamide-releasing activity of CD38. In fact, the
nicotinamide-releasing activity of the homogenates expressing
E150L/D151V-CD38 was at least 35 times lower than that of those
expressing WILD-CD38. This mutant was not catalytically inactive
however, as the nicotinamide-releasing activity of the homogenate
generated from the E150L/D151V-expressing cells was approximately
10-fold higher than that of the homogenates of the nontransfected
CD38-negative A20 control cells (NeoR). Interestingly, a more
conservative set of replacement amino acids at positions 150 and 151
(E150Q/D151N) did not dramatically alter the catalytic activity of CD38
compared with the CD38-WILD protein (70% of normal activity). Finally,
the replacement of the cysteine residue at amino acid position 123 with
a lysine residue (C123K-CD38) increased the CD38-mediated
nicotinamide-releasing activity approximately 2-fold over the activity
of WILD-CD38 (Table I).
The nicotinamide-releasing activity of the cell homogenates was
normalized for the amount of total protein and CD38 protein expressed
per cell (correction factor, Table I, column 2). The amount
of CD38 expressed by each cell was represented by the mean fluorescence
intensity value, which was determined by staining the cells with
polyclonal anti-CD38 Abs that contain multiple epitope
specificities (Fig. 2). The adjusted activities were expressed as
nanomoles of product produced per minute per arbitrary unit of CD38
(Table I, column 4). Even after the enzyme activity of each
of the homogenates was adjusted to reflect the amount of CD38 present,
the relative activity of each of the CD38 mutant molecules did not
alter significantly compared with the activity of the CD38 wild-type
protein (compare fold activity in column 2 to column
4 in Table I). Importantly, the greatly reduced catalytic activity
of E150L/D151V-CD38 could not simply be explained by either reduced
total cellular protein content or a drastic reduction of CD38 protein.
Next, to test whether any of the mutant forms of CD38 were altered in
their ability to produce the calcium-mobilizing second messenger,
cADPR, the cell homogenates were tested in assays using
[14C-adenosine]NAD+ as a substrate. The
formation of [14C]ADPR (NAD+ glycohydrolase
activity) and [14C]cADPR (ADP-ribosyl cyclase activity)
was individually measured 27 , and the ratio of cyclization to
hydrolysis was calculated (i.e., the cADPR/ADPR ratio; Table II). Using this assay, we found that
wild-type CD38 produced approximately 1000 times more ADPR than
cADPR. This was not unexpected, as recombinant soluble murine CD38 has
been shown to produce exceedingly small quantities of cADPR 13 . The
amount of cADPR produced by the homogenates generated from the CD38
cytoplasmic tail mutant cell line (µATG-CD38) and the CD38
transmembrane domain mutant cell line (µTFR-CD38) was essentially
equivalent to that produced by WILD-CD38. The amount of cADPR produced
by HA-CD38 and C123K-CD38 homogenates was increased over that produced
by WILD-CD38, but was still 20–50 times less than the amount of ADPR
produced. Interestingly, the E150Q/D151N-CD38 homogenate produced much
larger quantities of cADPR relative to ADPR. In contrast, no detectable
cADPR could be measured in either the E150L/D151V-CD38 or the CD38
negative (NeoR) homogenates. To ensure that we were not at the limits
of detection of our assay, the experiments were repeated using 10-fold
more (5.0 x 106 dpm) labeled
[adenine-2,8-3H]NAD+. However, even when NeoR
and E150L/D151V had catabolized >80% of the 3H-labeled
NAD+ to ADPR, no [3H]cADPR could be detected
(not shown), demonstrating that the cADPR/ADPR ratio for NeoR and
E150L/D151N is certainly no higher than that for wild-type CD38 and, if
anything, is somewhat lower. Importantly, the very low residual
NAD+-converting activity of E150L/D151V-CD38 that was
observed in the nicotinamide release assay (Table I) must be primarily
due to NAD+ glycohydrolase activity and cannot be
attributed solely to ADP-ribosyl cyclase activity.
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. The cyclase activity of the CD38 cytoplasmic and
transmembrane mutant homogenates (µATG-CD38 and µTFR-CD38) was
approximately equivalent to that of WILD-CD38. The cyclase activities
of HA-CD38, E150Q/D151N-CD38, and C123K-CD38 were very elevated (20- to
140-fold increased) relative to that of WILD-CD38. In contrast, the
cyclase activities of the homogenates prepared from the CD38 negative
NeoR clone and the E150L/D151V-CD38 clone were at least 40- to 400-fold
lower than that of the wild-type enzyme. Given that we were unable to
measure cADPR production by these homogenates, the actual cyclase
activity is probably significantly lower than the minimal calculated
activity given in Table II. Finally, the cyclase activity in each of
the homogenates was normalized for CD38 and total protein content
(Table II, third column). Again, even after these
adjustments, the cyclase activity of the different CD38 mutants did not
change substantially relative to that of the CD38 wild-type control
(see fold differences in parentheses in columns 2 and
3 in Table II). The cytoplasmic tail, transmembrane domain, and HA binding sites can be replaced without impairing CD38-mediated signal transduction
From the previous experiments it was clear that we had
produced a panel of cell lines that expressed CD38 mutant molecules
with varied enzymatic properties. This panel included CD38 molecules
with wild-type enzymatic activities (µATG-CD38, µTFR-CD38), a CD38
molecule with greatly reduced catalytic activity (E150L/D151V), and
molecules with increased ADP-ribosyl cyclase activity (E150Q/D151N,
C123K, and HA-CD38). We next tested the effects of these mutations on
CD38-mediated signaling. To be able to directly compare the
anti-CD38-mediated response of all of the CD38-transfected cell
lines generated, all the different mutant mixed clones were stimulated
with an optimal dose of cross-linked polyanti-CD38 for 6–8 h. The
supernatants were collected, and IL-2 production was measured by
bioassay. The experiment was repeated seven times, and in each
experiment the anti-CD38-mediated IL-2 response of WILD-CD38 was
set at 1.0, and the amount of IL-2 produced by each of the cell lines
expressing mutated forms of CD38 was calculated relative to the
wild-type response. The IL-2 response from each of the mutants from all
seven experiments is shown as a scatter plot in Fig. 4. As expected, none of the mixed clones
produced IL-2 constitutively, and all of the mixed clones produced
similar amounts of IL-2 after PMA and Ca2+ ionophore
treatment (not shown). Extending our previously published results 20 ,
we found that complete replacement of the cytoplasmic tail of CD38
(µATG-CD38) had no deleterious effect on CD38-mediated signaling. In
fact, the response of the µATG-CD38-expressing clone to anti-CD38
was consistently improved over that of WILD-CD38 (Fig. 4). When both
the cytoplasmic tail and the transmembrane domain of CD38 were replaced
(µTFR-CD38), there was again no effect on anti-CD38-mediated IL-2
induction, with the average response being equivalent to that of
WILD-CD38 (Fig. 4). When the putative HA binding sites of CD38 were
eliminated, the average anti-CD38-mediated response was reduced to
75% that of WILD-CD38; however, in about half the experiments the
response was equivalent to that of WILD-CD38 (Fig. 4). Although the
HA-CD38 IL-2 response was somewhat lower than the wild-type response,
it was easily measurable and was at least 10-fold increased over the
IL-2 detection threshold (<0.2 U/ml) and the CD38-negative control
clone (NeoR) in all experiments. Thus, from these experiments it
appeared as though the cytoplasmic tail, the transmembrane domain, and
the HA binding sites of CD38 could be completely replaced without
dramatically altering CD38 signaling.
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Although none of the previously described mutations in CD38
appreciably affected signal transduction, an examination of cells
expressing CD38 extracellular mutant molecules revealed striking
defects in anti-CD38-mediated signal transduction. For example, the
anti-CD38-induced IL-2 response of the C123K-CD38- and
E150Q/D151N-CD38-expressing cell lines was reduced by 80–90% compared
with that of cells expressing wild-type CD38 molecules, with little
variance in the response from experiment to experiment. Thus, the
reduced response of cells expressing these CD38 extracellular domain
mutants could not simply be attributed to the health or condition of
the cells on any given day (Fig. 4). Taken together, these experiments
indicate that the extracellular domain of CD38, rather than its
membrane anchor or cytoplasmic tail, controls anti-CD38-mediated
signal transduction in B lymphocytes.
CD38-mediated coreceptor activity is regulated by its extracellular domain
Previously we demonstrated that cocross-linking of the BCR and
CD38 can augment activation responses in both A20 cells and normal B
cells 20, 34 . Since some of the mutations in the extracellular
enzymatic domain of CD38 greatly decreased CD38-mediated signaling,
we next evaluated CD38-mediated costimulatory activity in the cells
expressing mutant CD38 molecules. Mixed clones expressing µTFR-C38,
HA-CD38, the various extracellular domain point mutations, and
WILD-CD38 were stimulated for 8 h with suboptimal doses of
cross-linked anti-Ig alone (Fig. 5, open bars), cross-linked
anti-CD38 alone (striped bars), or cocross-linked anti-CD38 and
anti-Ig (filled dark bars). IL-2 production was measured and is
shown in Fig. 5. In all cases, no
detectable response was made to suboptimal cross-linking of either the
BCR alone or CD38 alone. Interestingly, all the mixed clones did make a
synergistic IL-2 response when CD38 and the BCR were coligated, but
there were differences among the various cell lines in the strength of
the IL-2 response. There was no appreciable change in the synergistic
response of the µATG-CD38 (not shown), µTFR-CD38, or HA-CD38 mixed
clones compared with that of WILD-CD38 (Fig. 4). In contrast, cells
expressing the other CD38 extracellular domain mutant molecules made an
IL-2 response that was reduced 50–75% compared with that of
WILD-CD38.
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These data demonstrate that the sequences associated with the
cytoplasmic tail and transmembrane domain of CD38 are not required for
CD38-mediated signaling and coreceptor activity. Additionally, the data
indicate that selected point mutations in the extracellular domain of
CD38 can impair signaling, suggesting that the extracellular domain is
instrumental in CD38-mediated signal transduction. To determine whether
there was any correlation between the ability to mediate signal
transduction and the ability to produce nicotinamide
(nicotinamide-releasing activity), ADPR (NAD+
glycohydrolase activity), or cADPR (ADP-ribosyl cyclase activity), the
relative signaling capacity of each of the mutants was plotted vs their
relative enzyme capacities (Fig. 6). In
each case, the signaling capacity and enzymatic activity of WILD-CD38
(filled circles) were set at 1.0. The signaling capacity and enzymatic
activity of the cells expressing the altered CD38 molecules (filled
squares) were then compared relative to those of wild-type CD38.
Interestingly, there was no correlation between the signaling capacity
of CD38 and its ability to produce nicotinamide, cADPR, or ADPR. Most
strikingly, cells expressing the catalytically impaired
E150L/D151V-CD38 molecules made an easily detectable IL-2 response
after anti-CD38 stimulation. In fact, the average response of this
cell line was 65% that of wild-type cells, with a range of responses
that varied between 10–110% of wild-type cells (Fig. 4).
Additionally, cells expressing CD38 mutant proteins that had increased
cADPR production (E150Q/D151N and C123K) did not signal better than
cells that were not capable of producing any cADPR (E150L/D151V) and,
in fact, signaled less well. Taken together, these data suggest that
while the extracellular domain of CD38 is required for signal
transduction, its nicotinamide-releasing activity, NAD+
glycohydrolase activity, and ADP-ribosyl cyclase activities are
unlikely to be required for anti-CD38-mediated signaling in B
cells. The implications of these findings for CD38-mediated signal
transduction in B lymphocytes is discussed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). CD38 can also
hydrolyze cADPR into ADPR (cADPR hydrolase activity) 13 and can
catalyze base exchange reactions (transglycosidation) between the
nicotinamide group of NAD+ or NADP+ and other
acceptor compounds 35, 36 (Fig. 3). Interestingly, at least two of
the products generated by the enzymatic activities of CD38 have been
shown to regulate intracellular calcium levels 8, 35, 36 , and it has
been hypothesized that these products may control CD38-mediated signal
transduction 1, 13, 16 . To directly test this hypothesis, we
introduced a number of mutations into the murine CD38 molecule and
assessed their impact on the enzymatic activities of CD38 and its
ability to mediate signal transduction. A comparison of the signaling
capacity and enzymatic capabilities of cell lines expressing the mutant
molecules relative to those expressing WILD-CD38 is provided in Fig. 6. From the presented data it is clear that complete replacement of the cytoplasmic tail and transmembrane domain of CD38 does not alter signaling or any of the measured enzyme activities, suggesting that the cytoplasmic tail and transmembrane domains of CD38 are not necessary for any of its described functions. Instead, the enzyme activity and signaling properties of CD38 appear to be controlled by its extracellular domain, as independent point mutations in the extracellular domain of CD38 could alter both enzyme activity and anti-CD38-mediated signaling. For example, all the measured enzyme activities of one mutant, E150L/D151V-CD38, were dramatically reduced. In particular, the nicotinamide-releasing capacity of homogenates of cells expressing this mutant molecule was only 1–3% the activity of homogenates of cells expressing wild-type CD38. Since the release of nicotinamide from NAD+ is the necessary first step for all the known enzymatic activities of CD38, it is clear that this molecule is significantly catalytically impaired. However, no commensurate effect on signaling was observed, as the anti-CD38-induced IL-2 response in cells expressing E150L/D151V-CD38 was reduced less than twofold compared with that in cells expressing wild-type CD38. Thus, the enzyme activities of CD38 do not appear to be absolutely required to mediate anti-CD38 signaling in vitro.
In agreement with this conclusion, soluble recombinant enzymatically
active CD38 was not sufficient to induce signaling in A20 cells, even
after addition of the stimulatory anti-CD38 Ab (not shown).
Additionally, the direct addition of purified cADPR, ADPR, or both
metabolites to cultures of CD38-negative and CD38+ A20
cells did not induce cytokine production (not shown). Thus, not only is
enzyme activity of CD38 insufficient to induce signaling, the data also
suggest that the products produced via the catalytic activity of CD38
do not induce cytokine production on their own or directly regulate
anti-CD38-mediated signaling. This conclusion was further
strengthened when we compared the signaling capacity of the mutant CD38
molecules with their capacity to produce the different enzymatically
generated products (Fig. 6). For example, we found that the E150Q/D151N
mutant was >100-fold better at producing cADPR than the wild-type
enzyme, yet the signaling capacity of this mutant was reduced by
>80%. In contrast, the cyclase activity of E150L/D151V-CD38 was at
least 50-fold lower than that of wild-type CD38, yet the signaling
capacity of this mutant was reduced <2-fold.
Interestingly, the mutants that made the most cADPR (C123K-CD38 and E150Q/D151N-CD38) were also the most compromised in their ability to mediate signal transduction, suggesting that cADPR production might negatively regulate CD38 signaling. However, we believe that this possibility is unlikely. First, one might expect that CD38 mutants that make less cADPR than the wild-type enzyme should signal better than the wild-type protein. The E150L/D151V mutant, which does not make any detectable cADPR, can mediate signal transduction, but does not signal more efficiently than the wild-type CD38. Additionally, only a 2-fold difference in cyclase activity was seen when HA-CD38 and C123K-CD38 were compared, yet there was a striking difference in the signaling capacity of these two mutants. In contrast, an 800-fold difference in cyclase production between HA-CD38 and E150L/D151V resulted in only a 15% difference in signaling. Thus, if there is a negative correlation between cADPR production and signaling, there would have to be a very sharply defined border between the amount of cADPR produced and the signaling capacity of the cells.
To eliminate the concern that the polyclonal anti-CD38 Ab used to
initiate CD38 signaling might alter the enzymatic activities of CD38 or
may not be able to bind to the mutant molecules efficiently, we
repeated the enzyme measurements in the presence and the absence of
polyclonal anti-CD38, but found no differences in reaction rates or
the measured cADPR/ADPR ratios (not shown). Additionally, as shown by
FACS analysis (Fig. 2), all the mutant CD38 molecules were recognized
by the polyclonal anti-CD38 Abs used in the stimulation
experiments. Finally, Biacore measurements of the affinity of the
polyclonal anti-CD38 for the soluble version of one of the most
repressed signaling mutants, E150Q/D151N-CD38, was identical with the
affinity measured for WILD-CD38, demonstrating that the mutant could be
efficiently bound by the polyclonal anti-CD38 Ab (not shown). Thus,
we were left with the conclusion that anti-CD38-mediated signaling
in B lymphocytes can occur independently of CD38 enzyme activity or
enzymatically generated products.
This conclusion, at first glance, is in apparent contradiction to our own earlier published observation that addition of purified cADPR to activated B cells enhanced their proliferative capacity 13 . However, upon closer examination, we believe that the two findings are not mutually exclusive. In the earlier experiments we found that B cells that had been induced to enter cell cycle by a number of different stimuli, including LPS, anti-CD40, anti-Ig, and anti-CD38 13 , were all further responsive to the addition of purified cADPR. Thus, cADPR, perhaps through its calcium-mobilizing capacity, acted as a growth enhancer for all activated B cells. Since CD38 can produce cADPR, this might argue that the enzymatic activity of CD38 can enhance the proliferation of replicating B cells. However, this does not imply that cADPR is required for CD38 to initiate its own signal transduction pathway. Thus, the enzymatic activities of CD38 may play an important role in producing metabolites, such as cADPR, that can alter cell signaling; however, these same metabolites do not appear to be directly required to induce anti-CD38-mediated activation and cytokine production in B lymphocytes.
How, then, can we explain the requirement for the extracellular domain
of CD38 to mediate signal transduction if cADPR, ADPR, and nicotinamide
production are not responsible? First, perhaps the other known enzyme
activities of CD38, such as its base exchange reaction or cADPR
hydrolase activity (Fig. 3), might produce or destroy metabolites that
regulate CD38-mediated signaling. Although this hypothesis might be
used to explain our results, we believe the E150L/D151V-CD38 mutant
argues against this possibility. For example, we were unable to detect
cADPR hydrolysis by E150L/D151V-CD38, demonstrating that this enzyme
activity was also impaired in the mutant (not shown). Additionally,
since the base exchange reaction requires the release of nicotinamide
from NAD+, and the nicotinamide-releasing capacity of
E150L/D151V-CD38 was only 1–3% that of wild-type CD38, it is
exceedingly unlikely that this mutant is able to produce any other
signaling metabolites.
An alternate model is shown in Fig. 7,
which hypothesizes that the structure or conformation of CD38’s
extracellular domain regulates CD38-mediated signal transduction via
CD38’s association with other signaling molecules. We have previously
demonstrated that anti-CD38-mediated signal transduction in B cells
is critically dependent on expression of the BCR. Additionally, the
data suggested that the interaction between CD38 and BCR must occur
extracellularly via an intermediary protein that we have called Ag
receptor-associated protein (ARAP) 20 . Thus, if the conformation of
the CD38 ectodomain was altered such that ARAP and CD38 could not
efficiently communicate, signal transduction would be predicted to be
greatly reduced. In agreement with this possibility, we know that the
two mutations that most affected signaling (C123K and E150Q/D151N) are
predicted to alter the conformation of the CD38 enzyme active site. The
C123K mutation is within the solvent-exposed hinge region of CD38 and
is believed to change the size of the CD38 active site by changing the
conformation of the extracellular domain 37 . In contrast, the
residues at positions 150 and 151 are predicted to be localized
spatially within the buried hydrophobic carboxyl-terminal pocket that
has been hypothesized to be the active site of this family of enzymes
37 . Interestingly, the only difference between the catalytically dead
E150L/D151V CD38 mutant and the altered enzyme profile E150Q/D151N-CD38
mutant was the choice of amino acid replacements at residues 150 and
151. Thus, it appears that these residues are involved in catalysis of
the oxocarbonium intermediate (Fig. 3). Finally, we know that mutations
within the active site of the enzyme also alter global protein
conformation, as at least one of the mutants, E150L/D151V-CD38, is no
longer recognized by the mAb anti-CD38 Ab NIMR-5 18 (data not
shown). Thus, it is at least possible that some of the mutations that
altered CD38 conformation also altered the capacity of CD38 to
associate efficiently with ARAP, and thus diminished
anti-CD38-mediated signaling.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Frances E. Lund, The Trudeau Institute, P.O. Box 59, Saranac Lake, NY 12983. E-mail address:
3 Current address: Anergen, Inc., 301 Penobscot Dr., Redwood City, CA 94063.
4 Abbreviations used in this paper: cADPR, cyclic adenosine diphosphate ribose; ADPR, adenosine diphosphate ribose; HA, hyaluronate; BCR, B cell Ag receptor; ARAP, Ag receptor-associated protein.
Received for publication September 1, 1998. Accepted for publication November 23, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, granulocyte-macrophage colony-stimulating factor and IL-10 cytokines after activation of human purified T lymphocytes upon CD38 ligation. Cell. Immunol. 173:192.[Medline]
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, Glu150 and
Asp151. The various mutants are diagrammed schematically
with replacement amino acids (*). To produce µATG-CD38, the
cytoplasmic tail of CD38 was replaced with the signaling inert mouse
IgM BCR cytoplasmic tail, and the transmembrane domain and
extracellular domain of CD38 were left intact. The µTFR-CD38 mutant
contains the cytoplasmic tail from the IgM BCR, the transmembrane
domain from the mouse transferrin receptor (31), and the wild-type CD38
extracellular domain. The HA-CD38 mutant eliminates all the putative HA
binding sites in murine CD38 (28), one of which is located in the
cytoplasmic tail and the other of which is located in the extracellular
domain. To eliminate the cytoplasmic tail HA site, the cytoplasmic tail
was truncated, the transmembrane domain was left intact, and two of the
three canonical lysine residues in the putative extracellular HA site
(amino acids 131 and 133) were mutated to serine and threonine,
respectively. The extracellular enzymatic domain mutants were generated
with a CD38 wild-type cytoplasmic tail and transmembrane domain and
point mutations within the extracellular domain at positions 123, 150,
and/or 151. These mutants are designated by their respective amino acid
replacements.
