(Received for publication, November 4, 1996, and in revised form, November 26, 1996)
From the Department of Biochemistry, Tohoku University School of Medicine, Sendai 980-77, Miyagi, Japan
CD38 catalyzes not only the formation of cyclic ADP-ribose (cADPR) from NAD+ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR. To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA. Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129. We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg. Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR. Furthermore, the mutants did not bind cADPR, whereas they still used NAD+ as a substrate to form cADPR and ADPR. These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38.
Cyclic ADP-ribose (cADPR)1 (1) induces
the release of Ca2+ from microsomes in a variety of tissues
and cells including pancreatic cells (2-10). cADPR is synthesized
from NAD+ by ADP-ribosyl cyclase, which was purified as a
soluble 29-kDa protein from Aplysia ovotestes (11-13). The
amino acid sequences of Aplysia ADP-ribosyl cyclases (14,
15) showed a high degree of homology with that of CD38 (16, 17), which
was reported to be a surface antigen of human lymphocytes (18). From
the experiments in which CD38 cDNA was expressed in mammalian
cells, CD38 was shown to catalyze not only the formation of cADPR from NAD+ but also the hydrolysis of cADPR to ADP-ribose (ADPR)
(19-21). We have demonstrated that ATP inhibited the hydrolysis,
resulting in the accumulation of cADPR (19). Furthermore, we produced transgenic mice overexpressing human CD38 in pancreatic
cells and
demonstrated that ATP, produced in the process of glucose metabolism,
increased the accumulation of cADPR to enhance the Ca2+
mobilization from the intracellular stores for insulin secretion in the
transgenic islets (22).
In the present study, we expressed human CD38 in Escherichia coli and purified it to homogeneity. Using the purified CD38, we found that Lys-129 of CD38 participated in cADPR binding and that ATP competed with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38.
We isolated a CD38 cDNA
from an insulinoma of a Japanese patient by reverse
transcriptase-polymerase chain reaction (PCR) (19) and used it for
functional analyses of CD38 (17, 19, 22). The cDNA sequence was
exactly the same as the CD38 sequence reported by Jackson and Bell (18)
except for two base substitutions (nucleotide 213, A C, and
nucleotide 215, C
A) (19). The substitutions were also found in the
corresponding region of the genome isolated from a Japanese patient
(23). The CD38 cDNA (19) encoding amino acids 45-300 (17, 23) was
amplified by PCR using two primers, one sense
(5
-cct
AGGTGGCGCCAGACGTGGAG-3
, nucleotide positions
202-221) and one antisense
(5
-gc
GCTCAGATCTCAGATGTGCA-3
, nucleotide positions
955-974). Nonspecific nucleotides are shown in lowercase letters, and
SmaI and XbaI restriction sites are underlined,
respectively. After digestion by SmaI and XbaI,
the PCR product was inserted into the unique
XmnI-XbaI site of the pMAL-p2 vector (New England
Biolabs), downstream and in-frame with the maltose-binding protein
(MBP) coding sequence. The accuracy of the PCR-derived recombinant CD38
construct was confirmed by sequencing. After induction of the fusion
protein with isopropyl-1-thio-
-D-galactopyranoside in
E. coli, the protein was purified by affinity chromatography on cross-linked amylose resin (New England Biolabs). To separate the
CD38 from MBP, the fusion protein was cleaved by Factor Xa (New England
Biolabs), and the CD38 was purified by gel filtration (Sephacryl
S-100HR, Pharmacia Biotech, Uppsala, Sweden). To reconstitute the
enzymic activity, the purified CD38 was refolded as described (24).
The site-directed mutants were
made according to the procedure described by Reikofski and Tao (25)
using an LA PCR In Vitro Mutagenesis Kit (Takara Shuzo Co.
Ltd., Otsu, Japan). The synthetic oligonucleotides used for
site-directed mutagenesis were as follows: 5-AGCAGAATA
AGATCTGGCC-3
(K129A) and
5
-AGCAGAATAA
AGATCTGGCC-3
(K129R), which correspond to
the region from nucleotides 445-465 of the CD38 cDNA (19, 23) and
where underlined nucleotides were altered.
ADP-ribosyl cyclase and cADPR hydrolase assays were performed as described (17, 19, 26) with CD38. Briefly, 50 ng of CD38 was incubated for 5 min at 37 °C in 0.1 ml of phosphate-buffered saline (pH 7.4) (26, 27) with 0.2 mM NAD+ containing 5 µCi of [32P]NAD+ (DuPont NEN) for ADP-ribosyl cyclase or with 0.2 mM cADPR containing 5 µCi of [32P]cADPR, prepared as described previously (3, 17, 19, 26) for cADPR hydrolase. Reaction products were analyzed by HPLC (17, 19, 26) using a flow scintillation analyzer (Flow-One Beta-525TR, Packard, Meriden, CT). The protein concentration was measured by the method of Bradford (28) using bovine serum albumin as a standard.
Modification of CD38 with FSBAThe reaction of CD38 with FSBA (Boehringer Mannheim) (29, 30) was performed at 30 °C in phosphate-buffered saline (pH 7.4). The concentrations of CD38 and FSBA were 100 nM and 1 mM, respectively.
Detection of FSBA ModificationThe amount of covalent modification of CD38 with FSBA was estimated by Western blot as described previously (5, 17, 19, 26) using an ECL detection system (Amersham Corp.). A polyclonal antibody against FSBA (Boehringer Mannheim) was used at a final concentration of 1 µg/ml diluted with 5% milk powder solution as a primary antibody. The band intensity of the FSBA-labeled CD38 was measured using NIH Image software.
Identification of FSBA-labeled Peptide0.2 mg of CD38 was labeled with 1 mM FSBA for 4 h and subjected to proteolysis by lysylendopeptidase (Wako Pure Chemical Industries, Osaka, Japan) as described previously (31). The lysylendopeptidase fragments were separated by reverse-phase HPLC using a µRP C2/C18 column (3.2 × 30 mm, Pharmacia Biotech) at a flow rate of 200 µl/min. An aliquot (10 µl) of each peak was blotted on a polyvinylidene difluoride membrane and analyzed for the presence of FSBA-labeled peptide by Western blot using an anti-FSBA antibody as described above. The peak containing the FSBA-labeled peptide was subjected to automated Edman degradation as described previously (31).
cADPR Binding Assay1.5, 3, and 6 nmol of wild type CD38, K129A-CD38, K129R-CD38, or bovine serum albumin were incubated for 10-30 min at 4 °C in 0.1 ml of phosphate-buffered saline (pH 7.4) with 10 nmol of cADPR containing 5 µCi of [32P]cADPR. During the incubation, neither CD38 nor its mutants converted cADPR to ADPR. The incubation mixture was fractionated at 4 °C by a gel filtration column (PD-10, Pharmacia Biotech). 0.5-ml fractions were collected, and an aliquot (10 µl) of each fraction was analyzed for radioactivity. Another aliquot (50 µl) of each fraction was blotted on a polyvinylidene difluoride membrane and analyzed for the presence of CD38 by Western blot using a monoclonal antibody against human CD38 (T16, Cosmo Bio Co., Ltd., Tokyo, Japan) (17, 19, 26) as described above.
Human CD38 was expressed in E. coli and purified as a
soluble protein without the hydrophobic membrane domain (see
"Experimental Procedures"). When NAD+ was used as a
substrate, the purified CD38 catalyzed the formation of cADPR and ADPR
(Fig. 1A). On the other hand, when cADPR was used as a substrate, the CD38 exhibited the cADPR hydrolase activity to
form ADPR (Fig. 1B) as described previously (17, 19). We then examined the effect of ATP on the hydrolysis of cADPR by CD38. ATP
inhibited the hydrolysis in a dose-dependent manner (data
not shown), which was consistent with the previous result obtained by
using the COS-7 cell membrane fraction, in which CD38 was expressed, as
an enzyme source (19). Kinetic analysis was performed with various
concentrations of cADPR in the presence or absence of ATP, and the data
were plotted in a double reciprocal manner. As shown in Fig.
2, in the absence of ATP, CD38 had a Vmax value of 470 ± 38 nmol/min/mg of
protein (n = 3) and a Km value of
53 ± 7.0 µM (n = 3) for cADPR. ATP
inhibited the hydrolysis of cADPR in a competitive manner, with a
Ki value of 4.8 ± 0.53 mM
(n = 3). Competitive inhibition of the cADPR hydrolysis by ATP suggests that ATP and cADPR bind to the same site of CD38.
To identify the binding site for ATP and/or cADPR, we then labeled the
purified CD38 with FSBA (29, 30). As shown in Fig. 3A, the CD38 was labeled in a
time-dependent manner, and cADPR and ATP completely
inhibited the incorporation of FSBA into CD38, indicating that the
binding site for ATP and/or cADPR was affected by FSBA. We next
subjected the labeled CD38 to lysylendopeptidase digestion, and the
resultant peptide fragments were separated by reverse-phase HPLC (Fig.
3B). Aliquots of all peaks were withdrawn, and the presence
of FSBA-labeled peptide was analyzed by Western blot. Only peak 22 was
immunoreactive (Fig. 3B, inset). Edman degradation of peak 22 gave a 7-amino acid sequence ILLWSRI with yields
of 0.9-143.4 pmol/cycle, and the phenylthiohydantoin-derivative was
not identified for the 8th cycle. The 7-amino acid sequence corresponded to the sequence ILLWSRI in the theoretical
lysylendopeptidase fragment (amino acid residues 122-129) in the CD38.
Because it is known that FSBA can act as an electrophilic agent in
covalent reactions with several classes of amino acids, including
serine, tyrosine, lysine, histidine, and cysteine (29), Lys-129 is
therefore implicated as the FSBA-labeled residue that escaped
identification by Edman degradation.
We introduced site-directed mutation into the CD38 cDNA by
replacing the lysine 129 codon with that of alanine or arginine. The
resulting cDNAs were then introduced into E. coli and
mutant CD38s (K129A- and K129R-CD38) were produced and purified. As
shown in Fig. 4A, when the mutant CD38s were
incubated with FSBA, the mutant proteins were not labeled with FSBA,
indicating that Lys-129 of CD38 is actually the site of FSBA labeling.
We next analyzed the enzymic activities of the mutant CD38s. As shown
in Table I, the mutants did not catalyze the hydrolysis
of cADPR to ADPR, whereas their catalytic activity to form cADPR and
ADPR from NAD+ still remained. In addition, the
FSBA-labeled CD38 catalyzed the formation of cADPR and ADPR from
NAD+ but not the hydrolysis of cADPR to form ADPR (Fig. 1).
We next tested the mutant CD38s for their ability to bind to cADPR. As shown in Fig. 4B, cADPR was eluted in a gel filtration
chromatography around fraction 16. After incubation with wild type CD38
at 4 °C, a new elution peak appeared around fraction 3 (Fig.
4B, Wild), in which most of the immunoreactivity
against CD38 was recovered (inset), indicating that the
elution peak represented the CD38-cADPR complex. On the other hand, no
peak around fraction 3 appeared in chromatograms using mutant CD38s
(Fig. 4B, K129A and K129R), whereas
the CD38 immunoreactivity was recovered around fraction 3 (inset). These results indicate that the wild CD38 did bind to cADPR but the mutant CD38s did not, and that Lys-129 participates in
the cADPR/ATP binding.
|
On the other hand, as shown in Fig. 5, the
NAD+ binding site of CD38 appears to be different from
Lys-129 because both the site-directed mutant and FSBA-modified CD38s
used NAD+ as a substrate to form cADPR and ADPR (Fig. 1 and
Table I). Furthermore, the formation of cADPR and ADPR from
NAD+ by CD38 was inhibited by nicotinamide in a competitive
manner with a Ki value of 290 ± 32 µM (n = 3), whereas ADPR (up to 5 mM) did not inhibit the formation of cADPR and ADPR from NAD+. These results indicate that the binding of
NAD+ to CD38 is achieved by the nicotinamide moiety rather
than by the adenosine moiety. This is consistent with the previous
results that CD38 catalyzed the formation of cyclic GDP-ribose from
NGD+ (nicotinamide guanine dinucleotide) but used hardly
any cyclic GDP-ribose as a substrate to form GDP-ribose (32-34).
We have proposed a model for insulin secretion by glucose via cADPR-mediated Ca2+ mobilization (35). In the process of glucose metabolism, millimolar concentrations of ATP are generated (22), inducing cADPR accumulation by inhibiting the cADPR hydrolase activity of CD38, and cADPR acts as a second messenger for intracellular Ca2+ mobilization from the endoplasmic reticulum for insulin secretion (3, 5, 19, 22, 35, 36). In the present study, it has been shown that Lys-129 of CD38 participates in cADPR binding and that millimolar concentrations of ATP compete with the cADPR binding site, inhibiting the cADPR hydrolase activity of CD38. Therefore, when mutations at or around Lys-129 occur in CD38, cADPR metabolism cannot be regulated by ATP, which is generated in the process of glucose metabolism. Such a mutation in CD38 could be a predisposing factor for diabetes mellitus.
We are grateful to Brent Bell for valuable assistance in preparing the manuscript for publication and to Hideo Kumagai for skillful technical assistance.