Sep 19, 1994 - 201 of human CD38, exhibited not only ADP-ribosyl cy- clase activity but also cADPR hydrolase. These results indicate that cysteine residues ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Communication
Vol. 269, No. 46, Issue of November 18, pp. 28555-28557, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.
Essential Cysteine Residuesfor Cyclic ADP-riboseSynthesis and Hydrolysis byCD38* (Received for publication, June 13, 1994, and in revised form, September 19, 1994)
Akira TohgoS, Shin Takasawa, Naoya Noguchi, Tetsuhiko Koguma, Koji Nata, Takako Sugimoto, Yasuhito Furuya, Hideto Yonekura, and Hiroshi Okamotos From the Department of Biochemistry, Tbhoku University School of Medicine, Sendai 98077, Miyagi, J a p a n
We have recently demonstrated that cyclic ADP-ribose (cADPR) serves as asecondmessengerforglucoseinduced insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370373) and that human leukocyte antigen CD38 has both ADP-ribosyl cyclase and cADPR hydrolase activities (Takasawa, S., Tohgo, A, Noguchi, N., Koguma, T., Nata, K, Sugimoto, T., Yonekura, H., and Okamoto,H. (1993) J. Biol. Chem. 268,26052-26054). Although the amino acid sequence of Aplysia ADP-ribosyl cyclaseexhibits a high degree of aminoacidsequence identity with that of CD38, the Aplysia enzyme shows only ADP-ribosyl cyclase but notcADPR hydrolase. Inthe present study, we introduced site-directed mutations to CD38 and found that C119K- and/or C201E-CD38 exhibited only ADPribosyl cyclase activity. Furthermore,Aplysia ADP-ribosyl cyclase into whichweintroduced the mutations K95C and E176C, which correspondto residues 119 and 201 of human CD38, exhibited not only ADP-ribosyl cyclase activity but also cADPR hydrolase. These results indicate that cysteine residues 119 and 201 in CD38 have crucial roles in the synthesis and hydrolysisof cADPR. Cyclic ADP-ribose (cADPR)l is generated in pancreatic islets by glucose stimulation, servingas a second messenger forCa2+ mobilization in the endoplasmic reticulum to secrete insulin (1, 2). cADPR is synthesized fromNAD+by ADP-ribosyl cyclase (3) and is hydrolyzed to ADP-ribose (ADPR) by cADPR hydrolase (4, 5 ) . Human leukocyte antigen CD38 and its rodent homologues have been demonstrated to have both ADP-ribosyl cyclase and cADPR hydrolaseactivities (6-10). Comparative analysis revealed that the amino acid sequences of Aplysia kurodai and Aplysia californica cyclases(11, 13),2 whichdo not
* This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture, Japan, the Japan Foundation for Applied Enzymology, and the Japan Diabetes Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. S Recipient of a fellowship fromthe Japan Society for the Promotion of Science. 8 To whom all correspondence should be addressed: Dept. of Biochemistry, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-77 Miyagi, Japan. Tel.: 81-22-274-1111(ext. 2211); Fax: 81-22-272-8101. The abbreviations used are: cADPR, cyclicADP-ribose;ADPR, ADPribose; HPLC, high pressure liquid chromatography. K. Nata, T. Sugimoto, A.Tohgo,T. Takamura, A. Matsuoka, T. Numakunai, K. Shikama, H. Yonekura, S. Takasawa, and H. Okamoto (1994) Accessionno. D30048, EMBL Data Base.
'
show cADPR hydrolase activity (11, 141, showed a high degree of amino acid sequence identity with that of CD38 (6, 15, 16) and that 10 cysteine residues are conserved between Aplysia enzymes and human CD38 (11, 16). From recent results of cDNA cloning of rat (6, 10) and mouse (17) homologues to human CD38, we found that another 3 cysteine residues (Cys-16, Cys-119, and Cys-201 in human CD38) a r e conserved among the mammalian CD38s, but that the amino acid residue corresponding to Cys-16 of CD38 is not in Aplysia enzymesand those corresponding to Cys-119 and Cys-201 of CD38 are Lys-95 and Glu-176, respectively, in both Aplysia enzymes (see Fig.1). Here we introduced site-directed mutations into the human CD38 cDNA to replace the cysteine residues, expressed them in COS-7 cells, and showed that the Aplysia type (C119K and C201E) CD38 exhibited only ADP-ribosyl cyclase activity b u t not cADPR hydrolase. Moreover, we introduced mutations of K95C and E176C to Aplysia ADP-ribosyl cyclase and found that the CD38 type Aplysia protein exhibited not only ADPribosyl cyclase but also cADPR hydrolase activity. EXPERIMENTAL.PROCEDURES Site-directedMutagenesis-The site-directed mutants were made accordingto the proceduredescribed by Kunkel et al. (18) using a Mutan-KTMmutagenesis kit purchased from Takara Shuzo Co. Ltd. (Otsu, Japan). The synthetic oligonucleotidesusedfor site-directed mutagenesis were as follows (see Fig. 1): 5"AGAGAGCCGGgGCAGGGTTTG-3' (C16A),5"AATCTTGTTGgAGGTACGGTC-3' (C119A), 5'-AATCTTG"TI'TAGGTACGGTC-3' (C119K), 5"AATCTTGTTTTTTAGGTACGGTC-3' (C119E), 5"AATCTTGTTGCGAGGTACGGTC-3' (C119R), 5'-GAATTCACCAgCCATGTGAGG-3' (C160A1,5"CCAGTCTGGGgAGATTGATAG-3'(C173A),5"GACCACATCAEGGCAGCTTCT-3'(CBOlA), 5'-GACCACATCmGGCAGCTTCT-3' (C201E), 5"GACCACATCmGGCAGCTTCT-3' (C201K), 5"GACCACATCAEGGCAGCTTCT-3' (C201D)forCD38: 5'-CAC'l"GTTgATGGCAGCGTC-3' (K95C), 5"CACATCTCC=ATGCACTGTGG-3' (E176C) for Aplysia kurodai ADP-ribosyl cyclase, whereunderlined nucleotides were altered. The mutant clones were selected after sequencing by the method of Sanger (19). Functional Expression of CD38-A pSV2 vector carrying either the wild type or mutant human CD38 cDNAwas introduced and expressed in COS-7 cells as described (6). Western Blot Analysis-Western blot analysis was carried out using an ECL detectionsystem (Amersham Corp.) as described previously(6). Monoclonal antibody against human CD38 was T16 (6,20) (Cosmo Bio Co., Ltd., Tokyo, Japan). The band intensity of the expressed CD38 was measured using a Bio-Rad model 620 video densitometer. Functional Expression of Membrane-anchoredAplysia ADP-ribosyl Cyclase-The 837-base pair fragment of Aplysia kurodai ADP-ribosyl cyclase cDNA(nuc1eotideresidues 136-973)' was ligated to the 3'-end of the 187-base pair fragment of CD38 cDNA (nucleotideresidue 63-249) (6, 15). The resultant 1-kilobase pair fragment of the chimera cDNA encoding the fusion protein composed of amino acid residues 1-59 of human CD38 (6, 15) and residues 29-282of Aplysia ADP-ribosyl cyclase' was subclonedinto the pSV2 vector and expressed in COS-7 cells as described (6). Enzyme Assays-ADP-ribosyl cyclase and cADPR hydrolase assays were performed as described (6) with COS-7 cell homogenatescontaining similar amounts of mutant enzyme proteins estimated by Western blot analysis. Briefly, the homogenate of COS-7 cells into which either the wild type or mutant CD38 expression vector, or several kinds of human CD38-Aplysia ADP-ribosyl cyclase chimera expression vectors had been introduced, was incubated for 10-20 min at 37 "C in 0.1 ml of phosphate-bufferedsaline (137 mM NaC1,2.7 mM KC1,8.1mM Na,HPO,, 1.5 mM KH'PO,,pH 7.4) (21) with 0.2 mM NAD+ containing 5 pCi of [32PlNAD+(DuPont NEN)forADP-ribosylcyclaseor with 0.2 mM CADPR containing 5 pCi of t3'PlcADPR, prepared as described previously (1,2,6)for cADPR hydrolase. Reaction products were analyzed by HPLC (2,6) using a flow scintillation analyzer (Flow-One Beta-525TR,
28555
28556
Essential Cysteine Residues in CD38 for cADPR Metabolism
QPEKVQTLEA ~ I H G G R E D s R D L ~ P T I K E L E S I I S K m I Q F S L I Y R D .F.$u h ! d q + W d i K g t * snatss*sh Y e V d z k t m i h i w d n k t f v M d i e g d snzistssssln *!artV-mi+ahrdd=i ? a m - t W t r v k v iil+rlg+ki i E ! d g a g S L I D e k L V k a X h f a i d h r e n p t a v l t k l i s d i -tKkitkb%v i t t f q l g q k i i E T t g a g S L 1& W k a K k fg+d_h.enpK svletlkadi
dinmkiqi
PEDsS%SEI m p Y r l n V
300 303 304
wpkkrlnt barefrlak rfyria
282
b a r e f q l a k ryyria
282
FIG.1. Alignment ofamino acid sequences of mammalianCD38s (ADP-ribosyl cyclase/cADPR hydrolase) and Aplysia ADP-ribosyl cyclases. Amino acid sequences were deduced from cDNA sequences. Amino acid residues are numberedon the right. Comparisons of rat and mouse CD38s and AplysiaADP-ribosyl cyclases with human CD38 were made basedon functional conservation;symbols (2)indicate residues that are identical to human CD38, and uppercaseletters and lowercase letters indicate residues that are conservative substitutions and non-conservative substitutions, respectively. Cysteine residues conserved among all the proteins are marked with dots. The two cysteine residues (Cys-119 and Cys-201 in humanCD38) found in mammalian proteins but not in those ofAplysia are marked with inverted triangzes. Sequence gaps resulting from optimization of alignment are indicatedby dashes. Human, human CD38 (6, 15);Rut, rat CD38 (IO); Mouse, mouse CD38 (17); A.cuZifornica, A. californica ADP-ribosyl cyclase (13); A.kurodai, A. kurodui ADP-ribosyl cyclase (Ref. 11; see also Footnote2).
Packard, Meriden, CT). The protein concentration was measured by the method of Bradford (22) using bovine serum albumin as a standard. RESULTS AND DISCUSSION
To investigate themolecular basis of cADPR metabolism, we compared the amino acid sequences of the human, rat, and mouse CD38s and Aplysia ADP-ribosyl cyclases. As shown in Fig. 1, the human, rat, and mouse CD38s, which have both ADP-ribosyl cyclase and cADPR hydrolaseactivities(6-lo), have conserved 13 cysteine residues (amino acid residues 16, 67, 82, 99, 119, 160, 173, 180, 201, 254, 275, 287, and 296 in human CD38). Three of the 13cysteines (amino acid residues 16, 119, and 201 in humanCD38) are notconserved in Aplysia ADP-ribosyl cyclases. Neither of the two Aplysia proteins, which do not havecADPR hydrolase activity but do have ADPribosyl cyclase activity (11, 14), have the region corresponding to Cys-16 of the human CD38. The corresponding amino acid residues of Cys-119 and Cys-201 in the human protein are lysine and glutamicacid, respectively, in both Aplysia proteins. These observations suggested that theamino acid residues 119 and 201 in CD38 and the corresponding residues in Aplysia proteins (residues 95 and 176) may account for the important differences in their enzyme activities. We introduced site-directed mutations into the human CD38 cDNA by replacing the 2 cysteine residues (Cys-119 and Cys201) with lysine and glutamic acid, as was found in theAplysia enzymes. The resulting cDNA was then expressed transiently in COS-7 cells. We thenincubatedthe homogenate of the COS-7 cells with [32P]NAD+ and analyzed the reaction products by anion exchange HPLC. The time courses of the changes of products are shown in Fig. 2. Although both the Aplysia type mutant CD38 (C119K- and C201E-CD38) and the wild type CD38 showed cADPR synthesizing activity, the timecourses of the cADPR concentrations were very different; the concentration of cADPR decreased in the lateperiod of incubation when the wild type CD38 was used, but sucha decrease didnot occur when the Aplysia type mutant CD38 was used and thecADPR hydrolyzing activity in the mutantwas scarcely detected (Fig. 2; see also C119WC201E in Fig. 3). The time courses of NAD+ consumption were comparable to the increases of cADPR plus ADPR in thereactions (data notshown). Theseresults indicate
0
10
Y
20
F,
Y
30
b o 40
Incubation Time (min) FIG.2. Time course of cADPR and ADPR formation. NAD+ was incubated (6) with the homogenate (10pg of protein) of COS-7 cells into which the wild type CD38 expression vector (0,a)had been introduced or with the homogenate(20 pg of protein) of COS-7 cells intowhich the C119K-lC201E-CD38 expression vector (0,U)had been introduced. The 0) formed in the0.1-ml incuamounts of cADPR ( 0 , O )and ADPR (D, bation assay (6)are expressed by nmol/ml in ordinates.
that Cys-119 and/or Cys-201 in CD38 play(s) an importantrole in cADPR hydrolysis. We next constructed several CD38 mutants in which the codon for Cys-119 and/or Cys-201 was altered (see "Experimental Procedures"). We made 11mutant CD38s in which the cysteine residuewas replacedby alanine, arginine,lysine, aspartic acid, or glutamicacid and one mutant inwhich 2 residues were replaced by lysine and glutamic acid, and introduced the muexpression of the tants intoCOS-7 cells. In all 12 mutants, the mutant protein was observed by immunoblot analysis3 and the levels of expression were estimated by densitometric analysis of the immunoblot. ADP-ribosyl cyclase and cADPR hydrolase activities were measured using the homogenate of COS-7 cells into which the mutants had been introduced as an enzyme source. The relativeactivities of each mutant asa percentage of the activity of the wild type CD38 are shown in Fig. 3. ADPribosyl cyclase activity was detected not only in the mutant with two residue replacements (C119K and C201E) but also in the mutant havingone cysteine replacement for the same residue asAplysia enzymes (C119K or C201E). We could not detect All the mutant proteins were expressed in the plasma membrane and microsome fractions of the COS-7 cells as was the wild type CD38 (61, and about50%of the CD38 enzyme activity inwild and mutantcell homogenates was detected using intact cells, suggesting that some of the enzyme protein was expressed in the surface.
28557
Essential Cysteine Residues in CD38 for cALIPR Metabolism ADP-Ribosyl Cyclase cADPR Hydrolase
TABLEI ADP-ribosyl cyclase and cADPR hydrolase activities of Aplysia ADP-ribosyl cyclase mutants NAD+ or cADPR was incubated with the homogenates of COS-7 cells into which the expression vectors had been introduced as described under“ExperimentalProcedures.” Values aremean -c S.E. (nmoV m i d m g protein) of triplicate experiments.
Wild type Aplysia ADP-ribosyl cyclase K95C mutant E176C mutant K95CE176C mutant
ADP-ribosyl cyclase
cADPR hydrolase
2.0 2 0.43 2.3 2 0.65 2.5 2 0.63 2.2 2 0.49
-
FIG.3. Relative ADP-ribosyl cyclase and cADPR hydrolase 2.1 2 0.39 activities of the site-directed mutants of CD38. Three separate a -, below detection (
