Eur. J. Biochem. 264, 439±445 (1999) q FEBS 1999

Inhibitor peptide SNP-1 binds to a soluble form of BST-1/CD157 at a2 : 2 stoichiometry

Atsushi Sato1, Sumie Yamamoto1, Naoko Kajimura2, Masayuki Oda3, Jiro Usukura4 and Hisato Jingami1

Departments of 1Molecular Biology, 2Structural Biology and 3Bioinformatics, Biomolecular Engineering Research Institute, (BERI), Suita, Osaka,

Japan; and 4Department of Anatomy, School of Medicine, Nagoya University, Japan

Recently we have identified a 15-mer peptide, SNP-1, by a random phage library that can bind to bone marrow

stromal cell antigen-1 (BST-1)/CD157 [Sato, A., Yamamoto, S., Ishihara, K., Hirano, T. & Jingami, H. (1999)

Biochem. J. 337, 491±496]. SNP-1 inhibits BST-1 ADP-ribosyl cyclase activity uncompetitively with a Ki value

of 180 ^ 40 nm. In this study we analysed biophysically the SNP-1 binding to a soluble form of BST-1 (sBST-1).

Equilibrium binding data of wild-type SNP-1 from surface plasmon resonance studies gave a Kd value of

500 ^ 35 nm. Titration calorimetry analysis showed that the binding reaction is exothermic at 20 8C. The values

of Kd = 211 nm, enthalpy change, DH = ±18.68 kcal´mol21, and saturated molar ratio of bound SNP-1 per

sBST-1, N = 0.8 mol´mol21 were obtained. On the basis of the molecular masses of SNP-1 and sBST-1

calculated by analytical ultracentrifugation, the stoichiometry of the binding was determined to be 2 : 2. Electron

microscopy also revealed the dimer form of sBST-1. To delineate the core residue of SNP-1 responsible for

binding, each amino acid residue has been replaced by alanine. A region from amino acid residues 7±12 appeared

to be critical for the SNP-1 binding to sBST-1. The substitution of the first residue, His, to Ala led to a reduction

in binding, suggesting that the N-terminal residue is also crucial.

Keywords: cyclic ADP-ribose; alanine scanning; titration calorimetry; analytical ultracentrifugation; rotary

shadowing.

Bone marrow stromal cell antigen 1 (BST-1) [1] was identifiedby expression cloning using monoclonal antibodies againstbone marrow stromal cell lines derived from rheumatoidarthritis patients. BST-1 is a 40±46-kDa glycosyl-phosphatidy-linositol-anchored membrane protein with amino acid sequencesimilarity to CD38 [2], a type II membrane protein. Bothproteins encode ADP-ribosyl cyclase and cyclic ADP (cADP)-ribose hydrolase activities in their extracellular regions [3,4].ADP-ribosyl cyclase produces cADP-ribose from NAD+; thecADP-ribose is degraded by cADP hydrolase. cADP-ribose, theproduct of the enzymatic reaction, is known to be a modulatorof calcium release from intracellular calcium stores indepen-dent of the inositol 1,4,5-trisphosphate system. cADP-ribosehas been reported to be involved in various biologicalphenomena [5]. The relation between the extracellular enzy-matic activity and the function of cADP ribose, includingsignaling pathways both upstream and downstream of themolecule is currently being investigated. Internalization ofCD38 has recently been reported [6,7] and this finding may be aprelude to the elucidation of the roles of the ectoenzymeactivities. On the other hand, cross-linking studies usingagonistic antibodies have raised the possibility that extracel-lular signals may be transmitted through CD38 and BST-1.CD31 has recently been determined to be a ligand of CD38[8,9]. Thus, these ectoenzymes may function as a membranereceptor themselves or as receptor-associated proteins. Geneknock-out mice lacking these membrane cyclases exhibit

interesting phenotypes: abnormalities in B-cell developmentand antibody production in BST-1-deficient mice [10] andimpairment of T-cell dependent antibody response in CD38-deficient mice [11].

The atomic structure of Aplysia ADP-ribosyl cyclase [12],which has amino acid sequence similarity to BST-1 and CD38,has been resolved: the cyclase formed both a monomer anddimer under two different crystallization conditions. Assumingsimilarity to the structure of the Aplysia cyclase, a model ofCD38 has been proposed. Recently we obtained the peptideinhibitor of BST-1 ADP-ribosyl cyclase from a phage librarydisplaying 15-mer random peptides [13]. We have reported thatthe peptide inhibitor, SNP-1, inhibits BST-1 ADP-ribosylcyclase uncompetitively with a Ki value of 180 ^ 40 nm bysolution kinetic studies. SNP-1 does not affect the CD38enzyme activities. To gain a better understanding of ecto-ADPribosyl cyclase of BST-1, a study looking at the binding of thepeptide inhibitor to a soluble form of BST-1 (sBST-1) wasperformed. Here we have determined the stoichiometry ofSNP-1 binding to sBST-1 by analytical ultracentrifugation andisothermal titration calorimetry. Furthermore, we have carriedout a study of the binding of an alanine-scanning mutant SNP-1to sBST-1 by surface plasmon resonance to gain the informa-tion about which amino acid residues are critical for binding.These data will help to understand the intriguing ecto-ADPribosyl cyclase of BST-1 and to design a more potent inhibitoragainst its enzyme activity.

M A T E R I A L S A N D M E T H O D S

Production and purification of sBST-1

Production of sBST-1 in baculovirus-infected insect cells andpurification of its protein using ion-exchange chromatography

Correspondence to H. Jingami, Department of Molecular Biology,

Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita,

Osaka 565-0874, Japan. Fax: + 81 6 6872 8219, Tel.: + 81 6 6872 8214,

E-mail: jingami@beri.co.jp

Abbreviations: BST-1, bone marrow stromal cell antigen-1; cADP, cyclic

ADP.

(Received 25 February 1999, revised 3 March 1999, accepted 10 June 1999)

440 A. Sato et al. (Eur. J. Biochem. 264) q FEBS 1999

and dye ligand (blue) affinity chromatography has beendescribed previously [13].

Peptide synthesis and purification

Peptides were synthesized by standard automated methods onan Applied Biosystems Model 430A peptide synthesizer. Thepeptides were purified by reverse-phase HPLC and weredetermined to be homogenous by analytical HPLC (morethan 95% pure) and electrospray ionization mass spectrometry.The amino acid sequence of SNP-1 is as follows: NH2-His-Ser-Gln-Ile-Ser-Gly-Lys-Tyr-Gln-Arg-Try-Leu-Lys-Asp-Ala-COOH [13].

BIAcoreTM analysis

sBST-1 (2000±3600 RU) was amine-coupled to a CM5sensorchip using an amine coupling kit in a BIAcore2000 Biosensor (Biacore K.K., Tokyo, Japan) in 10 mmacetate buffer, pH 5.0. SNP-1, alanine-substituted SNP-1and C-terminal modified SNP-1 at the indicated concentrationswere injected over the sBST-1 surfaces at a flow rate of40 mL´min21. Binding was monitored at 25 8C. The buffer usedwas 20 mm Mes (pH 6.0) containing 150 mm NaCl, 3.4 mmEDTA and 0.005% Tween 20. Immobilized sBST-1 wasregenerated with 10 mm HCl. Analyses were performed witha nonlinear fit (1 : 1 binding with mass transfer) method withbia evaluation 3.0 software and a linear curve fitting of theScatchard plots.

Sedimentation equilibrium ultracentrifugation

Apparent molecular masses were determined by sedimentationequilibrium with a Beckman XL-I ultracentrifuge at 25 8C. Theloading concentration of SNP-1 and sBST-1 in 50 mm Mes(pH 6.0) and 100 mm NaCl were 200 mm and 10 mm,respectively. The rotor speed for each sample is listed inTable 2. The density of the loading buffer was measuredgravimetrically (r = 1.008 g´mL21). Partial specific volumeswere calculated from the average mass of the partial specificvolume of the individual amino acids. An average value of0.630 mL´g21 was used for the partial specific volume of thecarbohydrate component. A value of 0.728 mL´g21 wasestimated from the cDNA sequence of unglycosylatedsBST-1. As the difference in molecular mass of sBST-1(33.5 kDa) determined by mass spectroscopy from thatestimated from the cDNA sequence (30.5 kDa) is consideredto be due to the mass of carbohydrate, 10% glycosylation wasassumed for sBST-1. The partial specific volume for glycosy-lated sBST-1 was calculated from the following equation:

�1 � 0:630� 9 � 0:728�=10 � 0:718mL´g21

The peptide absorbance was monitored at a wavelength of280 nm. Equilibrium was assumed if no change in distributionwas observed over intervals of 2 h. The sedimentationequilibrium data were analysed using the software originprovided with the instrument. The data were shown to fit to asingle species.

Isothermal titration calorimetry

Titration calorimetry was performed at 20 8C using a MicrocalOmega titration calorimeter (Microcal) as described byWiseman et al. [14]. The solutions of SNP-1 and sBST-1were separately dialysed against 20 mm Mes (pH 6.0). Five

microliters of 400 mm SNP-1 was injected into the calorimetercell containing 2.0 mL of 10 mm sBST-1 at 3.0 min intervals.Integration of the calorimetric data was performed using thesoftware origin provided with the instrument.

Fig. 1. BIAcoreTM analysis of SNP-1 binding to immobilized sBST-1.

(A) Sensorgrams showing the interaction of SNP-1 with immobilized sBST-1.

sBST-1 (2000 RU) was directly immobilized on the surface of sensorchip

CM5. SNP-1, at concentrations ranging from 400 nm to 1000 nm (lower to

upper curves: 400, 500, 600, 700, 800, 900 and 1000 nm), was injected onto

the sensorchip at a flow rate of 40 mL´min21. A control peptide at 1000 nm

was injected as a negative control (bottom curve). (B) Equilibrium binding

data (Req versus C) obtained in A. Req, RU of SNP-1 bound at equilibrium;

C, concentrations of SNP-1 injected. (C) Scatchard plot analysis of data in

(B). The equilibrium dissociation constant (Kd) was calculated from the

slopes of the fitted line (Kd = 500 ^ 35 nm). RU, resonance unit.

q FEBS 1999 ADP-ribosyl cyclase inhibitor peptide (Eur. J. Biochem. 264) 441

Low angle rotary shadowing

Glycerol was added to BST-1 preparations at concentrations ofup to 50% (v/v). The final concentration of the protein was1.0 mg´mL21. Thirty microliters of each preparation wassprayed onto a mica surface that had been freshly cleavedusing a painter's airbrush (Olympus Model SP-B,

diameter = 0.18 mm). Then, the mica was rapidly broughtinto a freeze-etching device equipped with a large turbo pump(FR 7000, Hitachi, Mito, Japan), dried for 10 min (roomtemperature) in vacuum (1 � 1026 Pa), and then cooled to2100 8C. Subsequently, specimens were rotary shadowed withplatinum by an electron gun positioned at an angle of 2.58 to themica surface and then followed by carbon evaporation.

Table 1. Kinetic rate constants of various SNP-1 derivatives binding to BST-1. ND refers to values that could not accurately be determined because both

association and dissociation constants are very rapid. Eq, equilibrium.

Peptide k on (m21´s21)

Kd (nm)

k off (s21)

Kd (nm)

Off/On Eq

SNP-1(±NAD+) 1.4 �^ 0.3 � 105 0.073 �^ 0.015 520 �^ 20 500 �^ 35

SNP-1(+ NAD+)a 1.4 � 105 0.091 650 350

Ala scanning peptidesa

#1 ND ND ND 2020

#3 1.6 � 105 0.066 410 450

#6 9.4 � 104 0.056 600 740

#13 1.3 � 105 0.060 460 530

#14 3.0 � 105 0.18 600 560

C-terminal modificationa

Lys + Biotin 1.4 � 105 0.11 790 810

a Representative data obtained from two independent experiments are shown.

Fig. 2. Sedimentation equilibrium analysis of SNP-1 alone and sBST-1 alone. Measurements were made using 200 mm SNP-1 (A) or 10 mm sBST-1 (B)

in 50 mm Mes (pH 6.0) and 100 mm NaCl at 25 8C. The data fit well to an ideal single-species model.

442 A. Sato et al. (Eur. J. Biochem. 264) q FEBS 1999

Shadowed films were removed from the mica by slowlysoaking the mica into water, and they were then mounted oncopper grids for observation. We found that low angle rotaryshadowing at such low temperature and high vacuum enhancedresolution because it reduced the particle size of the evaporatedplatinum. In the conventional method, the temperature is about20 8C and the vacuum is not as high [15,16]. Molecules suitablefor image analysis were found in the periphery of the residualdeposit in which both proteins and buffer constituents werecondensed. Conditions were set to detect approximately 1000molecules by dilution of samples and about 10% of moleculesfound were used for the image analysis. We did not observe anymolecules in the periphery of the deposit if buffers were driedand shadowed. Thus, buffer components appeared to be toosmall for the shadowing. We selected well-defined andnondamaged molecules at random. Under such criteria,transmission electron micrographs of these molecules weretaken on film at 100 000 � by tilting the grid at 108 forstereoscopic analysis.

R E S U LT S A N D D I S C U S S I O N

Kinetic measurement of SNP-1 with sBST-1 by BIAcoreTM

We analysed the interaction between SNP-1 and sBST-1 bysurface plasmon resonance (BIAcoreTM) in the absence ofNAD+. SNP-1 was applied to a CM5 sensorchip on whichsBST-1 was directly immobilized as shown in Fig. 1A. SNP-1binding increased with the amount of SNP-1 injected. Thespecificity of the interaction was demonstrated by using acontrol peptide with an amino acid sequence in reverse to thatof SNP-1. Injection of 1 mm control peptide did not show anysignificant response. Equilibrium binding data were plottedagainst the concentration of SNP-1 as shown in Fig. 1B.Scatchard analysis (Fig. 1C) resulted in calculation of a Kd

value of 500 ^ 35 nm.Table 1 shows the kinetics of association and dissociation of

SNP-1 with immobilized sBST-1 in the absence or presence ofNAD+. A value of kon = 1.4 ^ 0.3 � 105 m21´s21 was deter-mined from the initial reaction rates of SNP-1 binding to sBST-1in the absence of NAD+. A similar value of kon was obtained inthe presence of NAD+. The koff values were 0.073 ^ 0.015 s21

in the absence of NAD+ and 0.091 s21 in the presence ofNAD+. Thus, by BIAcoreTM, no significant difference wasobserved between the binding of SNP-1 in the absence ofNAD+ and that in the presence of NAD+. A value of520 ^ 20 nm for the Kd was calculated from measurementof the kon and koff in the absence of NAD+, which was in goodagreement with the Kd value of 500 ^ 35 nm obtained from theequilibrium binding data.

Sedimentation equilibrium of SNP-1 alone and sBST-1 alone

As a first step for determining the stoichiometry of SNP-1binding to sBST-1, we calculated the molecular masses of SNP-1and sBST-1 by analytical ultracentrifugation. We analyzed each

Fig. 3. Calorimetric titration of SNP-1 with sBST-1. The top panel

shows the raw heat signal for 20 injections of 5-mL aliquots of 400 mm

SNP-1 into the sample cell containing 2.0 mL of 10 mm sBST-1 at 20 8C.

The signal in cal´s21 for each injection was integrated and then the heat of

dilution for the titration of SNP-1 into buffer without sBST-1 was subtracted

from the y-axis value of each point of the curve (the lower panel).

Representative data obtained in two independent experiments are shown.

Fig. 4. Rotary-shadowed sBST-1. Gallery of electron micrographs of

sBST-1. Scale bar represents 20 nm.

Table 2. Molecular mass as determined by sedimentation equilibrium

analysis. Constants used for the analysis are partial specific volume of

0.724 mL´g21 for SNP-1 and 0.718 mL´g21 for sBST-1; r (the density of

the sample solution) = 1.008 g´mL21. Partial specific volume of sBST-1

was calculated with the assumption of 10% glycosylation for sBST-1.

Peptide Rotor speed (r.p.m.) Molecular mass (Da) Average

SNP-1 40 000

42 000

50 000

2020

1820

2030

1960 �^ 68.0

sBST-1 8000

12 000

16 000

64 000

65 600

67 300

65 600 �^ 950

q FEBS 1999 ADP-ribosyl cyclase inhibitor peptide (Eur. J. Biochem. 264) 443

sample by three different rotor speeds using the Beckman XL-Ias shown in Fig. 2A,B, and Table 2. Both sets of data wereshown to fit a single species. The molecular mass of SNP-1estimated from the protein sequence is approximately 1.8 kDa.The molecular mass of sBST-1 was determined to beapproximately 33.5 kDa by matrix-assisted laser desorption/ionization time of flight spectroscopic analysis using a Voyager

Elite spectrometer (PerSeptive Biosystems) (data not shown).Data from the sedimentation equilibrium showed m = 1960 ^68.0 Da for SNP-1 and m = 65 600 ^ 950 dalton for sBST-1,suggesting that SNP-1 is a monomer and sBST-1 is a dimer insolution. Gel filtration chromatography and light scatteringanalysis for sBST-1 also showed dimer formation (data notshown).

Fig. 5. Comparative analysis of alanine-substituted SNP-1 binding to immobilized sBST-1 in BIAcoreTM. sBST-1 (2800 RU) was directly immobilized

on the surface of sensorchip CM5. Alanine replacement peptides at the concentrations of 500 nm were injected onto the sensorchip at a flow rate of

40 mL´min21. (A) Sensorgrams showing the interaction of alanine-substituted SNP-1 with immobilized sBST-1 (lower to upper curve: #1 His!Ala, #6

Gly!Ala, #14 Asp! Ala, #13 Lys!Ala, prototype SNP-1 and #3 Gln!Ala). The other curves were considered as being not detectable at a concentration of

500 nm in BIAcoreTM. (B) Binding capacities of alanine-substituted SNP-1. The replacement of #1 His, #3 Gln, #6 Gly, #13 Lys and #14 Asp with Ala

showed binding capacities to immobilized sBST-1 at a concentration of 500 nm (C) Equilibrium binding and Scatchard plots of alanine-substituted SNP-1.

Equilibrium binding data (left panel) and Scatchard plot analyses (right panel) are shown. sBST-1 (2800 RU) was directly immobilized on the surface of

sensorchip CM5. Various concentrations of alanine-substituted peptides (1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mm of #1 His!Ala peptide; 0.4, 0.6, 0.8, 1.0, 1.2 and

1.4 mm of #3 Gln!Ala, #6 Gly!Ala and #14 Asp!Ala peptides; 0.4, 0.6, 0.8, 1.2 and 1.4 mm of #13 Lys!Ala peptide) were injected onto the sensorchip at

a flow rate of 40 mL´min21. Kd values were calculated from the slopes of the fitted line.

444 A. Sato et al. (Eur. J. Biochem. 264) q FEBS 1999

Titration calorimetry

Next, titration calorimetry was performed. SNP-1 (400 mm)was dropped into the reservoir cell containing 10 mm of sBST-1at 20 8C. The heat of exothermic reaction was observed. Theheat of each injection was integrated and analysed by nonlinearleast squares fitting of the data. The best fit curve line is shownin Fig. 3. The value of each parameter of the binding reactionwas given as follows: Kd = 211 nm; enthalpy change, DH =218.68 kcal mol21; and saturated molar ratio of bound SNP-1per sBST-1, N = 0.8 mol´mol21. The Gibbs energy change(DG = 28.95 kcal´mol21) and the entropy change (DS =23.32 � 1022 kcal´mol21´K21) were calculated from thevalue Kd and DH (DG = RT lnKd, DG = DH ± TDS). Withconsideration of the molecular masses obtained by analyticalcentrifugation, titration point, 0.8 means stoichiometry ofbinding to be 2 : 2. The Kd value obtained here is compatiblewith that obtained from the BIAcoreTM assay as shown above.The existence of a single titration point in the calorimetryexperiment implies that the binding event at one binding sitedoes not affect that at another site within a dimer sBST-1molecule.

Electron microscopy

We observed the purified sBST-1 by low-angle rotary-shadowing electron microscopy (Fig. 4). Electron micrographsindicate that an sBST-1 molecule consists of two similar parts.An image in which the two crescent parts face each othersymmetrically was obtained. Monomeric crescents were alsoidentified when the sample was denatured with 10 mmdithiothreitol (data not shown). These observations suggestthat sBST-1 exists as a dimer, consistent with the result of thebiophysical analyses above. Although the dimer interfacecannot be seen clearly, it is presumed that the cleft of eachcrescent faces each other, creating a pocket. Prasad et al. [12]reported from a crystallization experiment that the AplysiaADP-ribosyl cyclase forms a dimer and that pockets are formedbetween the two monomers. Thus, our rotary shadowing imageof BST-1 predicts a structure homologous to the Aplysiacyclase. Whether the dimer formation of BST-1 is responsiblefor its signal transduction or its enzyme activity remains to beelucidated.

Alanine scanning mutagenesis of SNP-1

To delineate the core residues of SNP-1 responsible for bindingto sBST-1, we performed alanine-scanning mutagenesis.

Binding of each mutagenized peptide at a concentration of500 nm to sBST-1 was analysed by surface plasmon resonanceas shown in Fig. 5A,B. Amino acid mutation at residues 2, 4, 5,and 7±12 of SNP-1 abolished the binding to undetectablelevels. The response of the His1!Ala peptide was reduced toabout 30% of that of the wild-type peptide. Mutations ofresidues, 3, 6, 13 and 14 did not significantly reduce the bindingcapacity. In order to compare the binding constants of alanine-substituted peptides, equilibrium binding of the five mutagen-ized peptides, #1, 3, 6, 13, and 14, to sBST-1 were analysed asshown in Fig. 5C. Scatchard transformation showed a fourfoldincrement in the Kd value of the #1 peptide. Other mutagenizedpeptides, #3, 13 and 14, showed Kd values similar to those ofthe prototype, while #6 peptide showed an intermediate value.Thus, amino acid residues 7±12 seem to be critical for thebinding of SNP-1 to sBST-1. Because the N-terminal residuealso appears to be important for binding, we next prepared theC-terminal modified SNP-1. This lysine-extended SNP-1 wasbiotinylated. The peptide maintains its binding capacity,although the Kd value is increased to 810 nm as shown inFig. 6. The biotinylated peptide is expected to be useful forcross-linking studies or determination of the peptide bindingsite. The binding kinetics of the mutagenized peptides byalanine and the C-terminal modified peptide showed Kd valuessimilar to those obtained from the equilibrium binding (seeTable 1).

Conclusion

The Kd value for SNP-1/sBST-1 interaction obtained by thesolid phase assay (BIAcoreTM) (Kdoff/on = 520 ^ 20 nm; KdEq =500 ^ 35 nm) agreed well with that obtained by a solutionbinding analysis (calorimetry) (Kd = 211 nm). The sedimenta-tion equilibrium analyses and calorimetric studies showed thatSNP-1 binds sBST-1 at a stoichiometry of 2 : 2. The dimerformation of sBST-1 was observed by electron microscopy.Alanine-scanning mutagenesis of the SNP-1 peptide unveiledthe core residues for its binding to sBST-1. These fundamentalanalyses will be helpful in further structural studies and inapplying the inhibitor peptide to biological systems.

A C K N O W L E D G E M E N T S

We thank Dr Masataka Kuroda for data analysis of calorimetric studies; Dr

Yoshito Abe and Dr Susan E. Tsutakawa for help of mass spectroscopic

analysis; Dr Yoshinori Harada for helpful hints on rotary shadowing and

calorimetry; Dr Haruki Nakamura and Dr Kosuke Morikawa for critical

Fig. 6. Equilibrium binding and Scatchard

plot of C-terminal modified SNP-1

(Lys + biotin) in BIAcoreTM. sBST-1 (3600 RU)

was directly immobilized on the surface of

sensorchip CM5. A C-terminal modified SNP-1

at concentrations ranging from 400 nm to

1400 nm was injected onto the sensorchip at a

flow rate of 40 mL´min21. Equilibrium binding

(left) and Scatchard plot analysis (right) are

shown.

q FEBS 1999 ADP-ribosyl cyclase inhibitor peptide (Eur. J. Biochem. 264) 445

reading of the manuscript. We are indebted to Dr Katsuhiko Ishihara and

Professor Toshio Hirano for helpful suggestions at the initial stage of this

work and discussion. We are grateful to Dr Yoshiro Shimura for suggestions

and encouragement.

R E F E R E N C E S

1. Kaisho, T., Ishikawa, J., Oritani, K., Inazawa, J., Tomizawa, H.,

Muraoka, O., Ochi, T. & Hirano, T. (1994) BST-1, a surface molecule

of bone marrow stromal cell lines that facilitates pre-B-cell growth.

Proc. Natl Acad. Sci. USA 91, 5325±5329.

2. Jackson, D.G. & Bell, J.I. (1990) Isolation of a cDNA encoding the

human CD38 (T10) molecule, a cell surface glycoprotein with an

unusual discontinuous pattern of expression during lymphocyte

differentiation. J. Immunol. 144, 2811±2815.

3. Hirata, Y., Kimura, N., Sato, K., Ohsugi, Y., Takasawa, S., Okamoto,

H., Ishikawa, J., Kaisho, T., Ishihara, K. & Hirano, T. (1994) ADP

ribosyl cyclase activity of a novel bone marrow stromal cell surface

molecule, BST-1. FEBS Lett. 356, 244±248.

4. Howard, M., Grimaldi, J.C., Bazan, J.F., Lund, F.E., Santos-Argumedo,

L., Parkhouse, R.M., Walseth, T.F. & Lee, H.C. (1993) Formation and

hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen

CD38. Science 262, 1056±1059.

5. Lee, H.C. (1998) Calcium signaling by cyclic ADP-ribose and NAADP.

A decade of exploration. Cell Biochem. Biophys. 28, 1±17.

6. Zocchi, E., Franco, L., Guida, L., Piccini, D., Tacchetti, C. & De Flora,

A. (1996) NAD+-dependent internalization of the transmembrane

glycoprotein CD38 in human Namalwa B cells. FEBS Lett. 396,

327±332.

7. Funaro, A., Reinis, M., Trubiani, O., Santi, S., Di Primio, R. &

Malavasi, F. (1998) CD38 functions are regulated through an

internalization step. J. Immunol. 160, 2238±2247.

8. Deaglio, S., Morra, M., Mallone, R., Ausiello, C.M., Prager, E.,

Garbarino, G., Dianzani, U., Stockinger, H. & Malavasi, F. (1998)

Human CD38 (ADP-ribosyl cyclase) is a counter-receptor of CD31,

an Ig superfamily member. J. Immunol. 160, 395±402.

9. Horenstein, A.L., Stockinger, H., Imhof, B.A. & Malavasi, F. (1998)

CD38 binding to human myeloid cells is mediated by mouse and

human CD31. Biochem. J. 330, 1129±1135.

10. Itoh, M., Ishihara, K., Hiroi, T., Lee, B.O., Maeda, H., Iijima, H.,

Yanagita, M., Kiyono, H. & Hirano, T. (1998) Deletion of bone

marrow stromal cell antigen-1 (CD157) gene impaired systemic

thymus independent-2 antigen-induced IgG3 and mucosal TD

antigen-elicited IgA responses. J. Immunol. 161, 3974±3983.

11. Cockayne, D.A., Muchamuel, T., Grimaldi, J.C., Muller-Steffner, H.,

Randall, T.D., Lund, F.E., Murray, R., Schuber, F. & Howard, M.C.

(1998) Mice deficient for the ecto-nicotinamide adenine dinucleotide

glycohydrolase CD38 exhibit altered humoral immune responses.

Blood 92, 1324±1333.

12. Prasad, G.S., McRee, D.E., Stura, E.A., Levitt, D.G., Lee, H.C. &

Stout, C.D. (1996) Crystal structure of Aplysia ADP ribosyl cyclase, a

homologue of the bifunctional ectozyme CD38. Nat. Struct. Biol. 3,

957±964.

13. Sato, A., Yamamoto, S., Ishihara, K., Hirano, T. & Jingami, H. (1999)

Novel peptide inhibitor of ecto-ADP-ribosyl cyclase of bone marrow

stromal antigen-1 (BST-1/CD157). Biochem. J. 337, 491±496.

14. Wiseman, T., Williston, S., Brandts, J.F. & Lin, L.N. (1989) Rapid

measurement of binding constants and heats of binding using a new

titration calorimeter. Anal. Biochem. 179, 131±137.

15. Tyler, J.M. & Branton, D. (1980) Rotary shadowing of extended

molecules dried from glycerol. J. Ultrastruct. Res. 71, 95±102.

16. Heuser, J.E. & Salpeter, S.R. (1979) Organization of acetylcholine

receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo

postsynaptic membrane. J. Cell Biol. 82, 150±173.