A Monoclonal Antibody Recognizes an Epitope in the First ...

Vol. 267, No. 6, Issue of February 25, pp. 4097-4101,1992 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc

A Monoclonal Antibody Recognizesan Epitope in the First Extracellular Loop of the Plasma Membrane Ca2+Pump* (Received for publication, September 3, 1991)

Marina S. FeschenkoSQ, Elena I. ZvaritchSQll, Francesco HofmannS, Michail I. Shakhparonovll, Nikolay N.Modyanovll, Thomas VorherrS,and Ernest0 CarafoliS** From the $Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), 8092 Zurich, Switzerland and the I(Shemyakin Institute of Bioorganic Chemistry, Union of Soviet Socialist Republics Academyof Sciences, 1I7871 GSP Moscow, Union of Soviet Socialist Republics

A monoclonal antibody against the human erythrocyte Ca2* pump(1E4) reacted with the enzyme in intact erythrocytes. Using trypsinized preparations of the pump the antibody only reacted with the N-terminal fragments of 33.5 and 35 kDa. The fragments span from the N terminus (35 kDa) or fromresidue 19 (33.5 kDa) to residue 314 of the hPMCA4 isoform of the pump. Exhaustive degradation with a number of agents produced smaller peptides which reacted with the antibody. Sequencing analysis on two chymotryptic fragments of 8.8 and 13.5 kDa identified the epitope in an -80-residue domain beginning with Gly-81. Twopeptides corresponding to the putative extramembrane portions of this region of the pump were synthesized. The antibody reacted with one of them, spanning residues Phe-121 to Gly-152 and containing the first putative external loop of the pump. Peptides corresponding to overlapping portions of this peptide were synthesized, leading to the location of the epitope in a 13-residue sequence (Glu-130 to Glu-142) inthe first predicted extracellular loop (Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Strehler, E. E., Fischer, R., Heim, R., Vogel, G., Mathews, S., StrehlerPage, M-A., James, P., Vorherr, T., Krebs, J., Penniston, J. T., and Carafoli, E. (1988) J. Biol. Chern. 263, 14162-14159).

enzyme contains 10 transmembrane domains connected by short loops on the external side of the cell. The main portion of the pump faces the cytoplasm: the C terminusof the pump can be conclusively located on the internal side of the membrane, since it contains the calmodulin binding domain (James et al., 1988). Valuable experimental information on the membrane topology of proteins canbe obtained with the help of monoclonal antibodies (mAb).’ In the case of the Ca2+ pump of the sarcoplasmic reticulum, which has homology to theCa2+pump of plasma membrane, a monoclonal antibody has unambiguously identified one luminal loop of the pump (Clarke et al., 1990; Mattews et al., 1990). Previous work on the interaction of mAbs against the plasma membrane Ca2’-ATPase with intact erythrocytes and with inside-out vesicles had shown that themajority of the antibodies interacted only with insideout vesicles, showing that the epitopes are mainly located on the cytoplasmic portion of the pump (Feschenko et al., 1991). This was hardly surprising, if one considers that about 80% of the total mass of the pump is located on the cytoplasmic side. However, one antibody (1E4) was found to bind exclusively to theexternal side of red cells, indicating the extracellular location of the epitope. This made antibody IE4 particularly interesting; in this study its interactionwith the plasma membrane Ca2+pump was thus investigated in detail to verify experimentally the transmembrane topology of the enzyme in the region surrounding the epitope. The work has led to the The Ca2’-ATPase is an integral membrane protein consist- unambiguous identification of the epitope in the first extraing of a single polypeptide chain of molecular mass -134 kDa. cellular loop of the plasma membrane Ca2’-ATPase. The complete primary structure of several enzyme isoforms MATERIALS AND METHODS AND RESULTS~ has been deduced (Shull and Greeb, 1988; Verma et al., 1988; Greeb and Shull, 1989; Strehler et al., 1990), and the main DISCUSSION functional andregulatory domains have been identified in the purified human erythrocyte enzyme (Filoteo et al., 1987; The membrane topology of ion-motive ATPases is largely James et al., 1987, 1988, 1989). The membrane topology of based on predictions from hydropathy plots and on analogies the ATPase has notbeen established experimentally, and the among the various pumps. In a limited number of cases solid number and orientation of the transmembrane helices have experimental evidence has supportedthe hypothetical models, been only predicted from hydropathy plots. According to the predictions (Shull and Greeb, 1988; Verma et al., 1988) the The abbreviations used are: mAb, monoclonal antibody; BSA, * T h e workwas made possible by Grant 31-25285.88 from the Swiss National Science 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 solelyto indicate this fact. 8 Permanent address: Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow, USSR. ll Supported by a long term fellowship from the Federation of the European Biochemical Societies. ** To whom correspondence should be addressed Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Universitatsstrasse 16, 8092 Zurich, Switzerland. Fax: 011-41-1-252-6323.

bovine serum albumin; DCC, dicyclohexylcarbodiimide; Fmoc, 9fluorenylmethyloxycarbonyl; Hepes, N-2-hydroxyethylpiperazineN”2-ethanesulfonic acid; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; hPMCA, human plasma membrane Ca2+-ATPase; PBS,phosphate-buffered saline; tBu, tertiary butyl; SDS, sodium dodecyl sulfate; TBTU, 2-(1H-benzotriazoll-y1)-1,1,3,3- tetramethyluronium tetrafluoroborate; TFA, trifluoroacetic acid. Portions of this paper (including “Experimental Procedures,” “Results,” and Figs. 1-7) are presented in miniprint at theend of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal thatis available from Waverly Press.

4097

Membrane Topology of the Ca2+Pump

4098

e.g. recent work has located the epitope for a monoclonal antibody against the sarcoplasmic reticulum Ca2+-ATPasein one of the predicted luminal loops of the enzyme (Clarke et al., 1990; Mattews et al. 1990) and hasconfirmed the putative topological model for the mid-portion of that ATPase. In other cases, however, the experimental results have led to propose alternative topological models.This has been the case for the a-subunit of the Na+/K’-ATPase, where work with proteolytic fragments coupled to experiments with antibodies against synthetic peptides corresponding to the C-terminal domain of the subunit (Ovchinnikov et al., 1988) has challenged the traditional view that the a-subunit has an even number of transmembrane domains, with both the N and C termini located intracellularly. Doubts on the 8-transmembrane domain model for the H’/K+-ATPase of gastric mucosa have also been recently raised by Sachs et al. (1989), based on uncertainties in the deductions from the hydropathy profiles. Clearly, experimental evidence supporting or denying the predicted secondary structure models of ion pumps would be of great help, and monoclonal antibodies are in principle convenient tools in thisdirection; the recent works by Clarke et al. (1990) and by Mattews et al. (1990) mentioned above are striking examples. The work presented here has originated from the analysis of the reactivity of the Ca2+pump in intact erythrocytes and in inverted resealed erythrocyte vesicles with a number of monoclonal antibodies against the pump (Feschenko et al., 1991). Of 14 antibodies tested, only one was found to react exclusively with the pump in intact erythrocytes. This could be expected from the existing topological models of the pump, according to which less than 10% of its mass faces the extracellular ambient. It is perhaps pertinent to mention at this point that the successful preparation of antibody 1E4 was greatly aided by the use of the immobilized ATPase antigen (see “Materials and Methods”). In a first series of attempt using the regularly solubilized ATPase as the antigenno antibodies reacting with the extracellular portion of the pump were obtained. Clearly, antibody 1E4 recognized an external epitope and was thus a convenient tool to verify the topology of the pump in the portion around it. The finding that the antibody only reacted with the N-terminal tryptic fragment(s) of the pump greatly restricted the zone in which to search for the epitope, since these fragments are only about 300 amino acid residues long. Fragment 33.5 contains the domain of the pump preceding the first putative transmembrane domain, the loop connecting putative transmembrane domains 1 and 2, and about 140 residues following putative transmembrane domain 2. Since only the second of the non-membranous domains of the fragment is predicted to be extracellular, it was logicalto suggest that theepitope was located there. The exhaustive proteolysis experiments which were then performed, and the sequencing analysis on the fragments which reacted positively with the antibody, confirmed that the epitope was indeed confined to a domain that contained putative

E130

FIG. 8. A scheme of the membrane topology of the ATPase showing the location of the newly identified epitope. Although the scheme shows the topology of the entirepump, the assignment of transmembrane domains 3-10 is based solely on hydropathy profiles.

extracellular loop 1. The work on the synthetic peptides that followed has led to theplacement of the epitope in a stretch of 13 amino acid residues in the C-terminal portion of the putative extracellular loop 1. This confirms experimentally the deduced topology of the pump in this region, i.e. the location of the first two transmembrane domains and of the first extracellular loop (Fig. 8). A necessary assumption in making this assignment is the intracellular location of the N terminus of the pump. The assumption is very plausible, based on the analogy with the sarcoplasmic reticulum pump, where the cytoplasmic location of the N terminus has been experimentally verified (Reithmeier and MacLennan, 1981). Interestingly,asynthetic peptide corresponding to that containing the epitope but derived from the sequence of isoform hPMCAlb instead of that of hPMCA4 failed to interact with antibody 1E4. The main difference in the two peptides (see Fig. 6) is the replacement of the sequence. ValAla-Thr-Thr-Pro (135-139) in the hPMCA4 isoform for the residues Ser-Val-Gly-Glu-Glu (141-145) in thehPMCAlb one; although a word of caution is in order when using this type of argument, the result suggests that the sequence Glu130 to Glu-142, which is unique for the hPMCA4 isoform, represents the antibody binding site or is an essential part of it. REFERENCES Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 Beatty, J. D., Beatty, B. G., and Vlakos, W. G. (1987)’ J. Immunol. Methods 100,173-179 Beatty, J. D., Beatty, B. G., Dlahos, W. G., and Hill, L. R. (1987)’ J. Immunol. Methods 100, 161-172 Caride, A. J., Gorski, J. P., and Penniston, J. T. (1988) Biochem. J. 255,663-670 Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990) J. Biol. Chem. 265,17405-17408 Diano, M., Le Bivic, A., and Hirn, M. (1987) Anal. Biochem. 166, 224-229 Engval, E., and Perlman, P. (1972) J. Immunol. 109, 129-132 Fazekas de Sr. Groth, S., and Scheidegger, D. (1970) J. Immunol. Methods 35, 1-6 Feschenko, M. S.,Zvaritch, E. I., Shakhparonov, M. I., Modyanov, N.N. (1991) Biologicheskie Membrany, in press Filoteo, A.G., Gorski, J. P., and Penniston, J. T. (1987) J. Biol. Chem. 262,6526-6530 Greeb, J., and Shull, G. E. (1989) J. Bwl. Chem. 264, 18569-18576 James, P., Zvaritch, E., Shakhparonov, M., Penniston J. T., and Carafoli, E. (1987) Biochem. Biophys. Res. Commun. 149, 7-12 James, P., Maeda, M., Fischer, R., Verma, A. K., Krebs, J., Penniston, J . T., and Carafoli, E. (1988) J. Bid. Chem. 263,2905-2910 James, P., Pruschy, M., Vorherr, T., Penniston, J. T., and Carafoli, E. (1989) Biochemistry 28,4253-4258 Knorr, R., Trzeciak, A., Bannwarth, W., and Gillessen, D. (1989) Tetrahedron Lett. 30, 1927-1930 Kohler, G. (1981) in ImmunologicalMethods (Lefkowits, I., and Perris, B., eds) p. 296, Academic Press, New York Laemmli, U. K. (1970) Nature 227,680-685 Matsudaira, P. (1987) J. Biol. Chem. 262, 10035-10037 Matthews, I., Sharma, R. P., Lee, A. G., and East, J. M. (1990) J. Biol. Chem. 265,18737-18740 Niggli, V., Adunyah, E. S., Penniston, J. T., and Carafoli, E. (1981) J. Biol. Chem. 256,395-401 Ovchinnikov,Yu. A., Luneva, N. M., Arystarkhova, E. A., Gevondyan, N. M., Arzamazova, N. M., Kozhich, A. T., Nesmeyanov, V. A,, and Modyanov, N. N. (1988) FEBS Lett. 227,230-234 Ploug, M., Jensen, A. L., and Barkholt, V. (1989) Anal. Biochem. 181,33-39 Reithmeier, R. A. F., and MacLennan, D. H. (1981) J. Bioi. Chem. 256,5957-5960 Sachs, G., Munson, K., Balaji, V. N., Awes-Fischer, D., Hersey, S. J., and Hall, K. (1989) J. Bioenerg. Biomembr. 21, 573-588 Schagger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368379

Membrane Topology of the Ca2+Pump

4099

Sheffield, J. B., Graff, D., and Li, M . P. (1987) Anal. Biochem. 1 6 6 , Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Pennis49-55 J. ton, T.,Strehler, E. E., Fischer, R., Heim, R., Vogel, G., Mathews, S., Strehler-Page, M-A., James, P., Vorherr, T., Krebs, J., and Shull, G. E., and Greeb, J. (1988) J. Biol. Chem. 2 6 3 , 8646-8657 Strehler, E. E., James, P., Fischer, R., Heim, R., Vorherr, T., Filoteo, Carafoli, E. (1988) J. Biol. Chem. 2 6 3 , 14152-14159 A. G., Penniston, J . T., and Carafoli, E. (1990) J. Biol. Chem. 2 6 6 , Zvaritch, E., James, P., Vorherr, T., Falchetto, R., Modyanov, N., 2835-2842 and Carafoli, E. (1990) Biochemistry 29,8070-8076

SUPPLEMENTARY MATERIAL T O A MONOCLONAL ANTIBODY RECOGNIZES AN EPITOPE IN THE FIRST EXTRACELLULAR LOOP OF THE PLASMA MEMBRANE C& PUMP M e n 8 S. Feiehcnto. Elclu 1. Zvaritch, Franssieo Hofmann. Mishail 1. Sh&hpmnov. Nikolay N. Modyanov. Thomas Vnhcn. Emcm Carafoli

4100

Membrane Topology of the Ca2+Pump

RESIII.TS

97 kDa 66 kDa 45 kDa 31 kDa

-R ..

-

200 kDa 116kDa 97 kDa 66 kDa

K

1 2 3

-- -3I

fralmenls o l Ihe ATPase.Thc purified pmtcase ID ATPase ralro o l 1:20. Thc fragmcnlr were wpamed by eleemphorerir tn 10 % SDS-gclr. rrmrfemed lo n i m ~ l l ~ l omcmbrancr w and stained either for protein or with the anubody lE4.Lmr 1. low molceularmrsr standards: hnc 2 and 3. tryptic f m p e n l ~stained with amido black and wnh Fig. 3. Interaction OI the IE4 antibody wilh lryplic

ATPase was digcacd wtth trypsin On ice.

81 a

andbody IE4,rcspe~~*vely.

45 kDa

1 2 3 4 5

8.2 kDa 30000

40000

1 A

6.3 kDa

-

w+

1

2

Ir) r%

2.5 kDa

-

0

0 0

20

40

60

60

100

n

20

1 40

60

80

100

Antibody concentration [pglrnl] Fig. 2. Anlibody binding

-3

to intact erylhrocytes and

to isolated e r y l h r o c y b membranes.Red

ECIIS (pan4 A! @rwarhcd (permeable) eylhmcyle plasma membranes (panel 8 ) were incubsled with varying concenmdonr of antibodies IE4 (I)or IGZ (In)as d c r n i k d i nthe Materials and Methods wetion. The immune cmplexes formed wre detceted wtth a iodinated xeondvy antibody.

3

4101

Membrane Topology of the ea2+Pump hPMCA4

17.2 kDa

hPMCA4

14.6 kDa 8.2 kDa 6.3 kDa hPMCA 1 b

2.5 kDa

1

2 3 1.2 h

E

1.0

N

0,

0.8

8

0.2 0

10

20