na,k-atpase extracellular surface probed antibody that enhances

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

Na,K-ATPase Extracellular Surface Probed Antibody That Enhances Ouabain Binding*

Val. 267, No. 19, Issue of July 5, pp. 13694-13701, 1992 Printed in U. S. A.

with a Monoclonal

(Received for publication, January 27, 1992)

Elena ArystarkhovaSB, Marine GasparianS, Nikolai N. Modyanovs, and Kathleen J. SweadnerSV 11 From the $Department of Neurosurgical Research, Massachusetts General Hospital, Boston, Massachusetts 02114, the Ishemyakin Institute of Bioorganic Chemistry, Russian Academy of Sciences, I 1 7871 Moscow, Russia, and the VDepartment of Cellular and Molecular Physiology, Haruard Medical School, Boston, Massachusetts 021 15

The Na,K-stimulated ATPase is inhibited by extracellular cardiac glycosides, which bind to the enzyme’s (Y subunit. We used a monoclonal antibody, VG4, as a probe of the extracellular surface. The antibody was specific for Na,K-ATPase and bound to intact cells. The epitope was mapped to the first extracellular loop (Hl-HZ) of a, using a combination of techniques in- cluding trypsinolysis, N-terminal sequence of a fragment containing the determinant, and analysis of the effects of species-specific sequence differences. The antibody inhibited Na,K-ATPase activity under cer- tain circumstances, indicating that the H1-HZ loop participates in conformational changes that are transmitted to the active site. Mutations in the H1-HZ loop have been shown by others to affect ouabain affinity. Ouabain and the antibody acted synergistically to inhibit the enzyme, which seemingly supported the hy- pothesis that the H1-HZ loop is an essential part of the cardiac glycoside binding site. Direct measurements of the binding of [3H]ouabain, however, indicated that VG4 enhanced rather than inhibited binding, presum- ably by promoting favorable conformation changes. The data suggest the possibility that the cardiac glycoside binding site may be intramembrane rather than extracellular.

Na,K-ATPase’ catalyzes the active transport of Na+ and K’ across the plasma membrane. The enzyme is specifically inhibited by cardiac glycosides, and early work established unequivocally that the inhibitors act from the outside surface of the cell (1). The enzyme comprises two subunits: a 112- kDa catalytic subunit, a, which has the binding site for ATP; and a 32-kDa glycoprotein subunit, 8, which is required for assembly and for targeting to the plasma membrane. There is much evidence that the a subunit spans the membrane multiple times, exposing the majority of its mass at the intracellular surface, whereas the p subunit spans the membrane only once, exposing the majority of its mass at the extracellular surface (2). It is a widely held premise that the cardiac

* This work was supported by National Institutes of Health Grant HL 36271 (to K. J. S.). 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.

11 To whom correspondence should be sent: Wellman 4, Massachu- setts General Hospital, Fruit St., Boston, MA 02114. Tel.: 617-726- 8579; Fax: 617-726-7526.

’ The abbreviations used are: Na,K-ATPase, (Na++K’)-stimulated adenosine triphosphatase; SDS, sodium dodecyl sulfate; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine; CAPS, ~3-[cyclohexylamino]-l-propanesulfonic acid.

glycoside binding site will be identified within the extracellular residues.

Cardiac glycoside affinity varies between animal species and between Na,K-ATPase isoforms (1, 3). Transfection experiments have established that the affinity is controlled by the a subunit, not the 8 subunit (4,5). Covalent derivatization with cardiac glycoside analogs has indicated that the primary binding site, which recognizes the aglycone, is in the a subunit, whereas a secondary site that binds a sugar group may involve both a and p or a proteolipid (for review see Ref. 6).

Cardiac glycoside derivatives that covalently modify the Na,K-ATPase with reasonably high yield show incorporation of label specifically into the N-terminal half of the a subunit (6). Results obtained with chimeras between high and low affinity Na,K-ATPases and between Na,K-ATPase and Ca2+- ATPase are in agreement that the N-terminal half determines cardiac glycoside affinity (7-10). There are four hydrophobic stretches of amino acids there, termed H1, H2, H3 and H4 (for review see Refs. 2 and 11); evidence that they are trans- membranous is that they are not released from the membrane after extensive proteolysis (12). Three defined sites in the N- terminal half of the a subunit are known to be intracellular: the N terminus itself; a proteolytic cleavage site between H2 and H3, and the site of covalent phosphorylation, 28 amino acids on the C-terminal side of H4. Although physical proof is lacking, there is little controversy that the two short segments that link hydrophobic segments H1 to H2 and H3 to H4 are extracellular. Consequently, much attention has been focused on these as the likely sites of cardiac glycoside binding (13, 14).

Monoclonal antibodies can be useful probes of enzyme structure and function if their epitopes can be identified. Here we have studied a monoclonal antibody, VG,, that binds to the extracellular surface of the Na,K-ATPase (11). Mapping studies indicate that it binds at the extracellular loop Hl-H2, yet functional studies indicate that it does not block the cardiac glycoside binding site.

MATERIALS AND METHODS

Antibodies-Monoclonal antibody VG, (subclass IgG2b) was purified on a protein A-Sepharose column (Pharmacia LKB Biotechnol- ogy Inc.), concentrated to 1 mg/ml, and stored in phosphate-buffered saline (PBS) with 0.1% bovine serum albumin. Monoclonal antibody I C , was mapped earlier to amino acids 887-904 (11). Monoclonal antibody McBX3 is largely specific for the a3 isoform in the rat (15) but cross-reacts well with a1 in the pig; it has recently been mapped to the C-terminal half of the a subunit.’ A peptide-directed antibody against KKKAKKERDMDELKK (near the N terminus of a l ) was the generous gift of W. J. Ball, Jr., University of Cincinnati (16).

Na,K-ATPase Preparations-Pig kidney Na,K-ATPase was used in all experiments unless otherwise noted and was purified by minor

* E. Arystarkhova and K. J. Sweadner, unpublished observations.

13694

Antibody Enhances Cardiac Glycoside Binding to Na,K-ATPase 13695

modifications of the method of Jdrgensen (12, 17). Crude microsome preparations (17) of kidney, brain, and electric organ were used for ELISA analysis of binding to the Na,K-ATPases of different species.

Gel Electrophoresis and Fragment Purification-For most experiments, gel electrophoresis was with the buffer system of Laemmli (41), and blots were stained with antibodies and alkaline phosphatase- conjugated secondary antibodies as described previously (18). When low M , peptides were to be separated, the Tricine buffer system of Schagger and von Jagow was used (19). Elution of fragments from gel pieces was by diffusion for 24 h in 0.4 M NH4HC03, pH 8.0, with 2% SDS and 1% 0-mercaptoethanol. The supernatants were made 90% in methanol and kept at -15 "C for 2-3 days, after which the precipitated protein was collected by centrifugation at 14,000 rpm for 20 min in a Sorvall SS34 rotor. The precipitates were resuspended in buffer and used as antigen in ELISA. Purification of epitope-containing fragments by high performance liquid chromatography and im- munoaffinity chromatography was attempted but was hampered by hydrophobicity.

Trypsinolysis-Three different trypsinolysis protocols were used, each based on previously described conditions tailored to achieve different degrees of digestion.

Limited digestion in K+ was adapted from the work of Jdrgensen and Farley (20). 140 pg of purified Na,K-ATPase was incubated with 6 pg of trypsin (Sigma type XU, 1-tosylamido-2-phenylethyl chloro- methyl ketone-treated) for 30 min at 37 "C in a medium containing 25 mM imidazole, 10 or 150 mM KC1, and 1 mM EDTA (Tris salt) (pH 7.5). The reaction was stopped with 30 pg of soybean trypsin inhibitor (Kunitz), and samples were electrophoresed on Tricine gels. The primary digestion products were identified by reaction with antibodies and eluted from the gels.

Extensive digestion to remove much of the non-membrane-span- ning protein was performed in two stages. Purified Na,K-ATPase was suspended at 1 mg/ml in 0.1 M NH,HCO, buffer (pH 7.4). Trypsin was added at a protein:trypsin ratio of 1OO:l for 10 min at 37 "C (T- 10 membranes) as described in (12). Proteolysis was stopped by the addition of a 10-fold excess of cold water, and the membranes were collected by centrifugation at 35,000 rpm for 90 min at 4 "C (Beckman Ti-45 rotor) and resuspended in the original volume of buffer. In the second stage, T-10 membranes were treated further with 300 mM 0- mercaptoethanol and 3 mM EDTA for 20 min at 37 "C in a nitrogen atmosphere to reduce disulfide bonds, and were collected again by centrifugation. They were resuspended in buffer and digested with trypsin at a starting protein:trypsin ratio of 40:l for 20 min at 37 "C, as described in (12) (SHT-20 membranes). The final digested membranes were again collected by centrifugation, and were suspended in sucrose/EDTA/Tris buffer.

Fingerprinting with trypsin plus soybean trypsin inhibitor was performed as described (21) by modification of the method of Cleve- land et al. (22).

Sequence Analysis-After extensive tryptic hydrolysis (SHT-201, membrane-associated fragments were dissolved by overnight incubation at room temperature in 2% SDS, 2.5% 0-mercaptoethanol, 1 mM EDTA, and 40 mM Tris-C1, pH 8.3. The polypeptides were precipitated with 90% methanol, 1% P-mercaptoethanol at -15 "C for 2 -3 days. The precipitate was dissolved in electrophoresis sample buffer, pH 8.3, applied to Tricine gels (19), and electrophoretically blotted t o polyvinylidene difluoride membrane (Immobilon-P, Millipore) in 10 mM CAPS, pH 11, 10% methanol. Bands were visualized with Ponceau S, and the lowest M , band with antibody reactivity was cut from the blot and washed extensively with Milli-Q water. Amino acid analysis and 10 cycles of Edman degradation were performed a t the Microchemistry Facility at Harvard University.

Immunoassay-Antibody binding was measured using an enzyme- linked immunosorbent assay (ELISA). Titers were measured on preparations of crude membranes or purified Na,K-ATPase adsorbed overnight to Nunc MaxiSorp Immunoplates. Washing and dilution of antibodies were with PBS containing 0.5% bovine serum albumin and 0.1% Tween 20. The secondary antibody was biotinylated goat anti-mouse Ig, and color development was with streptavidin-conjugated horse radish peroxidase (Bethesda Research Laboratories).

Enzyme Assays-Continuous monitoring of ATP hydrolysis was performed with the coupled assay (23) in a spectrophotometer with a thermostatically controlled cell a t 37 "C. The ATPase reaction medium contained 3 mM ATP (disodium salt), 130 mM NaCl, 20 mM KC1, 4 mM MgCl,, 1 mM phosphoenolpyruvate, 0.2 mM NADH, 30 mM histidine, pH 7.2, 50 units/ml pyruvate kinase, and 35 units/ml lactate dehydrogenase. The reaction was initiated by the addition of enzyme. The maximal effect of VG, antibody on ATPase activity was

attained at 10-fold weight excess over enzyme. The relatively high ratio might be caused by loss of antibody affinity during purification or after prolonged storage.

K+-stimulatedp-nitrophenylphosphatase activity was measured by continuously recording the increase in absorbance at 410 nm during reaction in a cuvette thermostatted at 37 "C (26). Na,K-ATPase was first preincubated with antibody in a medium containing 80 mM KC1, 14 mM NaCl (from the antibody stock solutions), 12 mM MgCl,, 0.2 mM EDTA, and 50 mM imidazole, pH 7.2. The reaction was then initiated by the addition ofp-nitrophenylphosphate to a final concentration of 10 mM.

Binding of [3H]Ouabain-Binding of [3H]ouabain was measured with purified Na,K-ATPase in two different liganding conditions: (a) 5 mM MgCl,, 5 mM Pi, 50 mM Tris-HC1, pH 7.4; or ( b ) 5 mM ATP, 5 mM MgC12, 100 mM NaCl, 50 mM Tris-HC1, pH 7.4. 1-10 pg of Na,K- ATPase was preincubated for 1 h at 37 "C with a 5-10-fold excess of purified VG4 or nonspecific IgG in the same medium. Then [3H] ouabain (28.9 Ci/mmol) and unlabeled ouabain were added to a final concentration of 2 X 10-'-10-6 M and a specific activity of 300-1,500 cpm/pmol, in a volume of 400 pl-1 ml. Incubation was for 2 h at 37 "C. The binding was terminated by the addition of ice-cold wash buffer containing 50 mM Tris maleate, pH 7.4, 5 mM MgCl,, 5 mM Tris phosphate, and the samples were filtered on Whatman GF/F glass fiber filters. Nonspecific binding was determined in parallel in the presence of M unlabeled ouabain.

RESULTS

Immunocytochemistry In this section, evidence is presented that VG4 is a legitimate

and specific probe of the extracellular surface of the Na,K- ATPase. VG, is one of a set of 11 monoclonal antibodies raised against purified membrane-bound Na,K-ATPase from pig kidney (11). The hybridomas were screened for binding to purified enzyme by ELISA; the dissociation constant was 1.4 x lo-' M. VG4 also bound to intact pig kidney embryonic (PKE) cells in culture (11). Antibodies that bind to a given antigen sometimes react spuriously with other proteins: here we used immunolocalization in kidney tissue sections to determine whether VG4 exclusively stained structures known to be rich in Na,K-ATPase (24) Fig. 1). The most intense staining was detected in basolateral membranes of distal convoluted tubules and the thick ascending limb, with less stain in proximal convoluted tubules. Collecting tubules were practically unstained. The same distribution of immunofluorescence was obtained with sections of fixed or unfixed human kidney (not shown). These data match the known cellular distribution of Na,K-ATPase in basolateral membranes of nephron structures (18, 24).

Immunofluorescence also allowed us to determine the dis- position of the VG4 epitope relative to the plasma membrane. As shown in Fig. 2, a and c, VG4 bound well to fixed and living cells of the pig kidney epithelial cell line LLC-PK1, outlining the accessible edges of the cells. This cell line makes an organized epithelium with tight junctions which prevent access of antibody to the basolateral surface in the middle of a confluent group of cells. To permit VG4 penetration to possible intracellular determinants, we permeabilized the cells with Triton X-100 after fixation with periodate/lysine/paraformaldehyde. Although antibody then had access to all of the cells, the intensity of staining was not improved (Fig. 2b). In contrast, monoclonal antibody llC9 was shown to not bind to fixed or living cells (Fig. 2, a' and c') but only to fixed cells after permeabilization (Fig. 26'). Similar results were obtained with Madin-Darby canine kidney epithelial cells and dissociated cells from the rat retina (not shown). All together, the data indicate an extracellular exposure of the VG4 epitope and intracellular exposure of the llC9 epitope (11).

13696 Antibody Enhances Cardiac Glycoside Binding to Na,K-ATPase

FIG. 1. Immunofluorescent localization of VG4 binding in kidney sections. Cryostat sections (10 pm) of periodate/lysine/ paraformaldehyde-fixed (40) rat kidney were affixed to gelatin-coated slides and treated with 0.3% Triton X-100 in PBS with 5% goat serum. They were then stained with VG, (20 pg/ml) followed by rhodamine conjugated goat-anti-mouse IgG (Cappel) essentially as described earlier for another Na,K-ATPase-specific monoclonal antibody, McKl (18). Panels a and a’ show renal cortex: g, glomerulus; pt , proximal convoluted tubule; dt, distal convoluted tubule. Panels b and b’ show outer medulla: tal, thick ascending limb. Panels a and b are phase contrast images; panels a‘ and b’ show the immunofluorescence. Secondary antibody alone and several other anti-Na,K-ATP- ase antibodies served as controls (not shown).

Structural Identification of the VG4 Epitope In this section, several lines of evidence are presented for

the identification of the VG4 epitope. No single experiment stands alone as proof of the epitope’s location, but all of the experiments are consistent with a single deduced site.

Although VG4 recognized native pig and rat kidney Na,K- ATPase very well, it failed to detect SDS-denatured enzyme on Western blots even at high concentrations of antibody. This could be ascribed either to an epitope structure compris- ing discontinuous segments of the polypeptide or to a masking effect of the detergent. Treatment of SDS-solubilized Na,K- ATPase with 90% methanol to remove SDS led to complete recovery of antibody binding without restoration of native Na,K-ATPase structure, implying that the epitope must be composed of contiguous amino acids. Since there was no cross- reaction with purified /I subunit, the epitope was concluded to be in the (Y subunit (11).

The transmembrane organization of the CY subunit (espe- cially its C-terminal portion) is still controversial. Neverthe- less, it is generally accepted that the extracellular moiety of the molecule includes only a small proportion of the whole (2). Fig. 3 diagrams predicted transmembrane helices. The most likely targets for VG, binding are short junctions between H1 and H2 or H3 and H4 transmembrane rods; a small loop connecting H5-H6; or uncertain portions of the C terminus following H7 or H8.

It is possible to cleave the a subunit roughly in half by light digestion with trypsin in the presence of K’ (20). The cleavage (at the site labeled T1 in Fig. 3) is a t Arg448, producing 42- kDa N-terminal and 55-kDa C-terminal fragments. Specific antibodies were used to identify the fragments on blots (data not shown; see Ref. 18 for an example), and the bands were eluted from duplicate portions of the gel for testing with VG4 in ELISA. A large fraction, 32.8%, of the immunoreactivity

FIG. 2. Immunofluorescent staining of LLC-PK1 cells with VGr and llC9 antibodies. LLC-PK1 cells (derived originally from pig kidney) were grown in Nunc chambered slides in Dulbecco’s modified Eagle’s medium with 10% horse serum. Panels a-c show staining with antibody VG4, whereas a’-c’ show staining with antibody llC9 under the same conditions. In a and a’, the cells were fixed for 15 min with periodate/lysine/paraformaldehyde fixative and washed three times (30 min total, PBS with 5% goat serum). In b and b’, the cells were fixed, but permeabilized with 0.3% Triton X- 100 for 12 min before staining. In c and c’, the cells were stained as living cells a t 4 “C. Incubation with antibody or rhodamine-conjugated secondary antibody and washing were done in PBS with or without 0.1-0.2% Triton X-100 as appropriate. Scale bar, 100 pm.

was recovered in the N-terminal fragment. A negligible amount of immunoreactivity (3.3%) was recovered in the C- terminal fragment.

Extensive tryptic digestion of purified, membrane-bound Na,K-ATPase was used to determine whether the epitope was associated with soluble or membrane-bound fragments. Diges- tion was in two stages, as described previously (12).

T-10-Trypsinolysis in the presence of ammonium ions leads to exhaustive digestion of the (Y subunit. The residual membrane-associated protein contains intact /I subunit and transmembrane helices of the CY polypeptide.

SHT-20”Reduction of disulfide bonds, followed by a second period of trypsinolysis, results in further cleavage of a between H5 and H6 and elimination of the hydrophilic part of the /I subunit, whereas hydrophobic stretches of both polypeptides remain associated with the membrane. The membrane-bound fragments that remain after digestion are diagrammed in Fig. 3; the numbers refer to the known amino acids at the beginning and end of each fragment (12). For the CY subunit, the membrane-associated products of the two digestion conditions differ only at the C-terminal end.

Strictly speaking, digestion does not go to completion in either of the conditions, but digestion of at least 90% of the a subunit was obtained. After digestion, membrane-associated fragments were separated from soluble fragments by centrifugation and tested for VG4 binding in ELISA. All of the digested membrane preparations bound antibody VG4 as well as the native enzyme, and the affinity was unaltered (Fig. 4).


T-10 6 8 1 4 9 2 5 G 4 7 768- 932

880 372

FIG. 3. Linear map of hydrophobic regions and trypsin- resistant fragments of the Na,K-ATPase. A linear map of the Na,K-ATPase a subunit is shown, with the hydrophobic amino acid stretches indicated as probable transmembrane segments, Hl-H8. The actual arrangement of H5 to the C terminus is still controversial; hence H7 is qualified with a question mark. The N terminus is known to be exposed to the cytoplasm, and so the short stretches of amino acids between HI and H2, and H3 and H4 are proposed to be exposed at the extracellular surface. The sequences shown are those of pig kidney a1 (12). TI is a site that is cleaved by trypsin under very gentle conditions. The bars below the linear map indicate the membrane-associated, trypsin-resistant fragments that are obtained after much harsher digestion. In T-10, digestion is for 10 min without prior reduction of the enzyme; the Na,K-ATPase @ subunit (not shown) is unaffected in this condition. In SHT-20, digestion is for an additional 20 min after reduction of disulfides. The @ subunit is digested, as well as some additional sites between H5 and H6. The amino acids a t the N-terminal end of each fragment were determined directly by Edman degradation; the amino acids a t the C-terminal ends were deduced both from the fragment size and, more rigorously, from the fact that the N termini of the next portions of the sequence were identified in the soluble peptide fraction (12). The fragment encompassing H1- H2 was deduced to contain the VG4 binding site based on experiments described below.

l.Ol

0.5

0.04 . * . I . . . . , , , . I...., , . . I....( 1:10Z 1:10’ 1:to‘ 1:rd

dilution of VG, antibody

FIG. 4. VG, binding to membrane-bound Na,K-ATPase before and after extensive trypsinolysis. Purified Na,K-ATPase was digested with trypsin with and without reduction of disulfide bonds as described under “Materials and Methods,” and soluble fragments were removed by centrifugation. The washed, membrane- associated residual material was adsorbed to plates for ELISA with dilutions of VG, antibody. 0, undigested enzyme; W, T-10 membrane; A, SHT-20 membrane. The extensive digestion had no effect on recovery or affinity of antibody binding.

The epitope therefore is likely to be in one of the smallest membrane-associated fragments diagrammed in Fig. 3.

We attempted to identify the smallest active fragment generated during SHT-20 trypsinolysis conditions by eluting bands from Tricine gels (Fig. 5). Immunoreactivity was seen in fragments of various molecular weights, indicating incom- plete digestion. The data are expressed as the percentage of immunoreactivity recovered relative to undigested a subunit. In this and two similar experiments, the smallest immunoreactive fragment was 8 kDa. The 8-kDa fragment visible with Coomassie Blue stain was subjected to sequence analysis (10 cycles of Edman degradation) after transfer to polyvinylidene difluoride membrane (Table I). The amino acid sequence Asp-Gly-Pro-Asn-Ala-Leu-Thr-Pro-Pro-Pro was de-

Immunoreactivity 0 u) z z

I I I

FIG. 5. Identification of the smallest immunoreactive fragment. As shown in the photograph of the gel, a sample of trypsin- digested membrane (SHT-20) (lane 2; 16 pg) was electrophoresed on a Tricine gel next to undigested Na,K-ATPase (lane 1 ; 10 pg) and M , markers (Sigma 17 S, lane 3); this gel system resolves peptides in the molecular mass range of 2-100 kDa. Protein was visualized with Coomassie Blue staining. Other gels prepared in the same way, but not stained, were cut into 5-mm pieces; protein was eluted and precipitated with methanol; and the eluted fragments were tested for immunoreactivity in ELISA. The bar graph shows the recovery of immunoreactivity (expressed in arbitrary units, estimated relative to the recovery of immunoreactivity in intact a subunit) in fragments of different Mr. The smallest reactive band, 8 kDa, is marked with the arrow. The 8-kDa band from additional gels was blotted to a polyvinylidene difluoride membrane and sequenced.

TABLE I Amino acid analysis of the N terminus of 8-kDa immunoreactive

fragment For each cycle of Edman degradation, the amino acids detected are

listed from the most abundant to the least abundant. The most abundant amino acids (boxed line) match the sequence at the beginning of the fragment encompassing transmembrane segments H1 and H2. An alternative sequence corresponding to a fragment encompassing H3 and H4 can be followed in the less abundant amino acids (connected squares).

Cycleaumbcr: 1 2 3 4 5 6 7 8 9 10

I D 0 P N IA l L T P P P I A D / M \ F D D N

termined with high confidence; this sequence matches amino acids 68-77 in the primary structure of the a subunit and represents the beginning of the peptide encompassing the H1-H2 transmembrane rods (12). The total length of this fragment is 81 amino acids, consistent with the peptide’s molecular weight estimated by electrophoresis. However, another partial sequence, Ile-Ala-Thr-Leu-Ala-X-Gly-X-X-X, could be followed in background amino acids. This corre- sponds to amino acids 263-272 of the a subunit, which means that the fragment containing H3-H4 may have been present in the eluted band. Although the ratio between primary and possible secondary sequence was better than 5:l and some of the minor peaks may be caused by random cleavage background, the possibility that the H3-H4 link is the target for VG., binding could not be excluded on the basis of these data alone.

Next, selective proteolytic cleavage was used to separate Hl-H2 from H3-H4. In principle, one could generate a large

13698 Antibody Enhances Cardiac Glycoside Binding to Na,K-ATPase

C-terminal fragment containing H3-H4 by digestion either with chymotrypsin or with trypsin in the presence of Na+ (20); this was attempted but did not proceed in high enough yield to be useful. A different cleavage technique, based on proteolytic fingerprinting (21), was used instead. Although trypsin is normally readily inactivated by SDS, in the presence of soybean trypsin inhibitor enough activity is paradoxically stabilized against denaturation to permit some digestion. Lim- ited trypsinolysis takes place during gel electrophoresis. As shown in Fig. 6, purified Na,K-ATPase was digested in this way, and nitrocellulose blots were stained with antibodies against the N terminus and a site in the C-terminal half. The smallest fragment that reacts with antibodies against the N terminus (marked with the asterisk) was eluted; it reacted strongly with antibody VG, (data not shown). Since the relative M, of the epitope-containing N-terminal peptide was 17 kDa, this implicates Hl-H2 but not H3-H4 as the site of the VG, antigenic determinant. The smallest N-terminal fragment containing H3-H4 would be predicted to have a molecular mass of 38-39 kDa.

Since the VG4 epitope is evidently composed primarily of contiguous amino acids, fine-specificity analysis could be used to help map the antigenic determinant within the a subunit. Complete sequences of the a1 subunits of pig, rat, Xenopus, Torpedo, and chicken are known, as well as those of rat a 2 and a3 (13). Fig. 7 A shows the alignment of amino acids in the Hl-H2 and H3-H4 loops. Binding of VG, to crude membrane preparations containing each of these Na,K-ATPases was compared by ELISA. As shown in Fig. 7B, the highest titer was with porcine enzyme. There was very little binding to chicken and Torpedo enzymes. The titers for Na,K-ATPase from Xenopus luevis, rat brain (which contains a 2 and a3 in greater abundance than al), and rat kidney enzyme lay in between. These data indicate structural differences in the VG4 epitope among the analyzed enzymes. The epitope should be quite homologous in pig and rat a1 enzymes, whereas the site

a 106

I 80

4 275

1 18.5

FIG. 6. "Fingerprint" mapping of the VG4 epitope. Digestion during gel electrophoresis was used to produce a 17-kDa fragment (marked with the asterisk) containing the intact N terminus and H1- H2. Lunes 1 and 3 contained undigested Na,K-ATPase. For digestion, Na,K-ATPase (245 pg) was dissolved in 490 pl of Laemmli sample buffer (41) mixed with 33 p1 of a stock solution containing both 0.2 mg/ml trypsin and 0.6 mg/ml soybean trypsin inhibitor (Kunitz), and 50-pl samples were applied immediately to 10% polyacrylamide Laem- mli gels for electrophoresis (lanes 2 and 4) . The blot containing lanes 1 and 2 was stained with a rabbit polyclonal antiserum (diluted 1 : l O O O ) against a synthetic peptide from the N terminus. The fragments visualized in lane 2 thus all contain the N terminus. The blot containing lanes 3 and 4 was stained with the monoclonal antibody McBX3 as a marker of a site within the C-terminal half. The 17-kDa fragment was eluted from comparable gels and found to be positive for VG, immunoreactivity by ELISA.

A m-rn M-H4

dilution of VG, antibody

FIG. 7. VG4 binding to Na,K-ATPase from different species. A, alignment of the sequences from the Hl-H2 and H3-H4 extracellular loops of the CY subunits of the indicated species and isoforms. B. ELISA was used to compare the affinity of VG4 for Na,K-ATPases from different sources. Crude microsomes (1 pg/well) from the kid- neys of pig (.), rat (O), chicken (A), and X. laeuis (O), or from rat brain (W) or electric organ from Torpedo californica (A) were adsorbed to plates and then incubated with dilutions of VGI.

in Torpedo electroplax and avian kidney a1 has to be quite different. Identification of substitutions in rat a 2 and/or a3 would support the assignment. Comparison of the primary structures revealed that Hl-H2 but not H3-H4 satisfies all the requirements; most notably, chicken and pig a1 have identical sequences in H3-H4 but very different titers. It is still conceivable that both of these short junctions could participate in formation of the antigenic determinant in an additive manner, but the data unambiguously indicate that the peptide encompassing Hl-H2 is important and sufficient for binding.

Effects of VG, Monoclonal Antibody on Functions of Na,K-ATPase

Although VG, is not capable of completely inhibiting the Na,K-ATPase, the following experiments demonstrate that it has subtle and complex effects on enzymatic activity.

Binding of VG, was tested by competition ELISA in the presence of ligands that produce known conformational states: Na+ ions alone (El); K+ ions alone (E2); Na+,Mg2+, and ATP (E2-P, "front door"); and Mg2+,Pi (E2-P, "back door"). A 3-fold greater VG, affinity was obtained in (M<,Pi) (Fig. 8), the condition that also produces the highest affinity for ouabain binding (25). Otherwise, there was little difference in binding affinity in the different ligand combinations tested.

We next determined whether VG, could inhibit ATP hydrolysis. In pilot experiments, it was noticed that more inhibition seemed to occur during short assays (5 min) than during longer ones (20 min) as measured by the liberation of inor- ganic phosphate. Consequently, the time course of inhibition was examined continuously with the coupled assay, after preincubation with either VG4 or control IgG. As shown in the Fig. 9, the reaction was initially partially inhibited by VG,, resulting in the appearance of a lag. However, steady-

13699 Antibody Enhances Cardiac Glycoside Binding to Na,K-ATPase 125 0.25

100 W 0.20 .- .v c a 75

0

X 50 4 - E 0.15

.- 2 2 x

0.10

25 0

0.05

0 0.

0.00 competing antigen. p g 0 3 6 9 12 15

FIG. 8. Competition assay for VG4 binding to Na,K-ATPase time, rnin in the presence of different ligands. VG, antibody (1:5,000) was F ~ ~ , 10. Effect of VG, on ouabain inhibition of ATP hydrol- first incubated in solution (50 mM Tris-HC1 buffer, 0.1% bovine ysis. purified N ~ , K - A T ~ ~ ~ ~ (1,700 pmol of p,/h/mg of protein) was serum albumin) with aliquots of Na,K-ATPase and different combi- preincubated with 5 x 10-7 ouabain and either a 10-fold of nations of ligands. 0, 140 mM NaCk 0, 140 mM KC1; 0, 100 mM nonimmune IgG or vG,. The differ in the order of the incu- NaC1l mM ATP, and mM MgC12; Or ., mM MgC12 and mM pi. bations. A , ouabain for 1 h followed by control IgG for 1 h. B, ouabain After at 37 O c 1 the antigen with bound antibody was removed by followed by VG,. C, control IgG followed by ouabain. D, VG, followed centrifugation, and the supernatants were tested for residual antibody by ouabain. ~ ~ ~ i b ~ d ~ VG, enhanced the inhibition by ouabain, and by ability to bind to Na,K-ATPase-adsorbed to Nunc immunoplates the effect was most pronounced when antibody was added first. (0.2 pg/well) in ELISA.

0.15

0 d m

E O.1°

d 0 0.05

0 0 3 6 9 12 15

time, min

FIG. 9. Inhibition of ATP hydrolysis by VG,. Purified Na,K- ATPase (0.5-1.0 pg, specific activity 1,700 pmol/h/mg protein) was preincubated with nonimmune IgG ( A and C) or VG, antibody ( B and D) (0.5-10 pg) a t 37 "C for 1 h in 140 mM NaCl, 20 mM KCl, 4 mM MgCl,, 30 mM histidine, pH 7.4. Enzymatic activity was subse- quently measured continuously a t 37 "C in the presence of 3 mM ( A and B ) or 150 p~ ( C and D) ATP. The absorbance a t 340 nm represents the change in concentration of NADH in the coupled assay. ATP hydrolysis was linear in A but became linear only after a lag in B. When ATP was rate limiting (C), the inhibitory effect of VG, was more pronounced (D). Variation in the concentrations of either K' (0-20 mM) or Mg2+ (0.1-4 mM) had no significant effect (data not shown).

state velocities of ATP hydrolysis were indistinguishable after 5 min either in the presence or absence of VG,. Inhibition by VG, was markedly accentuated when ATP was rate limiting; at 150 p~ ATP or less, enzyme that had been preincubated with VG, showed both a longer lag and a sustained level of inhibition of ATPase activity of up to 50% of that obtained with control IgG (Fig. 9).

Preincubation with VG, also enhanced the sensitivity of the enzyme to subsequent inhibition by ouabain (Fig. 10). The apparent KD for ouabain is between and M in the presence of 20 mM K+, in turnover conditions, and so partial inhibition was obtained with 5 X M ouabain. In this figure, the control curves show the rate of hydrolysis obtained after 1-h preincubation with ouabain and 1-h preincubation with control mouse IgG, in either order; essentially the same final hydrolysis rates were obtained. When enzyme was preincubated with ouabain first and then with VG, there was a slight enhancement of enzyme inhibition. When enzyme was preincubated with VG, first and then with ouabain, signifi- cantly more inhibition was seen at the same concentration of

ouabain. The concentration of ATP was 3 mM in these experiments, and so a sustained inhibition of activity by VG, alone was not expected. We can speculate that the antibody either increases the apparent ouabain affinity (which is not optimal under these conditions) or perhaps stabilizes an inhibited conformation after ouabain has dissociated.

K+-stimulated p-nitrophenylphosphatase activity is a ouabain-sensitive reaction carried out by the Na,K-ATPase (24, 26); like ouabain binding, it requires the E2 conformation. Stimulation of K+-stimulated p-nitrophenylphosphatase activity by VG, was seen, maximal (15-20%) a t a 20-fold weight excess of purified antibody over the enzyme (data not shown).

Effects of VG4 Monoclonal Antibody on Ouabain Binding Since the Hl-H2 loop has been implicated in ouabain

binding, the obvious question is whether VG4 and ouabain compete for the same site. [3H]Ouabain binding, after preincubation with VG4 or control IgG, was investigated under two different ligand conditions that are known to support binding (25). In both cases, VG, led to significant enhancement of bound radioactivity. In the presence of Na+,M? and 3 mM ATP binding was 196 f 29% of control ( n = 4); in the same conditions but with only 0.1 mM ATP binding was 215 k 16% of control ( n = 2). The decrease of ATP concentration from 3 to 0.1 mM did not affect the total amount of [3H]ouabain binding. In the presence of M$+ and Pi the binding in the presence of VG4 was 193 f 47% of control ( n = 6). Similar results were obtained in different experiments with concentrations of [3H]ouabain of 10-7-10-6 M.

The [3H]ouabain data were surprising, not only because an increase in binding was seen instead of the predicted decrease, but also because there appeared to be an increase beyond what was ostensibly saturating. This is illustrated in Fig. 11, in which the effect of VG, is seen as a function of the concentration of [3H]ouabain in (Mg2+,Pi) conditions. Sam- ples of enzyme with control IgG or no antibody had comparable levels of binding (1-2 nmol/mg protein when protein recovery was optimal), whereas samples preincubated with VG, had increased binding without any evident effect on binding affinity (KO lo-' M in these conditions). We have verified that no binding of [3H]ouabain by the antibody alone was detected, either when soluble antibody was passed through the glass filters without added Na,K-ATPase, or when antibody was first bound to fixed staphylococcus A, incubated with [3H]ouabain, and then filtered. These data give strong evidence that occupation of the Hl-H2 loop by

13700

1 1600

. m 1200 a

I5 0 800

400

0

Antibody Enhances Cardiac Glycoside Binding to Na,K-ATPase

A

1o"O 10-0 10" 10" 10-8

ouabain, M

FIG. 11. Effect of VGI on binding of [3H]ouabain. Binding of ["Hlouabain was measured after preincubation with VG4 (A), control IgG (m), or no antibody (0). This graph contains data points (each performed in duplicate) from three different experiments for VG, and control IgG and from two experiments for no antibody. The pooled data were fit to Michaelis-Menten equations by the program Fig. P (Biosoft).

VG4 antibody not only failed to block ouabain binding but promoted or stabilized binding instead.

DISCUSSION

The results should be considered from three points of view. First, mapping the epitope for VG, required multiple experi- mental approaches and ultimately was based on converging lines of evidence. Second, the functional effects of the antibody on both the ATPase activity and ouabain binding were difficult to explain mechanistically but certainly supported other evidence (construed from site-directed mutagenesis) that the epitope affects ouabain binding. Finally, the results suggest the possibility that the cardiac glycoside binding site is between transmembrane segments, rather than extracellular.

Epitope Mapping-VG, bound to appropriate structures in the kidney and is thus unlikely to give spurious results when used in a fluorescence assay with intact cells. The antibody bound to intact cells, and no further increase in binding was seen when the cells were permeabilized. In contrast, antibody llC9, which has been mapped previously to a portion of the Na,K-ATPase a subunit between H6 and H7 containing the sequence WINDVEDSYGQQWTYEQR ( l l ) , bound only when the cells were permeabilized, and thus its epitope is intracellular.

The mapping of the VG, epitope was accomplished by measuring antibody binding to identified protein fragments. The epitope must be within the first 448 residues because the antibody reacted with the N-terminal fragment produced by tryptic cleavage at T1. The binding of VG, was unaffected by extensive tryptic digestion, in which most of the protein except a few membrane-anchored fragments is removed (12). N-terminal sequence analysis of fragments eluted from elec- trophoretic gels supported the assignment of the epitope to a large fragment that is thought to comprise a hairpin with the first two transmembrane stretches, H1 and H2, linked by 12 amino acids exposed at the extracellular surface (the fragment labeled 68-149 in Fig. 3). Identification of immunoreactivity in a 17-kDa tryptic fingerprint fragment that contains the N terminus was consistent with this location. Since VG, did not react equally well with Na,K-ATPase from all species, comparison of sequence differences within the two candidate extracellular segments gave strong support to the assignment. T o date, there has been no explicit evidence that the Hl-H2 link is extracellular, although cytoplasmic sites have been identified on both sides of the proposed hairpin (2).

The evidence does not rule out unequivocally the possibility

that the antibody also makes contact with residues from other extracellular loops of a or even the p subunit, but it does imply that residues within the Hl-H2 link are essential and sufficient for antibody binding. When the ability of VG, to bind to different Na,K-ATPases was compared in ELISA, some deductions could be made about likely essential amino acids. The single replacement of Thr114 in pig a1 with methionine in Xenopus a1 reduced antibody affinity 25-fold. Re- placement of Glu1I5 with valine and G ~ u " ~ with asparagine in Torpedo a l , a s well as Glu'I6 with glycine in chicken a l , nearly abolished antibody binding altogether. Homologous substitutions of G ~ u " ~ , " ~ with aspartic acid in rat a3 appar- ently did not change antibody affinity substantially, since the binding to axolemma.(a mixture of a2 and a3) had an affinity only 2-fold lower than that of al . We would predict that rat a2, which replaces Thr114 with methionine like Xenopus a l , would have a lower affinity for the antibody than rat a3. Residues 114-117 thus seem to be the most crucial ones, although in the case of the glutamic acid residues, negative charge may be more important than chain length. On either side of this cluster, substitution in rat a1 of serine for Ala"* or proline for Gin"' reduces affinity slightly relative to that for pig al. For comparison, the residues found to have the most effect on ouabain affinity when altered by site-directed mutagenesis were at the border of Hl-H2, Gln"' and Asp'" (27-29).

Antibody Effects on Conformation, Activity, and PHJOua- bain Binding-The interaction of other monoclonal antibodies with the Na,K-ATPase has been observed to affect enzyme conformation and function, with unique consequences in each case (30-36). The puzzling effects of VG4 are not exceptionally odd when one considers that the Na,K-ATPase is a highly flexible enzyme, sensitive to every ligand it binds. At any given time, the thermodynamic energies of the protein/antibody interface may be more or less important than those of conformation changes originating elsewhere in the protein.

In the simplest analysis, an antibody could inhibit enzyme turnover by stabilizing enzyme conformation in either E l or E2: in the former case, ouabain binding would be inhibited, and in the latter it would be promoted. The effects of the antibody on enzyme activity were actually much more complex. Activity was largely inhibited after preincubation with the antibody, but recovered fully within 2-3 min of the addition of 3 mM ATP. We do not know whether the recovery of activity was caused by dissociation of the antibody or a conformational adjustment of the enzyme with the antibody still bound. Dissociation seems unlikely because antibody binding was detected in all of the tested conditions (Fig. 8), but it is possible that the enzyme passes through a transient state that is incompatible with binding. Alternatively, antibody-induced slowing of a normal conformation change is also possible: Urayama et al. (31) observed that monoclonal antibody 50c caused a slowing in the time course of fluorescence intensity changes in fluorescein isothiocyanate-labeled Na,K- ATPase treated with ouabain. The slowing resulted in a lag of 1-2 min.

When ATP was rate-limiting, there was a sustained inhibitory effect of VG, on ATP hydrolysis. If ATP (binding to a low affinity, allosteric second site) is required either to promote antibody dissociation or to promote a conformational change, it is possible that the time required for the enzyme to recover activity is much greater when ATP is limiting. The apparent linearity of the hydrolysis rates at steady state, however, suggests that the antibody might simply raise the apparent K , for ATP.

Antibody caused a small net stimulation of K+-stimulated


p-nitrophenylphosphatase activity. Since this activity, like ouabain binding, is believed to be carried out primarily when the enzyme is in the E2 conformation, the observation rein- forces the premise that binding favors this conformation.

In Fig. 10, ATP hydrolysis was inhibited more by a subsaturating concentration of ouabain after the enzyme had been preincubated with VG,, i.e. with the antibody bound, the enzyme behaved as if it had a higher affinity for the cardiac glycoside. Enhancement of [3H]ouabain binding (Fig. 11) was seen both at subsaturating and saturating ouabain concentrations, however. There is more than one possible explanation. VG, binding may in fact improve the KO when conditions for ouabain binding are suboptimal, as in the presence of K', but not in the presence of Mg2+ and Pi. Ouabain binding in nominally optimal conditions may also not be at true equilibrium (25), and the rate of approach to equilibrium may be increased by the antibody. Alternatively, some of the enzyme in SDS-purified Na,K-ATPase preparations may be inactive because of partial denaturation by the detergent. The specific activities of the preparations used were 700-1,700 pmol/h/mg of protein, values that are typical, but lower than the best values reported (17). Antibody binding may be sufficient to induce a conformation permissive for ouabain binding in otherwise "dead" or "damaged" enzyme. The optimal levels of bound [3H]ouabain obtained, with VG4, were 3-4 nmol/mg of protein, which equals the best values reported in the literature without antibody (25).

Location of the Cardiac Glycoside Binding Site-Extensive pharmacological studies have delineated which parts of the cardiac glycoside molecule are important for binding, and the binding site on the protein must encompass loci for the lactone ring, steroid, and one carbohydrate residue (14). Covalent derivatives of cardiac glycosides have consistently labeled the N-terminal 42-kDa trypsin fragment of the (Y subunit (6). Site-directed mutagenesis has shown that ouabain affinity is controlled by residues in extracellular loop Hl-H2, leading to the logical conclusion that this is an important part of the site (7,27-29). Since none of the mutations abolished ouabain binding, however, the evidence is consistent with the possibility that the extracellular H1-H2 loop plays a modulatory, rather than an essential role. McParland et al. (37), using a new covalent derivative, recently reported labeling of a fragment encompassing H3, H4, and the H3-H4 extracellular loop, although in very low yield. Mutations in H3-H4, however, have so far been without effect on ouabain binding (38).

When preincubated with purified Na,K-ATPase, VG, enhanced the subsequent binding of [3H]ouabain. If we accept the premise that an antibody that binds to residues in a ligand binding site should sterically impede binding, the conclusion is that the Hl-H2 loop is not likely to be part of the cardiac glycoside binding site, unless the antibody binds to only a portion of the loop, leaving other portions still free to participate in ouabain binding. It is more likely that the Hl-H2 loop affects ouabain binding by conformational changes that are transmitted to other parts of the protein. A different antibody against the extracellular surface, M45-80, has been mapped to the region encompassing H3, H4, and the H3-H4 extracellular loop by its ability to react with expression products of different-length DNA fragments (39). Like VG,, it was capable of partially inhibiting enzyme activity, and it too enhanced ouabain binding, rather than blocking it (30). In theory, it should be possible to produce other antibodies to the same sites which will reduce cardiac glycoside binding by

stabilizing a different conformation. Where, then, does the cardiac glycoside bind? The physio-

logical evidence that cardiac glycosides bind from the extracellular surface does not require that they bind to superficially exposed, hydrophilic residues. Analysis of structure-activity relationships has suggested that they bind in a water-free cleft (14). When one considers that cardiac glycoside binding is exceptionally slow and conformation-dependent (25), it is plausible that the drug may intercalate itself between transmembrane segments, thus profoundly interfering with enzyme activity.

Acknowledgments-We express thanks to Drs. Natalia M. Luneva, Olga E. Lakhtina, and Dennis Brown for valuable discussions and materials.

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na,k-atpase extracellular surface probed antibody that enhances

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