©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Partial Characterization of the Cytoplasmic Domain of Human Kidney Band 3 (*)

(Received for publication, March 27, 1995; and in revised form, May 25, 1995)

Cheng Chang Wang Ryuichi Moriyama Christian R. Lombardo Philip S. Low (§)

From the Department of Chemistry, Purdue University, West Lafayette, Indiana 47907

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The major anion exchanger in type A intercalated cells of the cortical and medullary collecting ducts of the human kidney is a truncated isoform of erythrocyte band 3 (AE1) that lacks the N-terminal 65 residues. Because this missing sequence has been implicated in the binding of ankyrin, protein 4.1, several glycolytic enzymes, hemoglobin, and hemichromes in erythrocytes, we have undertaken examination of the structure and peripheral protein interactions of this kidney isoform. The cytoplasmic domain of kidney band 3, kidney CDB3, was expressed in Escherichia coli and purified to homogeneity. The kidney isoform exhibited a circular dichroism spectrum and Stokes radius similar to its larger erythrocyte counterpart. Kidney CDB3 was also observed to engage in the same conformational equilibrium characteristic of erythrocyte CDB3. In contrast, the tryptophan and cysteine clusters of kidney CDB3 behaved very differently from erythrocyte CDB3 in response to pH changes and oxidizing conditions. Furthermore, kidney CDB3 did not bind ankyrin, protein 4.1, or aldolase, and expression of erythrocyte CDB3 was toxic to its bacterial host, whereas expression of kidney CDB3 was not. Taken together, these data suggest that the absence of the N-terminal 65 amino acids in kidney CDB3 eliminates the major function currently ascribed to CDB3 in erythrocytes, i.e. that of peripheral protein binding. The primary function of residues 66-379 found in kidney CDB3 thus remains to be elucidated.


INTRODUCTION

Although band 3 protein (AE1)()was first identified and characterized in the human erythrocyte, subsequent studies have revealed that band 3 or one of its anion exchanger homologues (AE2 and AE3) is found in most cells of the body(1, 2, 3, 4) . In the human, rat, and rabbit kidneys(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) , band 3 (AE1) is localized primarily to the basolateral membrane of a subset of intercalated cells of the cortical and medullary collecting ducts. At this location, band 3 is thought to facilitate the exchange of Cl for HCO, a process involved in pH regulation by the kidney.

Several lines of evidence demonstrate that kidney band 3 is homologous to erythrocyte band 3 but truncated at its N terminus. First, antibodies with epitopes C-terminal to residue 66 of erythrocyte band 3 have been found to recognize the kidney isoform, while antibodies with epitopes at the extreme N terminus of erythrocyte CDB3 do not. Second, sequence analysis of band 3 cDNA from human(12) , rat(14) , and mouse (15) kidney indicates that the major transcripts begin at exon 4, i.e. at Met-66 in the human sequence or Met-79 in the rodent. Third, analysis of band 3 genomic DNA reveals an alternative promoter containing TATA and CCAAT boxes within intron 3 that could be used to drive transcription of a truncated transcript(16) . Taken together, these data suggest that a major fraction of kidney AE1 is translated from a shorter message beginning at exon 4.

Previous research on the interaction of the cytoplasmic domain of band 3 (CDB3) with glycolytic enzymes and skeletal components of the red blood cell membrane has strongly implicated the region missing from kidney AE1 in peripheral protein binding. Thus, although a sequence near the membrane junction may weakly contribute to protein 4.1 association(17, 18, 19) , five independent experiments have established that residues at the acidic N terminus are strongly involved in stabilizing the 4.1 interaction(19) . Similarly, although ankyrin association with CDB3 has been mapped in part to regions surrounding a putative proline-rich central hinge(20, 21) , contacts with the extreme N terminus have also been strongly implicated. Thus, Fabs of monoclonal and polyclonal antibodies that map to residues 1-20 of band 3 block ankyrin binding, and prior association of ankyrin with band 3 obstructs phosphorylation of Tyr-8 and Tyr-21 of CDB3(21) . Evidence for an N-terminal interaction with glycolytic enzymes comes from the observation that peptides from the extreme N terminus bind and inhibit aldolase and glyceraldehyde-3-phosphate dehydrogenase with nearly equal affinity to that reported for full-length band 3(22, 23, 24) . Furthermore, phosphorylation of Tyr-8 and Tyr-21 within this region of band 3 blocks enzyme binding and the consequent inhibition of catalysis(25, 26, 27) . Finally, the acidic N terminus of CDB3 has also been implicated in the association of hemoglobin (28, 29) and hemichromes (30) with the erythrocyte membrane. Indeed, x-ray analysis reveals that the first 11 residues of band 3 extend 18 Å into the diphosphoglycerate binding cleft of deoxyhemoglobin and shift the oxygen dissociation curve to the right(29) . Thus, except for protein 4.2 (31) (whose site has not yet been localized), all peripheral protein ligands of erythrocyte band 3 appear to associate in part with the extreme N terminus of the polypeptide. Since a Glu-40 Lys-40 mutation in band 3 leads to a deficiency in the protein 4.2 content of affected erythrocytes and a consequent hereditary hemolytic anemia(32) , the possibility exists that even protein 4.2 may require this region of band 3 for membrane binding.

In view of the fact that residues 1-66 are absent in human kidney band 3(12) , the question naturally arises whether kidney CDB3 can perform any of the peripheral protein binding functions of erythrocyte CDB3. To directly examine this issue, we have expressed kidney CDB3 (residues 66-379 of the human erythrocyte sequence) and have examined its structure and peripheral protein interactions. We report here that bacterially expressed kidney CDB3 is isolated as a an elongated dimer with similar but not identical structural properties to erythrocyte CDB3. We further show that the kidney isoform lacks the ability to bind ankyrin, protein 4.1, and the glycolytic enzyme aldolase, raising significant questions regarding the function of this cytoplasmic domain. During preparation of this manuscript, Kopito and co-workers (33) reported that rat kidney band 3 (lacking residues 1-79) is also unable to bind ankyrin.


EXPERIMENTAL PROCEDURES

Materials

Reagents for the polymerase chain reaction and the corresponding thermal cycler were from Perkin-Elmer Corp. Glycolytic enzymes and protein molecular weight markers were obtained from Sigma. Growth media for Escherichia coli was from Difco. Isopropyl-1-thio--D-galactopyranoside and antibiotics (chloramphenicol and ampicillin) came from U. S. Biochemical Corp. DNA markers and DNA sequencing reagents were from Biological Research Laboratory, and nitrocellulose was purchased from Schleicher & Schuell. Electrophoresis reagents and horseradish peroxidase-conjugated anti-rabbit antibodies were from Bio-Rad. Restriction enzymes and T4 ligase were from New England Biolabs. E. coli BL21 (DE3) (pLysS) and the CDB3/T vector have been described previously(34) . DEAE-Sepharose CL-6B and Sephacryl S-300 were from Pharmacia Biotech Inc. I-Bolton-Hunter reagent was from Amersham Corp.

Methods

KCDB3/T Vector Construction

The 314-amino-acid KCDB3 cDNA (corresponding to residues 66-379 of the cytoplasmic domain of erythrocyte band 3) was amplified form a CDB3 expression vector (CDB3/T) using two primers and polymerase chain reaction methodology(34) . Briefly, primers were synthesized to yield an NdeI site at the 5` end and a HindIII site with a TCA stop codon at the 3` end of the amplified KCDB3/T gene. The primers used were 5`-TCTCATATGGACGAAAAGAACCAGCAGCAGCTG-3` for the 5` end and 5`-AGAAAGCTTTCAGAAGAGCTGGCCTGTCT-3` for the 3` end. The amplified 1-kilobase pair KCDB3 NdeI-HindIII fragment was then subcloned into a T expression vector (T), as published previously(34) .

Expression of Kidney CDB3 in E. coli

The crude ligase mixture containing the cloned plasmid (Fig. 1) was introduced into competent E. coli BL21 (DE3) cells, and the cells were proliferated. Initial identification of colonies containing KCDB3 was conducted by use of antibodies against different regions of CDB3(21) . Positive clones were then confirmed by sequencing of the whole KCDB3 cDNA using Sequenase 2.0 (U. S. Biochemical Corp.). Expression conditions were the same as for CDB3 clones(34) . Briefly, a saturated 10-ml culture of E. coli BL21 (DE3) (pLysS) containing the KCDB3/T vector was used to inoculate 4 liters of LB medium supplemented with ampicillin (50 µg/ml) and chloramphenicol (25 µg/ml). When the cell density reached A = 1.0, 1 mM fresh isopropyl-1-thio--D-galactopyranoside (final) was added. The culture was then transferred to room temperature (25 °C) for an additional 6 h of shaking. The cells were pelleted and resuspended in 50 ml of lysis buffer (20 mM Tris, pH 7.0, 1 mM EDTA, 1 mM EGTA, 10 mM benzamidine, 0.2% -mercaptoethanol, and 20 µg/ml PMSF) and then stored at -80 °C.


Figure 1: Construction of the kidney CDB3/T expression vector. The kidney CDB3 gene (corresponding to amino acids 66-379) of CDB3 was amplified from CDB3/T vector by polymerase chain reaction and then inserted into NdeI and HindIII sites of the T vector(49, 50) , as described under ``Methods.''



Purification of KCDB3 from E. coli

Purification of kidney CDB3 was similar to recombinant CDB3, except elution from the DEAE column occurred at a lower salt concentration(34) . Briefly, the frozen E. coli pellets were lysed by three freeze-thaw cycles for 10 min in liquid N followed by thawing in a 37 °C water bath and then sonicated for 2 min at medium power with a Branson Sonifier. The crude cell lysates were equilibrated in 50 ml of DEAE-Sepharose CL-6B with column buffer (10 mM phosphate buffer, pH 7.5, 1 mM EDTA, -mercaptoethanol, and 20 mg/ml phenylmethylsulfonyl fluoride). The DEAE-Sepharose CL-6B beads were extensively washed with column buffer and then poured into a chromatography column (10 2.5 cm). Proteins were eluted with a salt gradient ranging from 0.0 to 0.4 M NaCl in a 200-ml volume, and kidney CDB3 eluted at 0.27 M NaCl. The appropriate fractions were pooled and precipitated in 30% (NH)SO and then subjected to gel filtration chromatography on a Sephacryl HS-300 column (100 cm 2.7 cm) in phosphate-buffered saline buffer (150 mM NaCl, 5 mM phosphate buffer, pH 7.4, 0.2 mM dithiothreitol, 1 mM EDTA, and 20 µg/ml phenylmethylsulfonyl fluoride). The presence of kidney CDB3 was verified in eluted fractions by Western blotting.

Primary Structural Studies

20 µl purified kidney CDB3 (1 mg/ml) was hydrolyzed under vacuum for 22 h at 110 °C in 30 µl of boiling HCl (Pierce) and amino acid analysis was conducted on a Beckman amino acid analyzer 7300. N-terminal amino acid sequencing was conducted at the Purdue University Laboratory for Macromolecule Structure using a gas phase amino acid sequencer equipped with an on-line 120A PTH analyzer (Applied Biosystem 470, Foster City, CA).

Circular Dichroism

Kidney CDB3 was dialyzed in 50 mM sodium phosphate, 50 mM sodium borate, 70 mM NaCl buffer adjusted to the desired pH. Circular dichroism spectra were recorded on a JASCO J-600 spectropolarimeter using a final concentration of 21 µg/ml kidney CDB3 in a cell of 1 mm light path at 25 °C. The calibration and measurements were completed as described in a previous report(35) .

Other Structural Studies

Cysteine cross-linking was conducted according to Thevenin et al.(36) . Kidney CDB3 (1 mg/ml) was oxidatively cross-linked with 1 mMo-phenanthroline and 0.2 mM CuSO for 30 min at 24 °C in 5 mM NaHPO, pH 8.0. The reaction was terminated by the addition of EDTA to 1 mM and then directly analyzed by a nonreducing SDS-polyacrylamide gel electrophoresis for the presence of cross-linked products(37) . The Stokes radius of kidney CDB3 was measured using the methods previously published(34, 35) . The pH-dependent intrinsic fluorescence titration curves were also obtained as described previously(34, 35, 38) .

Peripheral Protein Binding Assays

Analysis of the ability of kidney CDB3 to inhibit glycolytic enzymes was conducted according to published procedures(34, 35) . Ankyrin or protein 4.1 binding to recombinant kidney CDB3 was measured by competitive inhibition of ankyrin or protein 4.1 binding to KI-stripped inside-out erythrocyte membrane vesicles (KI-IOVs)(19, 21) . Briefly, kidney CDB3 was incubated at room temperature with KI-IOVs (35 µg/ml) for 3 h prior to the addition of I-labeled protein 4.1 or ankyrin. Bovine serum albumin was added with labeled proteins to maintain the total protein concentration at 6.5 mg/ml, and the incubation was continued for 20 min. Bound and free protein 4.1 or ankyrin were then separated by pelleting the IOVs through phosphate-buffered saline buffer containing 20% sucrose and 2 mg/ml bovine serum albumin. The plastic microcentrifuge tubes were then frozen in liquid nitrogen, and the tips were cut off and counted for radioactivity in a -counter.


RESULTS

Expression of the Cytoplasmic Domain of Kidney Band 3

The amplified KCDB3 gene was inserted into NdeI and HindIII sites of a T vector (Fig. 1) and expressed in E. coli strain B21 (DE3) (pLysS), as described under ``Methods.'' Several lines of evidence suggest that the desired KCDB3 gene product was in fact obtained. First, the entire insert was sequenced and found to correspond to the nucleotide sequence of amino acids 66-379 of CDB3. Second, Western blot analysis revealed that antibodies with epitopes evenly spaced along CDB3 all cross-reacted with the KCDB3 gene product, except those with epitopes within the first 65 residues of CDB3(21) . Finally, Edman degradation of the first 11 amino acids of KCDB3 yielded a sequence corresponding to residues 66-76 of CDB3 (data not shown).

Structural Similarities between CDB3 and Kidney CDB3

Structural analyses have revealed both similarities and differences between CDB3 and kidney CDB3. Comparison of the circular dichroism spectra of the two isoforms showed only minor variations in -helical and -pleated sheet content (Fig. 2). Kidney CDB3 exhibited slightly lower ellipticity at 222 nm but comparable ellipticity at 208 nM. Within experimental error, they both contain approximately 35-40% -helix (35, 38) .


Figure 2: Circular dichroism spectrum of erythrocyte and kidney CDB3. Erythrocyte and kidney CDB3 (21 µg/ml) were dialyzed against 50 mM sodium phosphate, 50 mM sodium borate, 70 mM NaCl, pH 7.0, and scanned at 24 °C in a 1-mm cell in a JASCO J-600 spectropolarimeter. Calibration and calculation of ellipticity were conducted as reported previously(35) . Curve A (dashed line) is the control from cloned CDB3, and curve B displays the kidney CDB3 spectrum under the same conditions.



A second structural characteristic of erythrocyte CDB3 is its pH-dependent conformational equilibrium. Between pH 6 and 10, CDB3 has been shown to reversibly elongate, with an increase in Stokes radius from 55 to 66 Å(34, 35, 38) . Similar analysis of kidney CDB3 showed a Stokes radius increase from 56 to 66 Å over the same pH range (Fig. 3). To ensure that these values were not influenced by the choice of gel filtration media, the Stokes radius of cloned CDB3 was examined on the same column at the same pHs. As seen in Fig. 3, recombinant erythrocyte CDB3 also expands from an R 55 to 67 Å between pH 6.5 and 10.5. Based on these data, it can be concluded that kidney CDB3 undergoes a pH-dependent conformational change similar to erythrocyte CDB3. Furthermore, like erythrocyte CDB3(38) , the kidney isoform must have a highly elongated morphology, even though it lacks the N-terminal 65 amino acids of CDB3. Since erythrocyte CDB3 has been shown to be a dimer with an estimated axial ratio of 10, we hypothesize that kidney CDB3 is also an elongated dimer; however, a more definitive conclusion will have to await further structural analyses. Nevertheless, it should be noted that rat kidney AE1 has recently been expressed and found to be a dimer(33) , suggesting that the subunit stoichiometry of band 3 may remain unaltered by deletion of the N terminus. Most importantly, these data demonstrate that kidney CDB3 is well folded and capable of undergoing a native conformational transition.


Figure 3: Variation of the Stokes radius of erythrocyte and kidney CDB3 with pH. Purified kidney and erythrocyte CDB3 (1 mg/ml) were dialyzed overnight against 50 mM sodium phosphate, 50 mM sodium borate, 70 mM NaCl, 1 mM EDTA, 0.2 mM dithiothreitol, and 1 mM NaN at various pH values. The protein samples (2 ml) were then chromatographed on a Sephacryl S-300 column (2.7 117 cm) equilibrated in the same buffer. Stokes radius was calibrated using the following standards chromatographed on the same column: horse spleen apoferritin (R = 79 Å), bovine liver catalase (R = 52 Å), rabbit muscle aldolase (R = 46 Å), bovine serum albumin (R = 35 Å), and ovalbumin (R = 27 Å). The solidcurve (circles) displays the R of kidney CDB3, and the dashedcurve (squares) displays the R of cloned CDB3.



Structural Differences between Erythrocyte and Kidney CDB3

The above pH-dependent structural change in CDB3 (Fig. 3) has been previously studied by techniques other than gel filtration chromatography. Thus, the conformational equilibrium can also be monitored by native gel electrophoresis, sedimentation analyses, extrinsic fluorescence, ankyrin binding analysis, differential scanning calorimetry, and intrinsic tryptophan fluorescence(35, 38) . Because the Trp cluster in CDB3 lies between residues 75 and 105, we felt its environment might be impacted by deletion of residues 1-65, leading to a change in its sensitivity to the structural transition. To test this possibility, we compared the pH dependence of the Trp fluorescence of CDB3 and kidney CDB3. As shown in Fig. 4and as reported previously(34, 35, 38) , the intrinsic fluorescence of erythrocyte CDB3 more than doubles between pH 6 and 10, exhibiting inflections near pH values of 7.2 and 9.2 (Fig. 4, squares). In contrast, the intrinsic fluorescence of kidney CDB3 increases by only a factor of 1.6 over the same pH range (Fig. 4, solidcircles). Furthermore, no inflections are obvious over the entire kidney CDB3 titration curve. However, that the intrinsic fluorescence change is still the property of a native structural transition can be demonstrated by showing its disappearance upon heat denaturation of kidney CDB3 (Fig. 4, opencircles). Thus, the tryptophans near the N terminus of kidney band 3 would appear to respond differently to the structural equilibrium than the same residues in the larger erythrocyte counterpart.


Figure 4: Intrinsic fluorescence of CDB3 isoforms as a function of pH. Purified kidney CDB3 or erythrocyte CDB3 was dissolved in 50 mM sodium phosphate, 50 mM sodium borate, 70 mM NaCl, preadjusted to the desired pH. The final protein concentration in all cases was 37 µg/ml. The relative magnitude of the fluorescence emission at 335 nm ( = 290 nm) is plotted as a function of pH. The experimentally obtained points were fitted with a nonlinear least squares program to a double titration curve. The squares show the pH profile of recombinant CDB3, as described previously(34) . The closedcircles present the titration curve of kidney CDB3, and the opencircles (dottedline) display the corresponding data from heat denatured kidney CDB3.



A second distinction between erythrocyte and kidney band 3 lies in the ease with which each isoform is oxidatively crossed-linked by CuSO/o-phenathroline. CDB3 contains a cluster of cysteines arising from the colocalization of Cys-201 and Cys-317 from each subunit in a common pocket(36, 39, 40) . Because of this close proximity, an intersubunit disulfide bond can form under oxidizing conditions between Cys-201 of one protomer and Cys-317 of the other(36, 39, 40) . Alternatively, in isolated CDB3, an intrachain disulfide linkage (i.e. 201 and 317) is sometimes preferred (39, 40) . To explore whether kidney CDB3 might display the same disulfide bonding pattern, we compared the sensitivity of CDB3 and KCDB3 to the oxidant CuSO4/o-phenanthroline. As shown in Fig. 5, disulfide cross-linking of either natural or cloned erythrocyte CDB3 yields a dimer of 85 kDa molecular mass. In contrast, subjection of kidney CDB3 to the same oxidation results in no disulfide bonded product. Therefore, we conclude that the arrangement of cysteines in kidney CDB3 is different than in CDB3. Because a change in proximity between cysteines of only a few angstroms can strongly inhibit disulfide bond formation, these observations need not imply that major structural differences between the two isoforms exist.


Figure 5: Comparison of the sensitivity of CDB3 isoforms to oxidative cross-linking. Kidney CDB3 or CDB3 (0.5 mg/ml) in 5 mM sodium phosphate, pH 8.0, was cross-linked with 1 mMo-phenanthroline, 0.2 mM CuSO for 30 min. at 24 °C. The reaction was terminated by the addition of EDTA to 1 mM. Then, an equal amount of Laemmli sample buffer was added without -mercaptoethanol, and the samples were separated on a 10% SDS Laemmli gel. Lane1 contains protein molecular markers; lanes2 and 5 are CDB3 released from erythrocytes; lanes3 and 6 are the cloned CDB3; and lanes4 and 7 are cloned kidney CDB3. Lanes2-4 are non-cross-linked controls, while lanes5-7 contain the samples treated with o-phenanthroline/CuSO.



Comparison of Peripheral Protein Binding to CDB3 and Kidney CDB3

Glyceraldehyde-3-phosphate dehydrogenase, aldolase, and phosphofructokinase bind reversibly to the extreme N terminus of the cytoplasmic domain of erythrocyte band 3 both in vivo(22, 27, 41, 42) and in vitro(23, 24, 25, 26) . Importantly, upon binding, the glycolytic enzymes are inhibited. Ercolani et al.(7) have also observed that glyceraldehyde-3-phosphate dehydrogenase colocalizes with band 3 in kidney tissues. To determine whether such colocalization at the light microscope level might derive from a direct interaction, the abilities of CDB3 and kidney CDB3 to bind and inhibit aldolase were examined. As seen in Fig. 6, CDB3 isolated from erythrocyte membranes was the most potent inhibitor of aldolase. Recombinant erythrocyte CDB3 also inhibited catalysis, albeit at a somewhat elevated concentration. This difference in inhibitory potency has been previously attributed to the presence of a protonated amino group at the N terminus of recombinant band 3 that is N-acetylated in the natural red blood cell polypeptide(34) . In contrast, kidney CDB3 displayed no tendency to inhibit aldolase, even at high concentration (Fig. 6). While these observations do not exclude the possibility of a noninhibitory association between the two polypeptides, they at least require that the nature of the interaction between band 3 and aldolase in red blood cells is not duplicated with kidney CDB3.


Figure 6: Comparison of aldolase binding to erythrocyte and kidney CDB3. Increasing amount of cloned kidney CDB3, cloned CDB3 or erythrocyte-derived CDB3 were mixed with 2.2 µg of rabbit muscle aldolase (Sigma) in a total volume of 1 ml containing 3.5 mM hydrazine sulfate, pH 6.0, and 0.1 mM EDTA. After a 5-min incubation, the solution was added to a cuvette containing 0.1 ml of 12 mM fructose-1,6-bisphosphate plus 2 ml of the above hydrazine-EDTA buffer. The absorbance at 240 nm was then monitored continuously for 5 min, and aldolase activity was calculated. Percent of control aldolase activity is plotted against the amount of CDB3 added. Triangles, natural erythrocyte CDB3; squares, cloned erythrocyte CDB3; circles, recombinant kidney CDB3.



A second well characterized association of erythrocyte CDB3 is its interaction with protein 4.1(17, 18, 19) . As mentioned in the introduction, protein 4.1 has been reported to bind near the C terminus (18, 19) or N terminus of CDB3 (19) or possibly even at both sites (19) . To test whether kidney CDB3 maintains an affinity for protein 4.1, the abilities of erythrocyte-derived CDB3, recombinant CDB3, and kidney CDB3 to compete for protein 4.1 binding to KI-IOVs were compared. As shown in Fig. 7, native erythrocyte CDB3 and recombinant CDB3 competitively displace 4.1 from its red blood cell membrane binding site with roughly equal potency. In contrast, kidney CDB3 displays no ability to obstruct the endogenous 4.1-membrane interaction. These data thus suggest that absence of the N terminus in kidney CDB3 prohibits its association with protein 4.1.


Figure 7: Comparison of protein 4.1 binding to erythrocyte and kidney CDB3. KI-IOVs were incubated with increasing concentrations of CDB3 at room temperature in isotonic phosphate buffer, pH 7.4. for 3 h prior to I-protein 4.1 addition. Binding of I-labeled 4.1 was then quantitated as described previously(19) . Percent of uninhibited binding of I-protein 4.1 to KI-IOVs was plotted as a function of the amount of CDB3 added: triangles, natural erythrocyte CDB3; squares, cloned erythrocyte CDB3; and circles, recombinant kidney CDB3.



Probably the most critical interaction of CDB3 in red blood cells is its association with ankyrin, since this linkage establishes the major connection between the membrane skeleton and the bilayer(43, 44, 46) . Because ankyrin-membrane interactions are critical to maintenance of membrane polarity in epithelial cells (44, 45) and since ankyrin colocalizes with band 3 in kidney membranes(6) , it has been hypothesized that an ankyrin-kidney CDB3 association might retain band 3 on the basolateral side of a collecting duct plasma membrane(6) . To directly evaluate this possibility, we have compared the abilities of CDB3 and kidney CDB3 to compete for ankyrin binding to KI-IOVs. Analogous to the behavior of protein 4.1, ankyrin displayed moderate affinity for both native erythrocyte and recombinant CDB3, but little or no affinity for kidney CDB3 (Fig. 8). These results confirm the observations of Ding et al.(33) on rat kidney band 3 and document the importance of the extreme N terminus of band 3 in ankyrin binding. The relatively weak competition exhibited by the two CDB3 preparations (K = 400 nM) is consistent with previous observations(46, 47) , and probably derives from the inability of CDB3 to easily form tetramers; i.e. the oligomeric state believed to form the predominant association with ankyrin(48) .


Figure 8: Comparison of ankyrin binding to CDB3 isoforms. KI-IOVs (43 µg/ml membrane protein) were incubated 30 min on ice in the presence of various concentrations of cloned erythrocyte CDB3 (squares), native erythrocyte CDB3 (triangles), or cloned kidney CDB3 (circles). I-labeled ankyrin (4 µg/ml, final concentration) was then added, and the mixture was allowed to further incubate for 3 h on ice. The mixture was then layered on top of a 600-µl sucrose cushion (155 mM NaCl, 0.2 mM dithiothreitol, 1.2 mg/ml bovine serum albumin, 15% sucrose) and centrifuged at 45,000 g for 40 min. Pellets containing the KI-IOVs and bound I-ankyrin were collected and counted(21) .



N-terminal Truncation of CDB3 Reduces Its Toxicity to Its Expression Host

During the course of research on CDB3 expression in prokaryotes, it was observed that the CDB3 expression plasmid is toxic to its bacterial host(34) . Thus, to permit growth of the transfected DE3 strain of E. coli, the bacteria had to be co-transfected with a suppressor of CDB3 leak-through expression contained on the plasmid pLysS(49, 50) . Although this modification also lowered induced expression of CDB3, the modified strain at least survived until CDB3 could be induced with isopropyl-1-thio--D-galactopyranoside. Curiously, the same expression vector containing the kidney CDB3 gene in place of the CDB3 gene but lacking any suppression plasmid was not toxic to E. coli. While elimination of ankyrin, protein 4.1 or glycolytic enzyme binding capabilities could conceivably explain this difference in toxicity, we suspect the inability of kidney CDB3 to inhibit glycolysis in the bacteria may be primarily responsible. However, studies of the effect of cdb3 expression on E. coli glycolysis will have to be conducted to confirm this hypothesis.


DISCUSSION

We have presented data demonstrating that the cytoplasmic domains of erythrocyte and kidney band 3 exhibit similar secondary and tertiary structures but have no common affinities for peripheral protein ligands. Although the two isoforms have analogous CD spectra, Stokes radii, and pH-dependent conformational transitions, the characteristic high affinity binding sites for aldolase, protein 4.1, and ankyrin are present only on erythrocyte CDB3. These data emphasize the critical importance of the first 65 amino acids of CDB3 in establishing affinity for peripheral protein ligands. They also confirm our earlier mapping studies that assigned a major portion of the CDB3 binding sites for the above peripheral proteins to the first 65 residues(19, 21, 25, 26, 27, 29, 30) . Although direct binding data for hemoglobin, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, and hemichromes were not obtained in this study, we believe the behavior of aldolase is representative of these proteins, since they all compete for binding to the same site on CDB3. Whether protein 4.2 will also rely on residues 1-65 for affinity is unknown, but the fact that a charge reversal at residue 40 of CDB3 results in an 88% deficiency in red blood cell protein 4.2 would suggest this possibility(32) .

The loss of virtually all binding interactions upon removal of residues 1-65 of CDB3 would have been unexpected based solely on sequence comparisons of band 3 homologues from different species. Thus, among the various regions of the band 3 protein, the extreme N terminus is clearly the least conserved(1, 2, 3, 4) . Although glycolytic enzyme binding by erythrocyte band 3 may be unique to higher mammals(3) , ankyrin association is thought to be common to all species. What then is the conserved feature that specifies a high affinity ankyrin site? Primary structure analysis reveals only that all erythrocyte homologues are very acidic at their N termini(51, 52) . Importantly, other membrane-spanning anchors for ankyrin may have similar acidic stretches in their ankyrin binding domains(44, 21) , but these are also not homologous. Whether tertiary structural comparisons will disclose a folding pattern that is conserved among all ankyrin binding sites must await crystallographic analyses.

With the N terminus of CDB3 assigned a major role in peripheral protein binding, the question arises regarding the function of the remainder of CDB3. As mentioned earlier, ankyrin and possibly protein 4.1 require sequences distant from the band 3 N terminus to complete their noncontiguous binding sites(19, 21) . Thus, part of the role of residues 66-379 in band 3 function might be to provide essential ligation sites for stabilization of these interactions. Furthermore, as shown in Fig. 3, the major conformational transition of CDB3 resides in residues 66-379. Since this structural equilibrium controls ankyrin affinity(48, 53) , the contribution of this region to maintenance of red blood cell stability and shape should not be minimized. Finally, there are suggestions that CDB3 may also contribute to regulation of band 3 subunit stoichiometry (54) and control of anion transport(55) . While the responsible sequences for these putative functions have not been assigned, it is conceivable that the highly homologous stretches of CDB3 might be critically involved. Clearly, for the kidney isoform of band 3, these latter possible functions may well be most important.

Finally, how do we explain the colocalization of band 3 with ankyrin (6) and glyceraldehyde-3-phosphate dehydrogenase (7) in the kidney? As noted by Kopito and co-workers(33) , there are multiple homologues of ankyrin that may cross-react with the same red blood cell ankyrin antibody and there is evidence for transcripts of AE1 in the kidney that do not start at exon 4(14, 15, 56) . Therefore, it is conceivable that the observed kidney colocalization may reflect an interaction between full-length AE1 and erythrocyte ankyrin or between the truncated AE1 and a nonerythroid ankyrin(44) . Because of the seemingly absolute requirement for CDB3's acidic N terminus in glycolytic enzyme binding(22, 23, 24, 25, 26, 27, 41, 42) , we can only speculate that the glyceraldehyde-3-phosphate dehydrogenase colocalization arises from expression of some full-length AE1 or from coincident localization of another glyceraldehyde-3-phosphate dehydrogenase docking site, such as tubulin(57) .


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM24417. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 317-494-0239.

The abbreviations used are: AE, anion exchanger; CDB3, cytoplasmic domain of kidney band 3; KI-IOV, KI-stripped inside-out erythrocyte membrane vesicle.


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