(Received for publication, March 27, 1995; and in revised form, May 25, 1995)
From the
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. Although band 3 protein (AE1) 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 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.
Figure 1:
Construction of the kidney
CDB3/T
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
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
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 (
A second distinction between erythrocyte and kidney
band 3 lies in the ease with which each isoform is oxidatively
crossed-linked by CuSO
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
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
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
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).
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) .
(
)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.
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.
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
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 Vector
Construction
) 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.
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 Na
HPO
, 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.
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) .
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.
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.
= 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.
/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.
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.
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.
=
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) .
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.