Download Recombinant Sickle Hemoglobin Containing a Lysine

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Recombinant Sickle Hemoglobin Containing a Lysine Substitution
at Asp-85( a ): Expression in Yeast, Functional Properties, and Participation
in Gel Formation
By Juha-Pekka Himanen, Anthony M. Popowicz, and James M. Manning
Clinical modalities based on inhibition of gelation of HbS
are hindered by the lack of quantitative information on the
extent of participation of different amino acid residues in
the aggregation process. One such site is Asp-85(a), which
is involved in a parallel interdouble strand ionic interaction
with Lys-144(b) according to the crystal structure of HbS, but
electron microscopy does not specifically show Asp-85(a) as
a contact site for fiber formation. Using a yeast recombinant
system, we have substituted this site by Lys to abolish ion
pairing and to make a quantitative determination of its participation in aggregation. The purified double mutant was
shown to have the expected pI, the calculated molecular
weight, correct amino acid composition, and peptide map.
The recombinant double mutant has an oxygen affinity of
10 mm Hg, which is identical to that for HbA and HbS under
the same conditions; it also has high cooperativity with an
average n value of 2.7. The change in P50 in response to
chloride ions was about 25% less than that for HbA or HbS
and is ascribed to the introduction of a new positive charge
near one of the major oxygen-linked chloride binding sites
of hemoglobin. The gelation concentration of the double
mutant was measured by a new procedure (Bookchin et al,
1994); the maximal amount of soluble hemoglobin (Csat ) in
the presence of dextran indicated a decreased tendency for
gelation with a Csat of 53 mg/mL compared with 34 mg/mL
for HbS. This inhibitory effect is smaller than that of the
E6V(b)/L88A(b) (Csat , 67 mg/mL) and the E6V(b)/K95I(b)
(Csat , 90 mg/mL) recombinant hemoglobins. Thus, we would
classify Asp-85(a) as a moderate contributor to the strength
of the HbS aggregate. This wide range of gelation values
demonstrates that some sites are more important than others in promoting HbS aggregation.
q 1997 by The American Society of Hematology.
T
mutants or that cannot be modified by chemical procedures.
Indeed, results obtained with both natural and recombinant
Hbs might resolve differences between models of the HbS
polymer.7,8
Most of the earlier work with the recombinant Hb system
focused on the sites at or near the hydrophobic pocket of
the beta chain, which is the acceptor for the mutated Val
residue.18-20 Recently, we showed that a residue located at
the exterior of the Hb tetramer, (Lys-95[b]),21 reduces the
gelation nearly twice as much as a residue in the hydrophobic
pocket, (Leu-88[b]).20 This finding was in agreement with
the electron microscopy studies,8 but not with the crystal
structure of HbS,6 which shows no contacts between Lys95(b) and the amino acid side chains of adjacent tetramers.
Because the Lys-95(b) site is on the exterior of the Hb
tetramer, it is a potentially accessible site for compounds to
be targeted for inhibiting gelation, whereas the residues at
the hydrophobic pocket are buried within the Hb tetramer
and hence not likely candidates.
The studies presented in this report represent a continuation of a systematic effort that we initiated recently employing a yeast expression system for producing recombinant
HbS double mutants.20,21 These recombinant hemoglobins,
which have been characterized by a number of biochemical
criteria, are being used to obtain quantitative information on
the participation of particular amino acid side chains in the
gelation process to identify potential target sites for therapeutic interventions. In this report, we describe an HbS double
mutant where the second mutation is on the alpha chain,
Asp-85(a). Although several a- and b-chain sites have been
reported to participate in gelation through the study of natural mutants,22-24 no information exists on a-chain recombinant mutants. The Asp-85(a) site described in this study was
chosen because the extent of its participation in gelation has
not yet been determined. The electron microscopic studies8
indicate that Asp-85(a) is 5 to 8 A away from an adjacent
HbS tetramer, suggesting that it might be a contact site of
moderate strength in the aggregate. The crystal structure of
HE PRIMARY CAUSE for sickle cell anemia has been
known since 1956 when Ingram1 showed that the difference in electrophoretic behavior between HbS and HbA
described by Pauling et al2 was due to the replacement of a
single amino acid, ie, Glu r Val at position 6 of the bchain E6V(b). This point mutation leads to the initial strong
hydrophobic interaction between the adjacent Hb tetramers
as described in detail by Bunn and Forget.3 The subsequent
formation of long Hb fibers involves many sites of contact
as deduced initially from the effects of natural Hb mutants
with substitutions at other sites on the polymerization of
HbS4 and subsequently from the x-ray structure5,6 and also
by electron microscopy,7,8 although differences in interpretation of these models persist. Because this structural information is available, it is now feasible to study the overall
strength of various sites in sickle hemoglobin aggregates.9,10
An alternate approach, ie, chemical modification has provided information about the extent of participation of other
amino acid residues in the aggregation process.11-15 However,
with this approach there are many sites that are not accessible
to a given reagent. More recently, the use of recombinant
Hb16,17 has provided the advantage that any amino acid substitution can be made at sites not represented by the natural
From The Rockefeller University, New York, NY; and Northeastern University, Boston, MA.
Submitted May 3, 1996; accepted January 21, 1997.
Supported in part by Grant No. HL-18819 (to J.M.M.) from the
National Institutes of Health, Bethesda, MD, and by the Academy
of Finland, Helsinki (J.-P.H.).
Address reprint requests to James M. Manning, PhD, Northeastern University, Department of Biology, 360 Huntington Ave, Boston,
MA 02115.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
0006-4971/97/8911-0021$3.00/0
Blood, Vol 89, No 11 (June 1), 1997: pp 4196-4203
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RECOMBINANT SICKLE HEMOGLOBIN DOUBLE MUTANT
Fig 1. FPLC chromatography of E6V(b)/D85K(a) double mutant
on Mono Q column. The hemoglobin obtained from the yeast extract
was first purified on CM-52 cellulose as described in the text, applied
to Mono Q column, and eluted using a linear NaCl gradient in 20
mmol/L Tris-acetate buffer, pH 8.0.
4197
chloromethylketone (TPCK)-treated trypsin, dextran, diphosphoglycerate (DPG), and inositol hexaphosphate (IHP) were purchased
from Sigma (St Louis, MO). pBluescript II SK(/) was from Stratagene (La Jolla, CA). The construction of pGS189 and pGS389
plasmids is described elsewhere.16,21 All other reagents were of analytical purity.
Site-directed mutagenesis. To prepare the E6V/D85K(a) mutant, we first inserted the a-globin-coding gene from pGS189 to
pBluescript II SK(/) as a Sal I fragment. This fragment contains
the full-length human a-globin cDNA under transcriptional control
of a pGGAP promoter. The modified plasmid (pSK[/]a) was transformed in Escherichia coli BW313, and the uridine-containing single-stranded DNA was isolated from the supernatant of the bacterial
culture after infecting the cells with M13KO7 helper phage. The
oligonucleotide 5*-GTGCGCGTGCAGCTTGCTCAGGGCGGA-3*
was used to create the Asp-85 r Lys mutation by the method of
Kunkel.25 The underlined bases are those used to create the desired
mutation. The presence of the mutation was sought by screening
with partial sequencing of the mutation site. The mutated a-globin
region was subcloned into pGS189sickle, which contains the native
a-globin and the 6(b)Glu r Val mutated b-globin cDNAs,17 by
digestion with BssHII and BstEII enzymes to create incompatible
cohesive termini and thus to increase the percentage of the insert in
correct orientation. Finally, the a- and b-globin gene cassette was
isolated as a Not I fragment after digesting the newly synthesized
pGS189sickle-85(Lys) with Not I and Bgl I and inserted into pGS389
previously digested with Not I. The correct insertional direction was
verified by restriction mapping and the entire a-globin gene was
sequenced to establish that the Asp-85(a) r Lys was the only mutation.
Growth of yeast and purification of the mutant Hb. The yeast
cells were transformed by the pGS389sickle-85(Lys) plasmid using a
lithium acetate method.26 The transformants were selected and the
copy number of the plasmid was increased by growing the yeast on
deoxy HbS also implicates Asp-85(a) in interdouble strand
contacts,5,6 as it has several interactions including an ion
pairing with Lys-144(b). To quantitate its role in aggregation, we replaced Asp-85(a) with Lys by site-directed mutagenesis to completely abolish its ion interaction with Lys144(b). However, in view of a possibly unfavorable interaction between Lys-85(a) and Lys-144(b), it was of critical
importance to establish that the functional integrity of the
double mutant was not compromised. For measuring the
gelation concentrations, we have adapted a new and sensitive
procedure to make quantitative comparisons between
E6V(b)/D85K(a) and previously produced HbS double mutants and to evaluate its significance as a potential target
site for the development of chemical inhibitors against HbS
gelation.
MATERIALS AND METHODS
Reagents and plasmids. The restriction endonucleases, T4 polynucleotide kinase, alkaline phosphatase, DNA ligase, and Gene 32
Protein, T4 were from Boehringer Mannheim (Germany). The DNA
sequencing kit and the T7 DNA Polymerase (Sequenase Version
2.0) were obtained from US Biochemical Corp (Cleveland, OH).
The 35S-labeled deoxyadenosine triphosphate (dATP) was from DuPont NEN (Boston, MA). The oligonucleotides were synthesized by
Operon Technologies (Alameda, CA). CM-Cellulose 52 was from
Whatman, MonoQ from Pharmacia (Stockholm, Sweden), and highperformance liquid chromatography (HPLC) columns (C-4 and
400VHP575) from Vydac (Southborough, MA). Tosylphenylalanine
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Fig 2. Isoelectric focusing of the purified E6V(b)/D85K(a) Hb. A
gel from Isolab (pH 6-8) containing about 30 mg of protein was electrophoresed at 10 W for 45 minutes and stained by bromophenol
blue. The standard contains (from top to bottom): HbC, HbS, HbF,
and HbA.
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HIMANEN, POPOWICZ, AND MANNING
Table 1. Amino Acid Composition of the Mutant Peptide
Amino Acid
Found
Expected for Mutant Pentapeptide
Asx
Thr
Ser
Glx
Gly
Ala
Val
Leu
Phe
His
Lys
Arg
0.1
0.3
0.2
0.1
0.1
1.6
0
0.9
0.4
2.1
[1.0]
0.2
0
0
0
0
0
1
0
1
0
2
1
0
The peptide was isolated as shown in Fig 3. Lys was set equal to
[1.0].
Analytical methods. Mass spectrometric analysis was done on
a matrix-assisted laser desorption time-of-flight mass spectrometer
constructed at Rockefeller University, New York, NY and described
elsewhere.27,28 Fast Protein-Peptide-Polynucleotide Liquid Chromatography (FPLC) purification on Mono Q (Pharmacia, Uppsala, Sweden) was performed using 20 mmol/L Tris-acetate buffer, pH 8.0,
and an NaCl gradient from 0 to 1 mol/L. Isoelectric focusing, amino
acid analysis, and other procedures were performed as described
earlier.20,21,29,30 To isolate the a- and b-globin chains, a Vydac C-4
column was equilibrated with 37.6% acetonitrile in 0.1% trifluoroacetic acid and the sample was eluted with a linear gradient of
acetonitrile to 43.3%. The isolated a-globin chain was digested with
trypsin,21 and the resulting peptides were separated on a Vydac
400VHP575 strong cation exchange column by a linear gradient of
NaCl in 20 mmol/L sodium acetate /10% acetonitrile, pH 5.2.
Fig 3. Tryptic peptide maps of the a-chains. The a-chains of HbA
(A) and E6V(b)/D85K(a) (B) were isolated, carboxymethylated, and
digested with trypsin as described in the text. The resulting peptides
were separated on a Vydac 400VHP575 strong cation exchange column by NaCl gradient (0 to 100 mmol/L) in 20 mmol/L Na-acetate/
10% acetonitrile buffer (pH 5.2). The arrow shows the mutant pentapeptide.
a complete minimal medium first without uracil, then without uracil
or leucine. To express the E6V(b)/D85K(a) mutant hemoglobin, the
yeast was grown in yeast extract-peptone (YP) medium for 4 days
with ethanol as the carbon source.16 The promoter controlling the
transcription of the globin genes was induced by adding 3% galactose for 20 hours before harvesting of the yeast cells. The cells
were disrupted in a Bead Beater homogenizer (Biospec Products,
Bartlesville, OK) and the Hb double mutant was purified on a CMCellulose 52 column with a slight modification from Martin de Llano
et al,17 as described below.
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Fig 4. Oxygen binding curve of E6V(b)/D85K(a). The purified double mutant in 50 mmol/L bis-Tris acetate, pH 7.4, was concentrated
to 0.5 mmol/L, converted to the oxy form, and the oxygen binding
curve was measured at 377C by a modified Hem O Scan instrument.
The inset shows the calculation of the n value.
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RECOMBINANT SICKLE HEMOGLOBIN DOUBLE MUTANT
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Table 2. Influence of Effectors on Oxygen Affinity of Recombinant HbS and E6V(b)/D85K(a)
[DPG] mmol/L
E6V(b)/D85K(a)
HbS, P50
[IHP] mmol/L
[Cl] mmol L
0
0.3
0.4
0.6
0.2
0.4
100
500
10
10
—
17
17
—
23
24
20
16*
51
37*
13
15†
17
21†
The Hb concentration was 0.5 mmol/L in tetramer.
* The values are from Himanen et al.38
† The values are from Martin de Llano et al.29
Tetramer-dimer dissociation constant. This measurement was
performed on the liganded recombinant Hb on Superose-12 using a
Pharmacia FPLC system.31
Functional studies. The oxygen dissociation curves were determined at 377C on a modified Hem O Scan instrument (Aminco,
Silver Spring, MD) as described previously.21,29 Before the measurements, the Hb samples were dialyzed, converted to the oxy form,32
and concentrated using CentriPrep, Centricon and MicroCon ultrafiltration devices (Amicon; molecular weight cut-off of 10,000). The
final protein concentrations were verified by amino acid analysis on
a Beckman 6300 analyzer (Palo Alto, CA). When evaluating the
effects of allosteric modulators, the samples were in 50 mmol/L bisTris buffer, pH 7.4.
For determining the Bohr effect, the Hb samples were first dialyzed against H2O, concentrated to a final concentration of 1 mmol/
L and diluted with an equivalent amount of 100 mmol/L bis-Tris
buffers of different pH values before the measurement of the oxygen
dissociation curves. The final pH was verified by a microelectrode
(Microelectrodes Inc, Bowdoinville, ME).
Determination of Csat . The method used for determining the
gelation concentration (Csat ) of Hb is based on the decrease of the
solubility of HbS in the presence of dextran.33 The E6V(b)/D85K(a)
or HbS sample in the oxy form in 50 mmol/L potassium phosphate
buffer, pH 7.5 was mixed with dextran (100 mg/mL final concentration). Mineral oil was then layered on top and fresh sodium dithionite
solution (50 mmol/L final concentration) was added below the Hbdextran mixture anaerobically using a gas-tight syringe. The reaction
mixture was incubated at 377C for 30 minutes, mixed thoroughly,
and centrifuged in a microcentrifuge for 30 minutes. The clear supernatant was carefully separated from the aggregated Hb and its hemoglobin concentration measured by amino acid analysis on a Beckman
6300 amino acid analyzer. The difference between the Hb concentration of the supernatant (Csat ) and the initial Hb concentration is an
indication of the extent of gelling.
Molecular modeling. The molecular modeling was done on a
Silicon Graphics, Inc (Mountain View, CA) Power Indigo 2 computer using the molecular modeling program Insight II (Biosym/
MSI). The coordinates of human sickle hemoglobin (1HBS) were
obtained from the Brookhaven protein database.
directed mutagenesis. Presumably the high G/C content of
the oligonucleotide (see Materials and Methods), and its consequent strong tendency to form a hairpin loop (DG Å 00.6
kcal mol01) caused the mutation frequency to fall below the
detection limit when using the standard Kunkel method.25
By decreasing the oligonucleotide:template ratio to 50:3 and
performing the annealing reaction at a temperature change
of 957C to 207C over a period of 15 hours in the presence
RESULTS
Mutagenesis. Because no a-chain HbS mutants have
been produced using the yeast expression system, it was
necessary to create a plasmid containing the a-globin gene.
This was accomplished by taking advantage of the unique
Sal I site at the multiple cloning site of pBluescript II SK(/).
A Sal I fragment of the pGS189 extending downstream from
the end of the b-globin gene to the end of the a-globin gene
was ligated with pBluescript II SK(/) previously digested
with the same restriction enzyme. The single-stranded DNA
rescued from this plasmid was used as a template in site-
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Fig 5. The alkaline Bohr effect of E6V(b)/D85K(a). The purified
double mutant in oxy form was diluted with bis-Tris buffers of different pH values to a final concentration of 0.5 mmol/L Hb in 50 mmol/
L bis-Tris, and the P50 values were determined as in Fig 4.
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HIMANEN, POPOWICZ, AND MANNING
Fig 6. Gelation concentration (Csat ) of HbS and E6V(b)/D85K(a).
Oxy hemoglobin samples in 50 mmol/L potassium phosphate, pH
7.5, were mixed anaerobically with dextran and sodium dithionite,
incubated at 377C, and centrifuged. The hemoglobin concentrations
in the supernatant before (initial [Hb]) and after (equilibrium [Hb])
the incubation were determined by amino acid analysis. If the equilibrium [Hb] is lower than the initial [Hb], it represents the gelation
concentration of Csat of the Hb.
of Gene 32 Protein, the open circular, incomplete circular,
and the covalently closed circular double-stranded DNA
forms were obtained by the extension reaction as shown by
Yuckenberg et al34 (data not shown). The mutation frequency
was increased to 25%.
Purification. After subcloning the mutated DNA fragment into pGS389, which contains the human a- and bglobin cDNAs, the sickle hemoglobin double mutant,
E6V(b)/D85K(a) was expressed in yeast as described in
Materials and Methods. On a CM-Cellulose 52 column, it
adhered more avidly than HbS, as expected for a mutant
having an Asp to Lys surface mutation. For elution, a gradient of up to 25.5 mmol/L potassium phosphate instead of
15 mmol/L used for HbS17 was required.
The purified hemoglobin was rechromatographed on
Mono Q column from which it eluted as a single peak (Fig
1) without any indication of multiple forms of recombinant
hemoglobin reported earlier by others.35,36 Isoelectric focusing (Fig 2) of the purified E6V(b)/D85K(a) indicated an
isoelectric point (pI)-value close to 8.0.
Mass spectrometry. Matrix-assisted laser desorption
mass spectrometry was used to verify the molecular masses
of the a- and b-globin chains of the purified Hb double
mutant. The molecular mass (15,140.3) obtained for the achain by a time-of-flight method agrees well with the theoretical value of 15,139.5 mass units for the mutant a-chain.
The difference of 13.9 mass units between the measured
value and the calculated value of a wild-type a-chain of
HbA (15,126.4 mass units) is close to the calculated difference (13.1 mass units) between the molecular masses of Asp
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and Lys residues. The molecular mass obtained for the bchain (15,835.4 mass units) agreed with the calculated value
for HbS (15,838.2 mass units) within the error of the measurement.
Peptide mapping. For this analysis, we took advantage
of the creation of a new trypsin cleavage site at the 85a position. Digestion of the isolated a-chain with trypsin
produced the expected strongly basic pentapeptide (Leu-HisAla-His-Lys). Because a reversed phase column generally
used for peptide mapping was unable to separate this peptide
from the others, we used a strong cation exchanger (Vydac
400VHP575) for this purpose. At pH 5, the pentapeptide has
a net charge of /3 and separated as shown in Fig 3. In
comparison, the chromatogram obtained using the a-chain
of HbS lacked this peak. This pentapeptide was collected
and it had the expected amino acid composition (Table 1).
Functional properties. The oxygen binding properties of
E6V(b)/D85K(a) were determined under various conditions.
In the absence of added chloride, the double mutant showed
a typical sigmoidal oxygen equilibrium curve (Fig 4). The
P50 value was 10, which is the same as that for HbA, HbS,
and the K95I(b) recombinant Hb21 under the same conditions. The double mutant was cooperative with an average
Hill coefficient of 2.7. In the presence of increasing amounts
of chloride ions, its P50 value gradually increased to a maximum value of 17 mm/L Hg at a chloride concentration of 500
mmol/L (Table 2). Under similar conditions, we typically
observe an increase in the P50 value of HbS, HbA, and other
double mutants to a maximum value from 21 to 25 mm
Hg.20,29,30,37 Thus, E6V(b)/D85K(a) shows a somewhat diminished response to chloride. A possible reason for this
effect is discussed below.
The influence of two organic phosphate effectors, diphosphoglycerate (DPG) and inositol hexaphosphate (IHP), on
the P50 value of the double mutant D95K was also tested
(Table 2). Although the results in Table 2 show some variability with each Hb, with either allosteric effector the ratio
of effector:Hb concentrations at the point of maximum effect
was close to one for both the E6V(b)/D85K(a) mutant and
HbS. The Hill coefficients of E6V(b)/D85K(a) in the presence of various concentrations of chloride or DPG varied
between 2.3 and 2.8. Thus, the double mutant showed full
cooperativity. Small conformational changes in these recombinant Hbs cannot be rigorously excluded. However, a careful circular dichroism study of another HbS double mutant20
did not show evidence for such an effect.
Tetramer-dimer dissociation constant. The tetramer-dimer dissociation constant for the liganded recombinant
E6V(b)/D85K(a) was found to be 2.1 { 0.2 mmol/L. This
value is slightly higher than the 0.7 { 0.2 mmol/L found for
HbS, but because it is much less than the Hb concentration
(500 mmol/L to -2 mmol/L) used for the functional studies
such as the oxygen binding curve, the Bohr effect and the
gelation studies, the double mutant was predominantly tetrameric during these measurements.
Bohr effect. When the pH was increased from 6.8 to 7.4,
the P50 of the double mutant and of HbA decreased from
18.2 to 9.0. The slope of the line obtained by plotting the
log P50 values against pH gives the alkaline Bohr coefficient
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RECOMBINANT SICKLE HEMOGLOBIN DOUBLE MUTANT
4201
Fig 7. View along the central
dyad axis of deoxy HbS. The distances between the two Lys-99
e-NH2 groups, which is a major
chloride-binding site in the center of the axis, and between the
oxygen-linked chloride-binding
residue (Val-1[a]) and the newlycreated mutant residue (Lys85[a]) are shown.
(Fig 5). This value was calculated to be 00.34 (correlation
coefficient, r Å .994) for E6V(b)/D85K(a), which agrees
reasonably well with the value of 00.41 (r Å .995) obtained
for HbA.
Gelation. The gelation concentration of E6V(b)/
D85K(a) was measured in the presence of 100 mg/mL of
dextran by the method of Bookchin et al33 as described in
Materials and Methods. After incubation at 377C and centrifugation, the maximum solubility (Csat ) of HbS or of the
double mutant was obtained by measuring the Hb concentration in the supernatant. The Csat value of E6V(b)/D85K(a)
at three different initial Hb concentrations of 56, 64, or 127
mg/mL was between 50 and 55 mg/mL (Fig 6). An initial
Hb concentration of 31 mg/mL was below the Csat value and
thus, no change in the Hb concentration after incubation and
centrifugation was observed. The average Csat value of 53
mg/mL of E6V(b)/D85K(a) is clearly elevated compared
with the value of 34 mg/mL of HbS, but much lower than
the values for two other double mutants produced in our
laboratory and whose gelation was determined by the same
method, ie, E6V(b)/L88A(b) (67 mg/mL) and E6V(b)/
K95I(b) (90 mg/mL).38
DISCUSSION
Recombinant hemoglobins have been produced using several different expression systems. In earlier studies, we
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showed that the recombinant sickle hemoglobin produced in
the yeast expression system is indistinguishable by a number
of chemical and biochemical assays from native HbS isolated
from human red blood cells.29 The misfolding of hemoglobin
reported for the E coli expression39 is not apparent for the
hemoglobin expressed in yeast by any of the criteria that we
have used for characterization. The production and characterization of an E6V(b)/D85K(a) hemoglobin double mutant
is, to our knowledge, the first a-chain sickle Hb mutant
produced using recombinant DNA technology. The mutant
Hb was shown to have the predicted molecular mass, isoelectric point, and trypsin cleavage sites. Its oxygen affinity,
response to DPG, Hill coefficient, and the alkaline Bohr
effect were the same as the corresponding values for native
HbA. Thus, it is identical to natural HbA and HbS in these
properties. Possible minor local changes in the orientation
of amino acids around the D85K(a) mutant site with respect
to chloride binding are discussed below.
The replacement of Asp with Lys could have created
strong unfavorable contacts between the a-85 site and some
positively charged residues in its vicinity and thus have had
a large effect on the intratetrameric contacts and on the
functional integrity of the mutant hemoglobin. However, the
dissociation constant (Kd) for the tetramer-dimer equilibrium of the E6V(b)/D85K(a) double mutant, measured by
a chromatographic method developed in our laboratory,31
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HIMANEN, POPOWICZ, AND MANNING
showed only a slightly increased Kd value (2.1 mmol/L) in
comparison to HbS (0.7 mmol/L). For the functional properties of the double Hb mutant described in this study at a Hb
concentration of 0.5 mmol/L to 2 mmol/L, the extent of
dimerization of the double mutant is negligible.
The E6V(b)/D85K(a) double mutant had a slightly decreased response to chloride; in the presence of 0.5 mol/L
NaCl, the double mutant had a P50 of 17 mm Hg in comparison to 21 mm Hg for HbS (ie, a 20% reduction in the oxygenlinked chloride effect). Earlier studies by us and others have
shown that one of the oxygen-linked chloride-binding sites
is located at the a-a interface in the central dyad axis of Hb
and consists predominantly of amino acids Val-1(a) and
Arg-141(a).40,41 As shown in Fig 7, the newly created
D85K(a) mutation introduces the positive charge of Lys-85
10.8 Å apart from that of Val-1(a). In comparison, the distance between the two Lys-99 e-NH2 groups in the center
of the dyad axis, a major Cl0 binding region,42-44 is 10.6 Å,
small enough to be bridged by a Cl0 ion with a Van der
Waals radii of about 2.5 Å. Thus, the D85K(a) mutation
could create a new Cl0-binding site that could compete with
or diminish the oxygen-linked chloride effect at Val-1(a).
Interestingly, Fronticelli et al45 have recently reported a construction of a human Hb mutant having a similar, but opposite, Cl0 effect, where an A76K(b) mutation creates a new
positively charged cleft between Lys-8(b) and Lys-76(b)
and an increase in the effect of chloride ions on the oxygen
affinity.
Three natural alpha-85 Asp mutants, Hb G-Norfolk (Asp r
Asn),46 Hb Atago (Asp r Tyr),47 and Hb Inkster (Asp r
Val)48 have been described. Each has an increased oxygen
affinity, but no studies have been reported on their participation in gelation. Our results on the functional properties of
the E6V(b)/D85K(a) mutant do not indicate an altered P50
and furthermore do not give any indication that the threedimensional structure of the double mutant has been adversely affected by the substitution.
In this study, we have continued our efforts to understand
not only the amino acid residues involved in the formation
of HbS fibers, but also to measure the relative strength of
these interactions in a quantitative manner. For this purpose,
we have employed a new method33 for measuring the gelation concentrations of HbS variants, which takes advantage
of the drastic diminishing effect of dextran on the solubility
of HbS. Using this method, we showed that the gelation
concentration of the E6V(b)/D85K(a) double mutant was
elevated to 53 mg/mL as compared with 34 mg/m obtained
for HbS. This decreased tendency for gelation is consistent
with the x-ray studies,5,6 ie, in the HbS crystal the Asp-85(a)
residue forms an ion pair with Lys-144(b) of the adjacent
Hb tetramer, which is abolished in the D85K(a) mutant and
the gelation is consequently inhibited.
Our conclusion that Asp-85(a) contributes moderately to
the strength of the HbS aggregate is also consistent with,
but does not distinguish between the electron microscopic
models7,8 and furthermore establishes its quantitative participation in gelation. The model of Watowich et al8 suggests
some degree of participation of Asp-85(a) in the gelation,
as it is 5 to 8 Å apart from the adjacent tetramer. The model
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also shows that Lys-95(b) is involved in an intermolecular
contact of less than 5 Å. Such proximity is consistent with
our earlier results21 in which we showed that a replacement
of Lys-95(b) with Ile causes a drastic inhibition of gelation.
These results emphasize the significant differences between
various ionizable surface amino acids in stabilizing the HbS
aggregate and suggest that an appreciation of the quantitative
contribution of critical amino acid side chains to the aggregation process might show new and more important sites, thus
prompting further consideration of developing a therapeutic
modality directed at the HbS polymer itself.
ACKNOWLEDGMENT
We thank Adelaide Acquaviva for her expert help with the typescript and Drs Urooj Mirza and Brian Chait for the mass spectrometric analysis.
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From www.bloodjournal.org by guest on May 16, 2016. For personal use only.
1997 89: 4196-4203
Recombinant Sickle Hemoglobin Containing a Lysine Substitution at
Asp-85( α): Expression in Yeast, Functional Properties, and Participation in
Gel Formation
Juha-Pekka Himanen, Anthony M. Popowicz and James M. Manning
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