Inhibition of ßS-chain dependent polymerization by synergistic complementation of contact site perturbations of {alpha}-chain: application of semisynthetic chimeric {alpha}-chains

Sonati Srinivasulu1, Ashok Malavalli1, Muthuchidambaran Prabhakaran3, Ronald L. Nagel1,2 and A.Seetharama Acharya1,2,4

1 Division of Hematology, Department of Medicine and 2 Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300, Morris Park Avenue, Bronx, NY 10461 and 3 Structural Bioinformatics, San Diego, CA 92127, USA


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Mouse {alpha}1–30-horse {alpha}31–141 chimeric {alpha}-chain, a semisynthetic super-inhibitory {alpha}-chain, inhibits ßS-chain dependent polymerization better than both parent {alpha}-chains. Although contact site sequence differences are absent in the {alpha}1–30 region of the chimeric chain, the four sequence differences of the region {alpha}17-22 could induce perturbations of the side chains at {alpha}16, {alpha}20 and {alpha}23, the three contact sites of the region. A synergistic complementation of such contact site perturbation with that of horse {alpha}31–141 probably results in the super-inhibitory activity of the chimeric {alpha}-chain. The inhibitory contact site sequence differences, by themselves, could also exhibit similar synergistic complementation. Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin (LM) mutation [His20({alpha})->Gln], a contact site sequence difference, engineered into human–horse chimeric {alpha}-chain has been investigated to map such a synergistic complementation. Gln20({alpha}) has little effect on the O2 affinity of HbS, but in human–horse chimeric {alpha}-chain it reduces the O2 affinity slightly. In the chimeric {alpha}-chain, Gln20({alpha}) increased sensitivity of the ßß cleft for the DPG influence, reflecting a cross-talk between the {alpha}1ß1 interface and ßß cleft in this semisynthetic chimeric HbS. In the human {alpha}-chain frame, the polymerization inhibitory activity of Gln20({alpha}) is higher compared with horse {alpha}1–30, but lower than mouse {alpha}1–30. Gln20({alpha}) synergistically complements the inhibitory propensity of horse {alpha}31–141. However, the inhibitory activity of LM–horse chimeric {alpha}-chain is still lower than that of mouse–horse chimeric {alpha}-chain. Therefore, perturbation of multiple contact sites in the {alpha}1–30 region of the mouse–horse chimeric {alpha}-chain and its linkage with the inhibitory propensity of horse {alpha}31–141 has been now invoked to explain the super-inhibitory activity of the chimeric {alpha}-chain. The `linkage-map' of contact sites can serve as a blueprint for designing synergistic complementation of multiple contact sites into {alpha}-chains as a strategy for generating super-inhibitory antisickling hemoglobins for gene therapy of sickle cell disease.

Keywords: chimeric HbS/HbS polymerization/intermolecular contact sites/linkage map/protein engineering/super-inhibitory {alpha}-chain


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The propensity of mouse {alpha}-chain to inhibit the ßS-chain dependent polymerization reaction is primarily responsible for the failure of the transgenic mice expressing human sickle cell hemoglobin to exhibit fully the sickling phenotype (Rhoda et al., 1988Go; Fabry et al., 1992Go; Constantini et al., 1986Go). Our earlier investigations of the molecular basis of the inhibition of the polymerization revealed that mouse {alpha}1–30 segment containing eight sequence differences and mouse {alpha}31–141 segment with 11 differences inhibited the polymerization. Their inhibitory potential was considerably lower than that of the full-length mouse {alpha}-chain. Besides, the sum of the inhibitory potential of the two complementary segments of mouse {alpha}-chain does not account for the full inhibitory potential of the full length (intact) mouse {alpha}-chain (Roy et al., 1993Go). The segregation of the sequence differences results in the loss of some amount of polymerization inhibitory activity, reflecting the presence of a `linkage' or `functional complementation' of the polymerization inhibitory potential of the sequence differences of two segments of mouse {alpha}-chain to exhibit a higher inhibitory potential.

This concept of `linkage' of the polymerization inhibitory potential of sequence differences in the high inhibitory potential of horse and mouse {alpha}-chain is supported by the observation that the grafting of mouse {alpha}1–30 with the horse {alpha}31–141 segment resulted in the generation of a super-inhibitory chimeric {alpha}-chain. The polymerization inhibitory potential of the mouse–horse chimeric {alpha}-chain is even higher than that of both parent chains, horse and mouse {alpha}-chains. None of the eight sequence differences of mouse {alpha}1–30 are at implicated contact sites and in the horse {alpha}31–141 six of sequence differences (of a total of 15) are located at the implicated intermolecular contact sites. Thus the polymerization inhibitory sequence differences of horse {alpha}31–141 can establish a cross-talk with the sequence differences of mouse {alpha}1–30 even though they are not at the implicated contact sites to establish a `functional complementation' of their polymerization inhibitory activity (Nacharaju et al., 1997Go).

The {alpha}17-22 region of mouse {alpha}-chain carries four of the eight sequence differences of the mouse {alpha}1–30, namely at {alpha}17, {alpha}19, {alpha}21 and {alpha}22 (Table 1Go). Three implicated intermolecular contact sites of HbS are located in this region, namely at {alpha}16, {alpha}20 and {alpha}23. The perturbation of the microenvironment of the side chains of one or more of these three contact sites may result from the sequence differences of this region. This may be the basis of the inhibitory activity and/or synergy seen in the earlier studies. Consistent with this, the molecular modeling studies of the chimeric HbS containing the mouse–horse chimeric {alpha}-chain suggested altered side chain interactions at the {alpha}1ß1 interface in this chimeric HbS as compared with HbS. The sequence differences of the AB/GH corner of the {alpha}-chain facilitated a distinct stereochemistry for the interaction of the {varepsilon}-amino group of Lys16({alpha}) with the ß-carboxyl group of Asp-116({alpha}) (Nacharaju et al., 1997Go), an implicated intermolecular contact site. This ß-carboxylate {varepsilon}-amino group interaction, in turn, perturbs the accessibility of the side chain of Lys16({alpha}) to the solvent and hence its participation in the polymerization process. Since Lys16({alpha}) is an implicated intermolecular contact site, we had hypothesized that this perturbation of the {alpha}-chain AB/GH corner represents, at least in part, the molecular basis of the high polymerization inhibitory activity of mouse–horse chimeric {alpha}-chain. Similar perturbation of the two other contact sites of the region, His20({alpha}) and Glu23({alpha}), is also conceivable, although it was not readily apparent in the earlier modeling studies. Accordingly, we now hypothesize that the propensity of mouse {alpha}1–30 to establish functional synergistic complementation (cross-talk) with the polymerization inhibitory propensity of horse {alpha}31–141 represents the perturbations of one or more of these three implicated contact sites [Lys16({alpha}), His20({alpha}) and Glu23({alpha})] because of the presence four sequence differences in this region of mouse–horse chimeric {alpha}-chains.


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Table I. Comparison of amino acid sequence differences in the {alpha} 1–30 segments human–horse, horse–human, mouse–horse and Le Lamentin–horse chimeric {alpha}-chains
 
Since the mouse {alpha}1–30 sequence differences at the AB corner of mouse–horse chimeric {alpha}-chain are not at contact sites, but cross-talk with the sequence differences of horse {alpha}31–141 (presumably with the contact site sequence differences), we have now asked the question of whether a contact site sequence difference of AB corner could exhibit a similar functional complementation with the polymerization inhibitory potential of sequence differences of horse {alpha}31–141, and, if it does, whether it can compare with that of mouse–horse chimeric {alpha}-chain. Hb Le Lamentin (LM) mutation [His20({alpha})->Gln] (Sellaye et al., 1982Go), a proven contact site sequence difference of this region (Benesh et al., 1982), has been now introduced into human–horse chimeric {alpha}-chains (Figure 1A and BGo), to map whether a synergistic complementation can occur between Gln20({alpha}) and the contact site sequence differences of horse {alpha}31–141. Human horse chimeric {alpha}-chain has six contact site sequence differences whereas LM–horse chimeric {alpha}-chain has seven sequence differences (Figure 1A and BGo). The {alpha}-globin semisynthetic reaction developed earlier in this laboratory (Sahni et al., 1989Go; Roy and Acharya, 1994Go) has been used to introduce Gln20({alpha}) into human and human–horse chimeric {alpha}-globin chains.



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Fig. 1. Molecular models of human–horse {alpha}-chimeric HbS with and without His20({alpha})->Gln mutation. A ribbon diagram of the tetramers, showing only the backbone, was used for the presentation of sequence differences and the identification sites of intermolecular contact site sequence differences. The {alpha}-chains are shown in green and the ß-chains are shown in blue. The position of sickle mutation is shown in yellow. The sequence difference of the chimeric {alpha}-chain of human–horse {alpha}-chimeric HbS is shown in red. An arrow pointing out from the indicated site of sequence difference provides the information on that site in the chimeric HbS. This information is provided for the sequence differences present in only one of the chimeric {alpha}-chains. The first letter represents the subunit of the tetramer and the number next to it identifies the position in the amino acid sequence of the chain. If a particular site of a sequence difference is at an implicated contact site of deoxy HbS fiber, the number is presented in red and it is placed in a red box. Within the brackets are given the amino acid sequence differences. The amino acids are identified by single-letter codes; the first letter identifies the amino acid residue in HbA and the second letter following the arrow identifies the amino acid residue in the chimeric {alpha}-chain. If the sequence difference is at an implicated molecular contact site and if there are solution studies confirming this, the amino acid residue that inhibits the polymerizarion when placed at this site is given outside the bracket in a blue box; the amino acid residue that inhibits the polymerization is presented inside this box in blue. (A) Chimeric HbS containing human–horse chimeric {alpha}-chain. (B) Chimeric HbS containing Le Lamentin–horse chimeric {alpha}-chain. The position of His20({alpha})->Gln is highlighted.

 
The studies reported here establish that LM mutation, a contact site sequence difference, exhibits synergy with the polymerization inhibitory propensity of horse {alpha}31–141. However, the synergistic complementation seen by the cross-talk of Gln20({alpha}) with the sequence differences of horse {alpha}31–141 is considerably lower than that generated by the cross-talk of the sequence differences of mouse {alpha}1–30 with the sequence differences of horse {alpha}31–141. The results suggest the contribution of the perturbations of the other contact sites of the {alpha}1–30 region of HbS in generating a super-inhibitory activity for the mouse–horse chimeric {alpha}-chain.


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Preparation of Hb samples and the globin chains

HbA and S were purified by DE 52 chromatography followed by CM 52 chromatography. The {alpha}- and ß-chains were separated according to the procedure described by Bucci (1981). Human {alpha}-globin were prepared by acid acetone precipitation. Horse Hb was purified on CM-cellulose as described previously (Nacharaju and Acharya, 1992Go). Horse {alpha}-globin was purified by CM 52 urea chromatography from total horse globin prepared by acid acetone precipitation.

Chemical synthesis of {alpha}1–30(His20->Gln)

The peptide corresponding to the 1–30 sequence of {alpha}-chain with one replacement at the 20th position (His->Gln) was chemically synthesized in the in-house Macromolecular Chemistry Laboratory. The synthetic peptide was purified by RP-HPLC. The authentication of the synthetic peptide was done by mass spectral analysis. Semisynthesis of chimeric {alpha}-globin and the assembly of the tetrameric structures and the purification of the assembled tetramers were carried out as described previously (Yipp et al., 1977; Roy and Acharya, 1994Go).

Mass spectral analysis of the purified semisynthetic {alpha}-globin

The mass spectral analysis was performed on an API-III triple-quadrupole mass spectrometer (Perkin-Elmer SCIEX, Thornhill, ON, Canada) using a SCIEX ionspray interface with nitrogen as the nebulizer gas. An ionspray voltage of about 3600 V and an orifice voltage of 85 V were used. The sample was infused into the mass spectrometer at 2 µl/min using a Harvard Apparatus syringe pump after diluting 1:1 with 50% acetonitrile–H2O + 0.1% acetonitrile.

Functional studies

The oxygen affinity of Hb samples was determined using a Hem-O-Scan at 37°C. These measurements were made in 50 mM Bis-Tris, 50 mM Tris–acetate buffer, pH 7.4. The concentration of the protein was 0.5 mM in tetramer. The oxygenation curves were generated in triplicate for each sample and P50 values could be determined with an accuracy of ±0.5. The polymerization of HbS and other semisynthetic Hbs was carried out in either 1.8 or 2.0 M phosphate buffer (pH 7.4) at 30°C. The kinetics of polymerization were performed by the method of Adachi and Asakura (1979).


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Purification and characterization of semisynthetic hemoglobins

The CM-cellulose chromatographic pattern of of Hb assembled from semisynthetic LM {alpha}-chain and ßS-chain is shown in Figure 2AGo. The main peak, eluting at ~220 ml, was identified as HbS-LM. RP-HPLC analysis of HbS-LM demonstrated the stoichiometry of semisynthetic {alpha}-globin and ßS-chain in HbS-LM (inset a). The experimental value for the mass of LM {alpha}-globin isolated by RP-HPLC was 15117 Da, compared with the calculated mass of this mutant {alpha}-chain of 15117.3 Da. The mass difference is consistent with the substitution of His20({alpha}) by a Gln residue. The homogeneity of semisynthetic HbS-LM was established by isoelectric focusing (Figure 2AGo, inset b). The semisynthetic HbS-LM electrofocused at a position intermediate between those of HbA and HbS. The loss of one positive charge (the His->Gln mutation) in HbS-LM relative to HbS should have compensated for the loss of one negative charge [the Glu6(ß)->Val mutation] of HbS and in electrofocusing HbS-LM and HbA should have identical patterns. The results reflect the fact that the pKa of His20({alpha}) is close to neutral pH (Sun et al., 1997Go), hence this residue will be protonated only partially around neutral pH, i.e. His20({alpha}) contributes less than one unit to the net charge of HbA and HbS. This difference also explains the elution of HbS-LM slightly ahead of the HbS from the CM 52 cellulose column.



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Fig. 2. CM-cellulose chromatography of semisynthetic hemoglobins. (A) Purification of semisynthetic HbS-LM. The reconstituted sample was purified by CM 52 cellulose column chromatography. The column (1.5x30 cm) was eluted with a linear gradient between 10 mM phosphate buffer (pH 6.5) and 15 mM phosphate buffer (pH 8.5), 250 ml each. The peak that corresponds to HbS-LM and the fractions pooled to isolate the chimeric HbS are indicated. The elution position of HbS is also indicated. Inset a shows the RP-HPLC analysis of the HbS Le Lamentin isolated. Inset b shows the isoelectric focusing pattern of the chromatographically purified sample of semisynthetic HbS Le Lamentin. (B) Purification of the semisynthetic {alpha}LHr2ßS2 The {alpha}-chain of this chimeric HbS is derived by splicing the {alpha}1–30 of Le Lamentin with {alpha}31–141 of the horse {alpha}-chain. The reconstituted sample was purified on a 1.5x30 cm CM-cellulose column. After loading the reconstituted sample, the column was eluted with a linear gradient of 250 ml each of 10 mM phosphate buffer, pH 6.5 and 15 mM phosphate buffer, pH 8.5. The peak corresponding to {alpha}L'H2ßS2 and the fractions pooled to isolate this chimeric HbS are indicated. The elution position of HbS is also indicated. The inset shows the isoelectric focusing pattern of purified chimeric HbS.

 
The chromatographic pattern of reconstituted semisynthetic HbS containing the chimeric {alpha}-chain ({alpha}LM–Hr) is shown in Figure 2BGo. The protein peak eluting at ~275 ml has been identified as a tetramer containing the chimeric {alpha}-chain. Mass spectral analysis of the chimeric {alpha}-chain gave the molecular mass of this {alpha}-chain as 15115 Da, the theoretical mass of this chimeric {alpha}-chain being 15115.5 Da. This confirmed the identity of the chimeric {alpha}-chain in the semisynthetic tetramer.

Isoelectric focusing established the homogeneity of the tetramer assembled (Figure 2BGo, inset a). The semisynthetic chimeric HbS electrofocused close to that of HbS. The presence of Gln at {alpha}20 partially compensates for the difference that existed in the electrofocusing patterns of HbS and the human horse chimeric {alpha}-chain containing tetramer. This difference in the isoelectric focusing pattern of HbS compared with that of the tetramer containing either horse {alpha}-chain or human–horse chimeric {alpha}-chain, is not readily explained based on their amino acid compositions. The conservatory substitutions of horse {alpha}31–141 should be inducing a different ionization behavior to one or more charged groups of semisynthetic chimeric HbS.

Oxygen affinity of semisynthetic hemoglobins

The intrinsic oxygen affinity of HbS-LM is comparable to that of HbA and the Hill coefficient is about 2.4 (Table IIGo), reflecting the limited influence of Gln20({alpha}) on the oxygen affinity. This result is consistent with the oxygen affinity of the naturally occurring HbA-LM and HbS-LM assembled in vitro from HbS and HbA-LM. Thus in the semisynthetic HbS-LM assembled in the present studies, the basic hemoglobin fold has been conserved. Further confirmation of the integrity of the basic Hb-fold of this semisynthetic HbS-LM has come from the influence of DPG in reducing the oxygen affinity of semisynthetic protein (Table IIGo). On the other hand, the O2 affinity of semisynthetic chimeric HbS (a tetramer containing the LM–horse chimeric {alpha}-chain, LM-Hr) is slightly lower than that of HbS (Table IIGo); the new chimeric HbS also binds oxygen cooperatively. The oxygen affinity of {alpha}LM–Hr2 ß2 is reduced by DPG much more than that with the chimeric HbS containing human horse chimeric {alpha}-chain. Thus, the propensity of LM mutation to reduce the oxygen affinity of the chimeric HbS seen in the absence of DPG, is magnified in the presence of DPG, the effector that modulates the oxygen affinity by interacting at the ßß cleft.


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Table II. Oxygen affinity of semisynthetic HbS
 
In contrast, the O2 affinity of horse Hb by itself shows a low sensitivity to DPG. Since the chimeric HbS containing the horse {alpha}-chain and human ßS-chain shows a normal response to DPG, the low sensitivity of horse Hb to DPG is apparently dictated by the horse ß-chain. The absence of His2(ß), a residue of the ßß cleft, is apparently the primary determinant of this phenomenon.

Polymerization of the semisynthetic proteins

The kinetics of polymerization of semisynthetic HbS-LM and HbS was studied in 1.8 M phosphate buffer, pH 7.4 (Figure 3Go). HbS-LM polymerized with a delay time longer than that of native HbS, a result consistent with the inhibitory effect of Gln20({alpha}). The delay time decreases as the concentration increases for HbS and HbS-LM [Figure 3AGo (inset a)]. At identical concentrations, the delay time of HbS-LM is nearly 1.7 times higher than that of HbS (Table IIIGo).



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Fig. 3. Polymerization of semisynthetic hemoglobins. (A) Kinetics of polymerization of HbS-LM: the polymerization was carried out in 1.8 M phosphate buffer, pH 7.4 at 30°C by the temperature jump method. The changes in the turbidity of the sample (a reflection of polymerization) was continuously monitored at 700 nm. The hemoglobin concentration was maintained at 0.462 mg/ml for these studies. Inset a shows the relationship between the logarithm of the reciprocal of the delay time and the logarithm of the initial Hb concentration. Inset b shows the solubility of the deoxy form of HbS and semisynthetic HbS Le Lamentin as a function of protein concentration. Horse–human chimeric HbS ({alpha}HrH2ßS2) was prepared as described earlier (Nacharaju et al., 1997). ({circ}, {alpha}H2ßS2; •, {alpha}L2ßS2). Csat was determined at 30°C by measuring the concentration of Hb in the soluble phase after the completion of the polymerization. The conditions were the same as those used for the kinetic studies. The delay times could be estimated with an accuracy of ±1 min. (B) Kinetics of polymerization of semisynthetic chimeric HbS: the kinetics of the polymerization were carried out in 2.0 M phosphate buffer, pH 7.4. The polymerization reaction was initiated by a temperature jump from 0 to 30°C. The turbidity changes were continuously monitored spectrophotometrically at 700 nm. The concentration of the protein was 0.8 mg/ml.

 

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Table III. Correlation of the polymerization inhibitory activity of His20({alpha})->Gln mutation with that the sequence differences of {alpha}1–30 segments of mouse and horse {alpha}-chains when they are placed in human or human–horse chimeric {alpha}-chain
 
The delay time–concentration correlation of the two samples is parallel (Figure 3AGo, inset a), suggesting that the overall mechanism of polymerization of deoxy protein has not been influenced by the substitution, although the solubility has increased by nearly 40%. The slope of the correlation line in both the cases is about 2.0, suggesting that the apparent size of the nucleus during the polymerization is also similar. The solubility of HbS and semisynthetic HbS-LM as a function of protein concentration is shown in Figure 3AGo (inset b). The increase in the solubility of deoxy HbS-LM seen in the present study using 1.8 M phosphate buffer is 35.7%, in reasonable agreement with the value of 27.3% obtained previously by the ultracentrifugation method using 100 mM phosphate buffer (physiological conditions).

The kinetics of the polymerization of ßS-chains hybridized with horse–human chimeric {alpha}-chain is also shown in Figure 3AGo. The increase in the delay time of the ßS-chain dependent polymerization reaction by His20({alpha})->Gln is higher than that of the three sequence differences of the {alpha}1–30 segment of horse {alpha}-chain (Table IIIGo). The polymerization behavior of HbS, {alpha}HHr2ßS2, {alpha}2->LM–HrßS2 and {alpha}2->LM–HrßS2 in 2.0 M phosphate buffer is shown in Figure 3BGo. The hybrid {alpha}LM–Hr2ßS2 exhibits a delay time which is noticeably higher than that of {alpha}Hr2ßS2. The relative increase in the delay time of the polymerization of {alpha}L–Hr2ßS2 as a result of introduction of the Le Lamentin mutation into the human–horse hybrid chain is larger (Table IIIGo) compared with that seen on introduction of the same mutation on to the human {alpha}-chain of HbS. As noted earlier, the polymerization inhibitory potential of LM mutation in the human {alpha}-globin frame is higher than the polymerization inhibitory potential of the three sequence differences of the {alpha}1–30 segment of horse {alpha}-chain in the same frame. The question then is whether this larger increase is a direct consequence of higher inhibitory potential or a consequence of synergistic anti- polymerization of Gln20({alpha}) with horse {alpha}31–141.

The inhibitory propensity of His20({alpha}) engineered into human horse chimeric {alpha}-chain (Table IIIGo) is higher than the three sequence differences of horse {alpha}1–30 when it is present as a part of the horse {alpha}-chain, but considerably lower than that of the mouse–horse chimeric {alpha}-chain. As noted earlier, the segregation of the sequence differences of horse and mouse {alpha}-chain into segments {alpha}1–30 and {alpha}31–141 results in the loss of some polymerization inhibitory potential. The higher inhibitory activity of human–horse chimeric {alpha}-chain with His20({alpha}), relative to the same mutation introduced into human {alpha}-chain, reflects a synergy of the polymerization inhibition of His20({alpha}) with horse {alpha}31–141. However, the additional inhibitory potential resulting from the synergistic complementation of Gln20({alpha}) with horse {alpha}31–141 is lower than that achieved by the eight sequence differences of mouse {alpha}1–30. Therefore, these studies establish the role of additional perturbations of the contact sites in the chimeric HbS containing the mouse–horse chimeric {alpha}-chain, that is achieved through the synergistic complementation of the sequence differences of mouse {alpha}1–30 with the sequence differences in horse {alpha}31–141.

Discussion

In our earlier studies of chimeric Hbs, both complementary segments were derived from the naturally occurring proteins. In the present study, one of the mutant segments needed was generated by chemical synthesis and hence represents a true semisynthetic protein. The semisynthetic mutant forms of HbS and HbS containing human–horse chimeric {alpha}-chain exhibit reversible, cooperative O2 binding and are sensitive to the presence of DPG, characteristic of the basic Hb fold. However, Gln20({alpha}) appears to reduce the O2 affinity of HbS only slightly both in the presence and absence of DPG.

His20({alpha}) of HbA has been identified recently as an alkaline Bohr group (Sun et al., 1997Go). This suggests that some differences in the O2 affinity of HbS-LM relative to HbS is to be expected. Gln20({alpha}) reduces the O2 affinity of the tetramer containing human–horse chimeric {alpha}-chain, though the influence of Gln20({alpha}) appears to be very limited when it is present in the human {alpha}-chain frame. With the semisynthetic chimeric HbS-LM, the Gln20({alpha}) mediated oxygen affinity lowering propensity is amplified in the presence of DPG, as compared with that of human–horse chimeric {alpha}-chain-containing tetramers. This reflects that in the presence of the interaction of DPG at the ßß cleft, the oxygen affinity-lowering propensity of Gln20({alpha}) is amplified by the presence of the sequence differences of horse {alpha}31–141. Hence one can conclude that the mutation of His20({alpha}) has an intrinsic potential to sensitize the ßß cleft of chimeric HbS, presumably a consequence of the loss of the positive charge at {alpha}20. This region of Hb exhibits strong electrostatic interactions that help to conserve the tertiary conformation of the chain and its interactions with the ß-chain generating the {alpha}1ß1 interface of the tetramer.

The GH corner of the {alpha}-chain represents the complementary region of the AB corner of the {alpha}-chain in its tertiary interactions. The LM–horse chimeric {alpha}-chain, carries three sequence differences in this region, namely at {alpha}111, {alpha}115 and {alpha}116. In human {alpha}-chain an Ala residue is present in {alpha}111, whereas it is a Val residue in horse; thus the substitution is conservative at best or a slight increase in the hydrophobicity of the region relative to that of LM {alpha}-chain. LM–horse chimeric {alpha}-chain has a sequence differences at {alpha}115. The Ala residue of human {alpha}-chain at {alpha}115 has been replaced by an Asn residue in the LM–horse chimeric {alpha}-chain. LM–horse chimeric {alpha}-chain carries an Asp residue at {alpha}116, instead of the Glu present in LM {alpha}-chain. Earlier molecular modeling studies have indicated that the presence of Asp at {alpha}116 introduces a perturbation in the orientation of the side chain of Lys16({alpha}), in human horse chimeric {alpha}-chain (Nacharaju et al., 1997Go). The complementation of this effect with that of Gln20({alpha}) may be responsible for the amplification of the low oxygen affinity-inducing propensity of the latter mutation when the ßß cleft is occupied by DPG.

This amplification process [sensitization of the ßß cleft in the presence of Gln20({alpha})] reflects that the interactions of Gln {alpha}20 with sequence differences of the GH corner is communicated to the ßß cleft. The communicated information remains latent until the ßß cleft interacts with the allosteric effector DPG; the interaction of DPG at the ßß cleft translates this structural information to the heme site as lowered oxygen affinity. Hence this represents a communication pathway of the new structural information of the {alpha}1ß1 interface to the heme site through the mediation of the ßß cleft. A similar intersubunit surface communication between {alpha}1ß1 interface and ßß cleft has been recently invoked in the case of Hb J-Sardegna to explain the differential sensitivity of this mutant protein to DPG relative to HbA. In this mutant, His50({alpha}) has been replaced by an Asp residue (Giardina, unpublished; abstract presented at the Asilomar Conference, 1998) and represents the perturbation of the electrostatic interaction of the region. We have recently mapped a cross-communication between the {alpha}1ß1 interface and DPG pocket in dictating a lower O2 affinity in the presence of DPG (A.S.Acharya, J.M.Ho and C.Friedman, manuscript, in preparation). Hence the {alpha}1ß1 interface does not appear to be inert in the Hb allostery as was previously thought.

As noted earlier, the AB/GH corner of the {alpha}-chain is involved in strong electrostatic interactions that stabilize the tertiary and quaternary interactions of Hb. His20({alpha}) and His112({alpha}), the basic residues of this region, are proximal in the three-dimensional structure of Hb. The NMR studies of Craescu et al. (1986) have implicated the presence of a strong electrostatic influence between a number of surface His residues that are not in direct contact. For example, the presence of Gln20({alpha}), the Le-Lamentin mutation appears to decrease the pKa of His112({alpha}). Crystallographic analysis suggest that these two residues are at least 6.6 Å apart. An indirect effect mediated through the Glu residues of this region [Glu23({alpha}), Glu27({alpha}) and Glu116({alpha})] apparently perturbs the hydrogen bond structure of this region in Hb-Le Lamentin. The presence of three GH corner mutations in horse GH corner presumably amplifies the perturbation of the structure of this region. Nonetheless, the plasticity of the ßß cleft buffers this perturbation until the cleft itself is challenged by the electrostatic interaction with the allosteric effector, DPG.

The role of His20({alpha}) as an inter-double strand axial contact in the deoxy HbS fiber has been well recognized based on crystallographic studies of the crystal (Wishner et al., 1975Go; Padlan and Love, 1985aGo,bGo; Cretagny and Edelstein, 1993) and validated as active in the polymer by solution studies (Rhoda et al., 1984Go). HbS-LM described here exhibits a solubility higher than that of HbS. Although in the present study the solubility data were obtained using a high-phosphate system, the relative increase in the solubility of the semisynthetic protein compared with that of HbS is nearly the same as that of the HbS-LM assembled by Rhoda et al. (1984) from naturally occurring mutants. NMR studies have also demonstrated that His20({alpha}) is one of the surface residues of HbS that is selectively influenced by the polymerization phenomenon (Russu and Ho, 1980Go).

The polymerization inhibition by Gln20({alpha}) is slightly higher than the effect of the three sequence differences of the {alpha}1–30 segment of horse {alpha}-chain, namely Pro4({alpha})->Ala, Gly15({alpha})->Ser and Ala19({alpha})->Gly placed in the human {alpha}-chain frame. Its inhibitory propensity is lower than that of mouse {alpha}1–30 placed in the human {alpha}-chain frame.

The situation is very distinct when the Gln20({alpha}) or the sequence differences of either horse or mouse {alpha}1–30 are present in the human–horse {alpha}-chain frame. The Gln20({alpha}), exhibits a higher level of inhibition relative to horse {alpha}1–30 when contiguous with horse {alpha}31–141. Thus, Gln20({alpha}) mimics the synergistic complementation phenomenon that we have detected earlier with horse {alpha}-chain, mouse {alpha}-chain and mouse–horse chimeric {alpha}-chain. However, the inhibitory activity of Gln20({alpha}) is not comparable to that of mouse {alpha}1–30 contiguous with horse {alpha}31–141. Thus, the perturbation of His20({alpha}) and its synergistic complementation with the inhibitory propensity of horse {alpha}31–141 by itself cannot explain the super-inhibitory activity of mouse–horse chimeric {alpha}-chain. Additional perturbations of the contact sites by the sequence differences of mouse {alpha}1–30 need to be invoked to explain the super-inhibitory activity of mouse–horse chimeric {alpha}-chain. Perhaps the microenvironments of two other contact sites (Kraus et al., 1966Go; Benesh et al., 1982) of the region [Lys16({alpha}) and Glu23({alpha})] are also perturbed by the four sequence differences of the AB corner of mouse–horse chimeric {alpha}-chain; the integration of these with the perturbations of the GH corner of mouse–horse chimeric {alpha}-chain and complementation with the other contact site sequence differences of mouse–horse chimeric {alpha}-chain has imparted a super-inhibitory activity to the mouse–horse chimeric {alpha}-chain.

All three intermolecular contact sites of the {alpha}1–30 region are located in the cis {alpha}ß dimer and participate in intra-double strand axial contact site interactions. Subtle perturbations of all three intra-double strand axial contact site interactions might have occurred in the mouse–horse chimeric {alpha}-chain, rather than limiting it to one site as implicated in the present study. The subtle perturbations of multiple sites may then be amplified by the sequence differences of the GH corner of mouse–horse chimeric {alpha}-chain better than the sequence difference of a single contact site. As noted earlier, the molecular modeling studies of human–horse chimeric {alpha}-chain reflected the perturbation of Lys16({alpha}) by the sequence differences of GH corner. The sequence differences of the AB corner of mouse–horse chimeric {alpha}-chain impose further restrictions on the accessibility of the {varepsilon}-NH2 group of Lys16({alpha}) at the protein surface. Synergistic complementation of the polymerization inhibitory propensity resulting from each of these three contact sites with the polymerization inhibitory potential of horse {alpha}31–141 has to be invoked to explain the `super-inhibitory potential' of mouse–horse chimeric {alpha}-chain. The results suggest that when a contact site perturbation is introduced into a molecule that already carries a set of polymerization inhibitory sequence differences, a synergistic influence could be coming from the potential to generate a higher subset of intermolecular contact site perturbations. Thus, the perturbation of all three intermolecular contact sites of the AB corner of {alpha}-chain by the four sequence differences of mouse AB corner in mouse–horse chimeric {alpha}-chain may be facilitating the generation of higher subset of polymerization inhibitory contact site perturbations.

The generation of a mouse–horse chimeric {alpha}-chain (Nacharaju et al., 1997Go) and Le Lamentin–horse chimeric {alpha}-chain with a higher inhibitory potential than the horse {alpha}-chain demonstrates the potential use for a `linkage map' of the polymerization inhibitory sequence differences of intermolecular contact sites. We suggest that a simultaneous perturbation of multiple contact sites is a powerful strategy to neutralize the ßS-chain dependent polymerization. The `linkage map', identifying the contact site perturbation that can synergistically complement, is distinct from the contact site map that identifies the chemical nature of the side chain at a given contact site that exhibits the maximum polymerization inhibitory propensity, that is being mapped by site-directed mutagenesis. A `linkage map' of polymerization inhibitory sequence difference of multiple intermolecular contact sites will provide a blueprint for the design of a super-inhibitory {alpha}-chain and hence of super-inhibitory Hb for gene therapy of sickle cell disease.


    Acknowledgments
 
This work was supported by National Institutes of Health Grants Hl-38665, HL-55435 and HL-58512. We also thank Dr Bookchin for encouraging us in the semisynthetic studies of HbS.


    Notes
 
4 To whom correspondence should be addressed Back


    References
 Top
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 Introduction
 Materials and methods
 Results
 References
 
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Received July 6, 1998; revised July 28, 1999; accepted August 5, 1999.





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