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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Abstract |
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Keywords: chimeric HbS/HbS polymerization/intermolecular contact sites/linkage map/protein engineering/super-inhibitory -chain
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Introduction |
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This concept of `linkage' of the polymerization inhibitory potential of sequence differences in the high inhibitory potential of horse and mouse -chain is supported by the observation that the grafting of mouse
130 with the horse
31141 segment resulted in the generation of a super-inhibitory chimeric
-chain. The polymerization inhibitory potential of the mousehorse chimeric
-chain is even higher than that of both parent chains, horse and mouse
-chains. None of the eight sequence differences of mouse
130 are at implicated contact sites and in the horse
31141 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
31141 can establish a cross-talk with the sequence differences of mouse
130 even though they are not at the implicated contact sites to establish a `functional complementation' of their polymerization inhibitory activity (Nacharaju et al., 1997
).
The 17-22 region of mouse
-chain carries four of the eight sequence differences of the mouse
130, namely at
17,
19,
21 and
22 (Table 1
). Three implicated intermolecular contact sites of HbS are located in this region, namely at
16,
20 and
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 mousehorse chimeric
-chain suggested altered side chain interactions at the
1ß1 interface in this chimeric HbS as compared with HbS. The sequence differences of the AB/GH corner of the
-chain facilitated a distinct stereochemistry for the interaction of the
-amino group of Lys16(
) with the ß-carboxyl group of Asp-116(
) (Nacharaju et al., 1997
), an implicated intermolecular contact site. This ß-carboxylate
-amino group interaction, in turn, perturbs the accessibility of the side chain of Lys16(
) to the solvent and hence its participation in the polymerization process. Since Lys16(
) is an implicated intermolecular contact site, we had hypothesized that this perturbation of the
-chain AB/GH corner represents, at least in part, the molecular basis of the high polymerization inhibitory activity of mousehorse chimeric
-chain. Similar perturbation of the two other contact sites of the region, His20(
) and Glu23(
), is also conceivable, although it was not readily apparent in the earlier modeling studies. Accordingly, we now hypothesize that the propensity of mouse
130 to establish functional synergistic complementation (cross-talk) with the polymerization inhibitory propensity of horse
31141 represents the perturbations of one or more of these three implicated contact sites [Lys16(
), His20(
) and Glu23(
)] because of the presence four sequence differences in this region of mousehorse chimeric
-chains.
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Materials and methods |
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HbA and S were purified by DE 52 chromatography followed by CM 52 chromatography. The - and ß-chains were separated according to the procedure described by Bucci (1981). Human
-globin were prepared by acid acetone precipitation. Horse Hb was purified on CM-cellulose as described previously (Nacharaju and Acharya, 1992
). Horse
-globin was purified by CM 52 urea chromatography from total horse globin prepared by acid acetone precipitation.
Chemical synthesis of 130(His20
Gln)
The peptide corresponding to the 130 sequence of -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
-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, 1994
).
Mass spectral analysis of the purified semisynthetic -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% acetonitrileH2O + 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 Trisacetate 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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Results |
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The CM-cellulose chromatographic pattern of of Hb assembled from semisynthetic LM -chain and ßS-chain is shown in Figure 2A
. The main peak, eluting at ~220 ml, was identified as HbS-LM. RP-HPLC analysis of HbS-LM demonstrated the stoichiometry of semisynthetic
-globin and ßS-chain in HbS-LM (inset a). The experimental value for the mass of LM
-globin isolated by RP-HPLC was 15117 Da, compared with the calculated mass of this mutant
-chain of 15117.3 Da. The mass difference is consistent with the substitution of His20(
) by a Gln residue. The homogeneity of semisynthetic HbS-LM was established by isoelectric focusing (Figure 2A
, 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(
) is close to neutral pH (Sun et al., 1997
), hence this residue will be protonated only partially around neutral pH, i.e. His20(
) 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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Isoelectric focusing established the homogeneity of the tetramer assembled (Figure 2B, inset a). The semisynthetic chimeric HbS electrofocused close to that of HbS. The presence of Gln at
20 partially compensates for the difference that existed in the electrofocusing patterns of HbS and the human horse chimeric
-chain containing tetramer. This difference in the isoelectric focusing pattern of HbS compared with that of the tetramer containing either horse
-chain or humanhorse chimeric
-chain, is not readily explained based on their amino acid compositions. The conservatory substitutions of horse
31141 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 II), reflecting the limited influence of Gln20(
) 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 II
). On the other hand, the O2 affinity of semisynthetic chimeric HbS (a tetramer containing the LMhorse chimeric
-chain, LM-Hr) is slightly lower than that of HbS (Table II
); the new chimeric HbS also binds oxygen cooperatively. The oxygen affinity of
LMHr2 ß2 is reduced by DPG much more than that with the chimeric HbS containing human horse chimeric
-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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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 3). HbS-LM polymerized with a delay time longer than that of native HbS, a result consistent with the inhibitory effect of Gln20(
). The delay time decreases as the concentration increases for HbS and HbS-LM [Figure 3A
(inset a)]. At identical concentrations, the delay time of HbS-LM is nearly 1.7 times higher than that of HbS (Table III
).
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The kinetics of the polymerization of ßS-chains hybridized with horsehuman chimeric -chain is also shown in Figure 3A
. The increase in the delay time of the ßS-chain dependent polymerization reaction by His20(
)
Gln is higher than that of the three sequence differences of the
130 segment of horse
-chain (Table III
). The polymerization behavior of HbS,
HHr2ßS2,
2
LMHrßS2 and
2
LMHrßS2 in 2.0 M phosphate buffer is shown in Figure 3B
. The hybrid
LMHr2ßS2 exhibits a delay time which is noticeably higher than that of
Hr2ßS2. The relative increase in the delay time of the polymerization of
LHr2ßS2 as a result of introduction of the Le Lamentin mutation into the humanhorse hybrid chain is larger (Table III
) compared with that seen on introduction of the same mutation on to the human
-chain of HbS. As noted earlier, the polymerization inhibitory potential of LM mutation in the human
-globin frame is higher than the polymerization inhibitory potential of the three sequence differences of the
130 segment of horse
-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(
) with horse
31141.
The inhibitory propensity of His20() engineered into human horse chimeric
-chain (Table III
) is higher than the three sequence differences of horse
130 when it is present as a part of the horse
-chain, but considerably lower than that of the mousehorse chimeric
-chain. As noted earlier, the segregation of the sequence differences of horse and mouse
-chain into segments
130 and
31141 results in the loss of some polymerization inhibitory potential. The higher inhibitory activity of humanhorse chimeric
-chain with His20(
), relative to the same mutation introduced into human
-chain, reflects a synergy of the polymerization inhibition of His20(
) with horse
31141. However, the additional inhibitory potential resulting from the synergistic complementation of Gln20(
) with horse
31141 is lower than that achieved by the eight sequence differences of mouse
130. Therefore, these studies establish the role of additional perturbations of the contact sites in the chimeric HbS containing the mousehorse chimeric
-chain, that is achieved through the synergistic complementation of the sequence differences of mouse
130 with the sequence differences in horse
31141.
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 humanhorse chimeric -chain exhibit reversible, cooperative O2 binding and are sensitive to the presence of DPG, characteristic of the basic Hb fold. However, Gln20(
) appears to reduce the O2 affinity of HbS only slightly both in the presence and absence of DPG.
His20() of HbA has been identified recently as an alkaline Bohr group (Sun et al., 1997
). This suggests that some differences in the O2 affinity of HbS-LM relative to HbS is to be expected. Gln20(
) reduces the O2 affinity of the tetramer containing humanhorse chimeric
-chain, though the influence of Gln20(
) appears to be very limited when it is present in the human
-chain frame. With the semisynthetic chimeric HbS-LM, the Gln20(
) mediated oxygen affinity lowering propensity is amplified in the presence of DPG, as compared with that of humanhorse chimeric
-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(
) is amplified by the presence of the sequence differences of horse
31141. Hence one can conclude that the mutation of His20(
) has an intrinsic potential to sensitize the ßß cleft of chimeric HbS, presumably a consequence of the loss of the positive charge at
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
1ß1 interface of the tetramer.
The GH corner of the -chain represents the complementary region of the AB corner of the
-chain in its tertiary interactions. The LMhorse chimeric
-chain, carries three sequence differences in this region, namely at
111,
115 and
116. In human
-chain an Ala residue is present in
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
-chain. LMhorse chimeric
-chain has a sequence differences at
115. The Ala residue of human
-chain at
115 has been replaced by an Asn residue in the LMhorse chimeric
-chain. LMhorse chimeric
-chain carries an Asp residue at
116, instead of the Glu present in LM
-chain. Earlier molecular modeling studies have indicated that the presence of Asp at
116 introduces a perturbation in the orientation of the side chain of Lys16(
), in human horse chimeric
-chain (Nacharaju et al., 1997
). The complementation of this effect with that of Gln20(
) 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()] reflects that the interactions of Gln
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
1ß1 interface to the heme site through the mediation of the ßß cleft. A similar intersubunit surface communication between
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(
) 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
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
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 -chain is involved in strong electrostatic interactions that stabilize the tertiary and quaternary interactions of Hb. His20(
) and His112(
), 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(
), the Le-Lamentin mutation appears to decrease the pKa of His112(
). 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(
), Glu27(
) and Glu116(
)] 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() 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., 1975
; Padlan and Love, 1985a
,b
; Cretagny and Edelstein, 1993) and validated as active in the polymer by solution studies (Rhoda et al., 1984
). 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(
) is one of the surface residues of HbS that is selectively influenced by the polymerization phenomenon (Russu and Ho, 1980
).
The polymerization inhibition by Gln20() is slightly higher than the effect of the three sequence differences of the
130 segment of horse
-chain, namely Pro4(
)
Ala, Gly15(
)
Ser and Ala19(
)
Gly placed in the human
-chain frame. Its inhibitory propensity is lower than that of mouse
130 placed in the human
-chain frame.
The situation is very distinct when the Gln20() or the sequence differences of either horse or mouse
130 are present in the humanhorse
-chain frame. The Gln20(
), exhibits a higher level of inhibition relative to horse
130 when contiguous with horse
31141. Thus, Gln20(
) mimics the synergistic complementation phenomenon that we have detected earlier with horse
-chain, mouse
-chain and mousehorse chimeric
-chain. However, the inhibitory activity of Gln20(
) is not comparable to that of mouse
130 contiguous with horse
31141. Thus, the perturbation of His20(
) and its synergistic complementation with the inhibitory propensity of horse
31141 by itself cannot explain the super-inhibitory activity of mousehorse chimeric
-chain. Additional perturbations of the contact sites by the sequence differences of mouse
130 need to be invoked to explain the super-inhibitory activity of mousehorse chimeric
-chain. Perhaps the microenvironments of two other contact sites (Kraus et al., 1966
; Benesh et al., 1982) of the region [Lys16(
) and Glu23(
)] are also perturbed by the four sequence differences of the AB corner of mousehorse chimeric
-chain; the integration of these with the perturbations of the GH corner of mousehorse chimeric
-chain and complementation with the other contact site sequence differences of mousehorse chimeric
-chain has imparted a super-inhibitory activity to the mousehorse chimeric
-chain.
All three intermolecular contact sites of the 130 region are located in the cis
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 mousehorse chimeric
-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 mousehorse chimeric
-chain better than the sequence difference of a single contact site. As noted earlier, the molecular modeling studies of humanhorse chimeric
-chain reflected the perturbation of Lys16(
) by the sequence differences of GH corner. The sequence differences of the AB corner of mousehorse chimeric
-chain impose further restrictions on the accessibility of the
-NH2 group of Lys16(
) 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
31141 has to be invoked to explain the `super-inhibitory potential' of mousehorse chimeric
-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
-chain by the four sequence differences of mouse AB corner in mousehorse chimeric
-chain may be facilitating the generation of higher subset of polymerization inhibitory contact site perturbations.
The generation of a mousehorse chimeric -chain (Nacharaju et al., 1997
) and Le Lamentinhorse chimeric
-chain with a higher inhibitory potential than the horse
-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
-chain and hence of super-inhibitory Hb for gene therapy of sickle cell disease.
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Acknowledgments |
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Notes |
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References |
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Received July 6, 1998; revised July 28, 1999; accepted August 5, 1999.