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Examples of chemical modification and recombinant DNA approaches with hemoglobin
TCB 1996
2:109-111
Examples of Chemical Modification and Recombinant DNA Approaches with Hemoglobin J.M. MANNING Rockefeller University, New York
R~sum~ M o d i f i c a t i o n b i o c h i m i q u e de l ' h 6 m o g l o b i n e et production d'h6moglobine recombinante : e x e m p l e s Les d6riv6s d'h6moglobine sont obtenus par traitement de l'h6moglobine avec un produit poss6dant les propri6t6s n6cessaires ~ l'obtention d'un substitut du sang. La technique de production d'h6moglobine recombinante a 6t6 mise au point pour la recherche de substituts du sang. L'int6r~t des m6thodes de modification biochimique et de production d'h6moglobine recombinante est 6valu6 de mani6re ~ d6terminer la solution ~ utiliser de pr6f6rence pour obtenir un produit de substitution cliniquement utile et non toxique. Chacune de ces m6thodes pr6sente des avantages par rapport ~ l'autre. Des exemples d6crivant chacune d'elles sont donn6s plus loin.
Introduction
Chemical modification
Modified cell-free hemoglobin derivatives have been under study as potential red cell substitutes for nearly three decades [1]. These derivatives are prepared by treatment of hemoglobin with some chemical reagent to yield a product that possesses a property considered desirable for a blood substitute. Recombinant DNA technology has been developed for hemoglobin in general and for blood substitute research in particular. At present, both the chemical and the recombinant DNA approaches are being evaluated in order to determine the preferable route to a practical, clinically useful, and non-toxic blood substitute. It is quite possible, of course, that some combination of these will eventually be used. Each has some advantages over the other since their capabilities are different. These approaches will be treated separately with some emphasis on studies from the author's laboratory.
The strategy of identifying the site{s} of interaction of the natural reversibly-bound allosteric regulator, carbon dioxide, and then modifying this same site covalently in order to attain a permanently lowered oxygen affinity is illustrated by chemical modification. In attempts to identify the CO2 binding sites, we used a stable analog of CO2, the carboxymethyl ICm) group, which was introduced at the N-terminal amino groups under mild conditions at neutral pH by treatment of hemoglobin with sodium glyoxylate in the presence of sodium cyanoborohydride [2, 4]. The Cm group at the N-terminus of each chain leads to a lower oxygen affinity, which is analogous to the effect of CO2 on Hb. It is known that the N-terminus of the a-chain was one of the major chloride binding sites [5 and refs. therein]. A second major site of chloride binding was estabhshed as the region that binds 2,3-DPG in a cleft between the two/~-chains in the deoxy conformation [5,6 and refs. therein]. However, there still remained a residual 20-25% chloride-induced lowering of the
Correspondence to: ]. M. Manning,NortheasternUniversity,Boston, MA 02115, USA.
109
110
J.M. MANNING
oxygen affinity that was not acccounted for by these two sites. A n e w procedure, which is referred to as regionrandom chemical modification, was employed for their identification [7, 8]. In region-random chemical modification, the objective is to identify both the major and the minor chloride binding sites; the entire reaction mixture is analyzed so that nothing is discarded. The complexity of working with a mixture of modified products also means that the objective is not to assign a specific hemoglobin function to a particular site, as is usually the case, but rather to make a quantitative assessment of all the amino acid side chains that are involved in the process. By this approach, two functional chloride binding sites per fl-chain and three functional sites per flchain were identified. Some of these comprised the two well-known regions described above, and the Lys-99 ((x) previously identified by Winslow and his colleagues as a functional chloride binding site [9]. Molecular modelling showed the relationship of these other chloride binding regions to the other two major sites. Unexpectedly, as described below, the location of this third chloride binding region is very much related to current blood substitute research. This third functional chloride binding region was found in the central dyad axis of hemoglobin. When viewed in this manner, a relationship becomes apparent, i.e. the sites are aligned along the sides connecting the major oxygen-linked sites and appear to form a channel connecting them. Furthermore, there is a symmetrical relationship between them in deoxy hemoglobin that disappears upon oxygenation. It is possible that the binding of chloride at these sites helps to hold the central dyad axis in its more open configuration by repulsion of the negatively-charged chloride ions. Rather than the previously held notion that these are specific chloride binding sites, it is preferable to view them as chloride binding regions with overlapping functions. This suggestion has also been advanced by Perutz et al. [10]. Our working hypothesis is that any constraint that prevents the constriction of the dyad axis leads to a low oxygen affinity of hemoglobin.
Recombinant Hemoglobins Expression in the yeast S. cerevisiae was designed with the objective of avoiding fusion proteins. A synthetic (x and ~-globin cDNA sequence was incorpo-
rated on a single plasmid together with yeast promoters [11]. The system produced equal amounts of (x and fl-globin chains and utilized the endogenous yeast hem e to produce a soluble hemoglobin tetramer. The expression of the hemoglobin in yeast was somewhat lower than in E.coli but the system has the advantage that the protein is processed and folded correctly [12, 13]. This conclusion was reached after a variety of analysis comparing natural HbS from sickle red cells and recombinant HbS expressed in yeast and purified by cation exchange chromatography and HPLC. According to the following criteria, both hemoglobins are identical: tryptic peptide mapping, sequencing of the mutant peptide, circullar dichroism, mass spectrometry, carboxypeptidase digestion, and HPLC separation of the globin chains. Functional analyses showed that Pso and n values were completely superimposable. Bunn and Jandl showed that the tetramer-dimer equilibrium of hemoglobin leads to the rapid loss of infused hemoglobin from the plasma and that chemical cross-linking stabilizes the tetramer and leads to a prolonged plasma retention. In our studies, we have shown with a highly purified Hb cross-linked between the two (x-chains, DIBS-Hb, has a much longer plasma retention than Cm-Hb even though the oxygen affinity of the former is much higher than that of the latter. Hence, plasma retention is related only to molecular mass and not to oxygen affinity. A recombinant mutant hemoglobin with Ash-102 (fl) replaced by an Ala [called N102A (fi)] was recently prepared using the yeast expression system [14]. The side chain of Asn-102 (fi) is part of an important region of the alfl2 interface that undergoes large structural changes in the transition between the deoxy and oxy conformations. Alanine was chosen as the replacement because its methyl side chain cannot participate in hydrogen bond formation thought to be important at this site, yet it is small enough not to disrupt the subunit interface. Its oxygen binding curve indicated a very low Pso of 42 mmHg in the absence of chloride. In the presence of added chloride, its oxygen affinity was further reduced only slightly to a Pso of 49 mmHg. Thus, the results with N102A (fl} indicate that the maximal decrease in oxygen affinity may already have been achieved and that chloride is not necessary for this purpose.
EXAMPLES OF CHEMICAL MODIFICATION AND RECOMBINANT DNA APPROACHES
In general, the strategy of using both approaches may eventually lead to a practical and clinically useful blood substitute.
Acknowledgments Supported in part by NIH Grant HL-48018 and U.S. Army Contract DAMD17-94-V-4010.
References [1] Winslow R.M. (1992) Hemoglobin-based red cell substitutes. Johns Hopkins University Press. Baltimore. [2] DiDonato A., Fantl W.J., Acharya A.S. and Manning J.M. (1983) Selective carboxymethylation of the amino groups of hemoglobin. Effect on functional properties. J. Biol. Chem., 258, 11890-11895. [3] Fantl w.J., DiDonato A., Manning J.M., Rogers P.H. and Arnone A. (1987) Specifically carboxymethylated hemoglobin as an analogue of carbamino hemoglobin: Solution and x-ray studies of carboxymethylated hemoglobin and x-ray studies of carbamino hemoglobin. J. Biol. Chem., 262, 12700-12713. [4] Fantl w.J., Manning L.R., Ueno H., DiDonato A. and Manning J.M. (1987) Properties of carboxymethylated, crosslinked hemoglobin A. Biochemistry, 26, 5755-5761. [5] Nigen A.M., Manning J.M. and Alben J.O. (1980) Oxygenlinked binding sites for inorganic anions to hemoglobin, jr. Biol. Chem., 255, 5525.
111
[6] Arnone A. (19721 X-ray diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhaemoglobin. Nature, 237, 146-149. [7] Ueno H. and Manning J.M. (1992) The functional, oxygenlinked chloride binding sites of hemoglobin are contiguous within a channel in the central cavity. J. Prot. Chem., 11, 177-185. [8] Ueno H., Popowicz A.M. and Manning J.M. (1994) Random chemical modification of the oxygen-linked chloride binding sites of hemoglobin: Those in the central dyad axis may influence the transition between deoxy- and oxy-hemoglobin. J. Prot. Chem., 12, 561-570. [9] Vandegriff K.D., Medina F., Marini M. and Winslow R.M. (1989) Equilibrium oxygen binding to human hemoglobin crosslinked between the c~chains by bis (3,5 dibromosalicyl) fumarate. ]. Biol. Chem., 264, 17824-17833. [10] Perutz M.F., Shih D.T.-b and Williamson D. (1994) The chloride effect in human haemoglobin. A new kind of allosteric mechanism. J. Mol. Biol., 239, 555-560. [11] Wagenbach M., O'Rourke, Vitez L., Wieczorek A., Hoffman S., Durfee S., Tedesco J. and Stetler G. (1991) Synthesis of wild-type and mutant human hemoglobins in Saccharomyces cerevisiae. BioTechnology, 9, 57-61. [12] Martin de Llano J.J., Jones W., Schneider K., Chait B.T., Rodgers G., Benjamin L.J., Weksler B. and Manning J.M. (1993) Biochemical and functional properties of recombinant human sickle hemoglobin expressed in yeast, f. Biol. Chem., 268, 27004-27011. [13] Martin de Llano J.J., Schneewind O., Stetler G. and Manning J.M. (1993) Recombinant sickle hemoglobin in yeast./:'roe. Natl. Acad. Sei., 90, 918-922. [14] Yanase H., Manning L.R., Vandegriff K., Winslow R.M. and Manning J.M. (1995) A recombinant human hemoglobin with asparagine-102 (fi) substituted by alanine has a limiting low oxygen affinity, reduced marginally by chloride. Protein Sei., 4, 21-28.
2:109-111
Examples of Chemical Modification and Recombinant DNA Approaches with Hemoglobin J.M. MANNING Rockefeller University, New York
R~sum~ M o d i f i c a t i o n b i o c h i m i q u e de l ' h 6 m o g l o b i n e et production d'h6moglobine recombinante : e x e m p l e s Les d6riv6s d'h6moglobine sont obtenus par traitement de l'h6moglobine avec un produit poss6dant les propri6t6s n6cessaires ~ l'obtention d'un substitut du sang. La technique de production d'h6moglobine recombinante a 6t6 mise au point pour la recherche de substituts du sang. L'int6r~t des m6thodes de modification biochimique et de production d'h6moglobine recombinante est 6valu6 de mani6re ~ d6terminer la solution ~ utiliser de pr6f6rence pour obtenir un produit de substitution cliniquement utile et non toxique. Chacune de ces m6thodes pr6sente des avantages par rapport ~ l'autre. Des exemples d6crivant chacune d'elles sont donn6s plus loin.
Introduction
Chemical modification
Modified cell-free hemoglobin derivatives have been under study as potential red cell substitutes for nearly three decades [1]. These derivatives are prepared by treatment of hemoglobin with some chemical reagent to yield a product that possesses a property considered desirable for a blood substitute. Recombinant DNA technology has been developed for hemoglobin in general and for blood substitute research in particular. At present, both the chemical and the recombinant DNA approaches are being evaluated in order to determine the preferable route to a practical, clinically useful, and non-toxic blood substitute. It is quite possible, of course, that some combination of these will eventually be used. Each has some advantages over the other since their capabilities are different. These approaches will be treated separately with some emphasis on studies from the author's laboratory.
The strategy of identifying the site{s} of interaction of the natural reversibly-bound allosteric regulator, carbon dioxide, and then modifying this same site covalently in order to attain a permanently lowered oxygen affinity is illustrated by chemical modification. In attempts to identify the CO2 binding sites, we used a stable analog of CO2, the carboxymethyl ICm) group, which was introduced at the N-terminal amino groups under mild conditions at neutral pH by treatment of hemoglobin with sodium glyoxylate in the presence of sodium cyanoborohydride [2, 4]. The Cm group at the N-terminus of each chain leads to a lower oxygen affinity, which is analogous to the effect of CO2 on Hb. It is known that the N-terminus of the a-chain was one of the major chloride binding sites [5 and refs. therein]. A second major site of chloride binding was estabhshed as the region that binds 2,3-DPG in a cleft between the two/~-chains in the deoxy conformation [5,6 and refs. therein]. However, there still remained a residual 20-25% chloride-induced lowering of the
Correspondence to: ]. M. Manning,NortheasternUniversity,Boston, MA 02115, USA.
109
110
J.M. MANNING
oxygen affinity that was not acccounted for by these two sites. A n e w procedure, which is referred to as regionrandom chemical modification, was employed for their identification [7, 8]. In region-random chemical modification, the objective is to identify both the major and the minor chloride binding sites; the entire reaction mixture is analyzed so that nothing is discarded. The complexity of working with a mixture of modified products also means that the objective is not to assign a specific hemoglobin function to a particular site, as is usually the case, but rather to make a quantitative assessment of all the amino acid side chains that are involved in the process. By this approach, two functional chloride binding sites per fl-chain and three functional sites per flchain were identified. Some of these comprised the two well-known regions described above, and the Lys-99 ((x) previously identified by Winslow and his colleagues as a functional chloride binding site [9]. Molecular modelling showed the relationship of these other chloride binding regions to the other two major sites. Unexpectedly, as described below, the location of this third chloride binding region is very much related to current blood substitute research. This third functional chloride binding region was found in the central dyad axis of hemoglobin. When viewed in this manner, a relationship becomes apparent, i.e. the sites are aligned along the sides connecting the major oxygen-linked sites and appear to form a channel connecting them. Furthermore, there is a symmetrical relationship between them in deoxy hemoglobin that disappears upon oxygenation. It is possible that the binding of chloride at these sites helps to hold the central dyad axis in its more open configuration by repulsion of the negatively-charged chloride ions. Rather than the previously held notion that these are specific chloride binding sites, it is preferable to view them as chloride binding regions with overlapping functions. This suggestion has also been advanced by Perutz et al. [10]. Our working hypothesis is that any constraint that prevents the constriction of the dyad axis leads to a low oxygen affinity of hemoglobin.
Recombinant Hemoglobins Expression in the yeast S. cerevisiae was designed with the objective of avoiding fusion proteins. A synthetic (x and ~-globin cDNA sequence was incorpo-
rated on a single plasmid together with yeast promoters [11]. The system produced equal amounts of (x and fl-globin chains and utilized the endogenous yeast hem e to produce a soluble hemoglobin tetramer. The expression of the hemoglobin in yeast was somewhat lower than in E.coli but the system has the advantage that the protein is processed and folded correctly [12, 13]. This conclusion was reached after a variety of analysis comparing natural HbS from sickle red cells and recombinant HbS expressed in yeast and purified by cation exchange chromatography and HPLC. According to the following criteria, both hemoglobins are identical: tryptic peptide mapping, sequencing of the mutant peptide, circullar dichroism, mass spectrometry, carboxypeptidase digestion, and HPLC separation of the globin chains. Functional analyses showed that Pso and n values were completely superimposable. Bunn and Jandl showed that the tetramer-dimer equilibrium of hemoglobin leads to the rapid loss of infused hemoglobin from the plasma and that chemical cross-linking stabilizes the tetramer and leads to a prolonged plasma retention. In our studies, we have shown with a highly purified Hb cross-linked between the two (x-chains, DIBS-Hb, has a much longer plasma retention than Cm-Hb even though the oxygen affinity of the former is much higher than that of the latter. Hence, plasma retention is related only to molecular mass and not to oxygen affinity. A recombinant mutant hemoglobin with Ash-102 (fl) replaced by an Ala [called N102A (fi)] was recently prepared using the yeast expression system [14]. The side chain of Asn-102 (fi) is part of an important region of the alfl2 interface that undergoes large structural changes in the transition between the deoxy and oxy conformations. Alanine was chosen as the replacement because its methyl side chain cannot participate in hydrogen bond formation thought to be important at this site, yet it is small enough not to disrupt the subunit interface. Its oxygen binding curve indicated a very low Pso of 42 mmHg in the absence of chloride. In the presence of added chloride, its oxygen affinity was further reduced only slightly to a Pso of 49 mmHg. Thus, the results with N102A (fl} indicate that the maximal decrease in oxygen affinity may already have been achieved and that chloride is not necessary for this purpose.
EXAMPLES OF CHEMICAL MODIFICATION AND RECOMBINANT DNA APPROACHES
In general, the strategy of using both approaches may eventually lead to a practical and clinically useful blood substitute.
Acknowledgments Supported in part by NIH Grant HL-48018 and U.S. Army Contract DAMD17-94-V-4010.
References [1] Winslow R.M. (1992) Hemoglobin-based red cell substitutes. Johns Hopkins University Press. Baltimore. [2] DiDonato A., Fantl W.J., Acharya A.S. and Manning J.M. (1983) Selective carboxymethylation of the amino groups of hemoglobin. Effect on functional properties. J. Biol. Chem., 258, 11890-11895. [3] Fantl w.J., DiDonato A., Manning J.M., Rogers P.H. and Arnone A. (1987) Specifically carboxymethylated hemoglobin as an analogue of carbamino hemoglobin: Solution and x-ray studies of carboxymethylated hemoglobin and x-ray studies of carbamino hemoglobin. J. Biol. Chem., 262, 12700-12713. [4] Fantl w.J., Manning L.R., Ueno H., DiDonato A. and Manning J.M. (1987) Properties of carboxymethylated, crosslinked hemoglobin A. Biochemistry, 26, 5755-5761. [5] Nigen A.M., Manning J.M. and Alben J.O. (1980) Oxygenlinked binding sites for inorganic anions to hemoglobin, jr. Biol. Chem., 255, 5525.
111
[6] Arnone A. (19721 X-ray diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhaemoglobin. Nature, 237, 146-149. [7] Ueno H. and Manning J.M. (1992) The functional, oxygenlinked chloride binding sites of hemoglobin are contiguous within a channel in the central cavity. J. Prot. Chem., 11, 177-185. [8] Ueno H., Popowicz A.M. and Manning J.M. (1994) Random chemical modification of the oxygen-linked chloride binding sites of hemoglobin: Those in the central dyad axis may influence the transition between deoxy- and oxy-hemoglobin. J. Prot. Chem., 12, 561-570. [9] Vandegriff K.D., Medina F., Marini M. and Winslow R.M. (1989) Equilibrium oxygen binding to human hemoglobin crosslinked between the c~chains by bis (3,5 dibromosalicyl) fumarate. ]. Biol. Chem., 264, 17824-17833. [10] Perutz M.F., Shih D.T.-b and Williamson D. (1994) The chloride effect in human haemoglobin. A new kind of allosteric mechanism. J. Mol. Biol., 239, 555-560. [11] Wagenbach M., O'Rourke, Vitez L., Wieczorek A., Hoffman S., Durfee S., Tedesco J. and Stetler G. (1991) Synthesis of wild-type and mutant human hemoglobins in Saccharomyces cerevisiae. BioTechnology, 9, 57-61. [12] Martin de Llano J.J., Jones W., Schneider K., Chait B.T., Rodgers G., Benjamin L.J., Weksler B. and Manning J.M. (1993) Biochemical and functional properties of recombinant human sickle hemoglobin expressed in yeast, f. Biol. Chem., 268, 27004-27011. [13] Martin de Llano J.J., Schneewind O., Stetler G. and Manning J.M. (1993) Recombinant sickle hemoglobin in yeast./:'roe. Natl. Acad. Sei., 90, 918-922. [14] Yanase H., Manning L.R., Vandegriff K., Winslow R.M. and Manning J.M. (1995) A recombinant human hemoglobin with asparagine-102 (fi) substituted by alanine has a limiting low oxygen affinity, reduced marginally by chloride. Protein Sei., 4, 21-28.