Model mice for Presbyterian hemoglobinopathy (Asn(beta108)-->Lys) confer hemolytic anemia with altered oxygen affinity and instability of Hb

Hb Presbyterian is a variant hemoglobin that carries Lys at Asn-108 of beta-globin. This variant Lys(beta108) residue enhances the stability of Hb in the deoxy-state, conferring the low affinity for oxygen-binding in vitro. In the present study, we generated mutant mice carrying the Presbyterian mutation (Asn(beta108)-->Lys) at the beta-globin locus by a targeted knock-in strategy. Heterozygous mice showed the expression of Hb Presbyterian in 27.7% of total peripheral blood without any hematological abnormalities, which well mimicked human cases. On the other hand, homozygous mice exclusively expressed Hb Presbyterian in 100% of peripheral blood associated with hemolytic anemia, Heinz body formation, and splenomegaly. Hb Presbyterian showed instability in an in vitro precipitation assay. Erythrocytes from homozygous mice showed a shortened life span when transfused into wild-type mice, confirming that the knocked-in mutation of Lys(beta108) caused hemolysis in homozygous mice. This is the first report on the hemolytic anemia of unstable hemoglobin in an animal model. These results confirm the notion that the higher ratio of an unstable variant beta-globin chain in erythrocytes triggers the pathological precipitation and induces hemolysis in abnormal hemoglobinopathies.     

Effect of amino acid at the beta 6 position on surface hydrophobicity, stability, solubility, and the kinetics of polymerization of hemoglobin. Comparisons among Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6)

Surface hydrophobicity, stability, solubility, and kinetics of polymerization were studied using hemoglobins with four different amino acids at the beta 6 position: Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6). The surface hydrophobicity increased in the order of Hb C, Hb A, Hb Machida, and Hb S, coinciding with the hydrophobicity of the amino acid at the beta 6 position. Solubility of the oxy-form of these hemoglobins decreased in relation to increases in their surface hydrophobicity, suggesting that the solubility is controlled by the strength of hydrophobicity of the amino acid at the beta 6 position. The solubility of the oxy-form of these hemoglobins is always higher than that of the deoxy-form. There is a similar linear relationship between the solubility and surface hydrophobicity among deoxyhemoglobins A, C, and Machida. However, the solubility of deoxy-Hb S deviated significantly from the expected value, indicating that the extremely low solubility of deoxy-Hb S is not directly related to the hydrophobicity of the beta 6 valine. Kinetic studies on the polymerization of deoxy-Hb Machida revealed a distinct delay time prior to polymerization. This confirms our previous hypothesis that beta 6 valine is not responsible for the delay time prior to gelation. The kinetics of the polymerization of 1:1 mixtures of sickle and non-sickle hemoglobins were similar to those of pure Hb S, suggesting that only one of the two beta 6 valines is involved in an intermolecular contact. In mixtures of equal amounts of Hb S and Hb A, Hb C, or Hb Machida, half of the asymmetrical AS, SC, and S-Machida hybrid hemoglobins behaved like Hb S during nucleation, while the other half behaved like the non-sickle hemoglobin.     

Additive effects of beta chain mutations in low oxygen affinity hemoglobin betaF41Y,K66T.

In order to decrease significantly the oxygen affinity of human hemoglobin, we have associated the mutation betaF41Y with another point mutation also known to decrease the oxygen affinity of Hb. We have synthesized a recombinant Hb (rHb) with two mutations in the beta chains: rHb betaF41Y,K66T. In the absence of 2, 3-diphosphoglycerate, additive effects of the mutations are evident, since the doubly mutated Hb exhibits a larger decrease in oxygen affinity than for the individual single mutations. In the presence of 2,3-diphosphoglycerate, the second mutation did not significantly increase the P(50) value relative to the single mutations. However, the kinetics of CO binding still indicate combined effects on the allosteric equilibrium, as evidenced by more of the slow bimolecular phase characteristic of binding to the deoxy conformation. Dimer-tetramer equilibrium studies indicate an increase in stability of the mutants relative to rHb A; the double mutant rHb betaF41Y, K66T at pH 7.5 showed a K(4,2) value of 0.26 microM. Despite the lower oxygen affinity, the single mutant betaF41Y and double mutant betaF41Y,K66T show only a moderate increase of 20% in the autoxidation rate. These mutations are thus of interest in developing a Hb-based blood substitute.     

Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-linked polyubiquitin chains

AMPK (AMP-activated protein kinase)-related kinases regulate cell polarity as well as proliferation and are activated by the LKB1-tumour suppressor kinase. In the present study we demonstrate that the AMPK-related kinases, NUAK1 (AMPK-related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4), are polyubiquitinated in vivo and interact with the deubiquitinating enzyme USP9X (ubiquitin specific protease-9). Knockdown of USP9X increased polyubiquitination of NUAK1 and MARK4, whereas overexpression of USP9X inhibited ubiquitination. USP9X, catalysed the removal of polyubiquitin chains from wild-type NUAK1, but not from a non-USP9X-binding mutant. Topological analysis revealed that ubiquitin monomers attached to NUAK1 and MARK4 are linked by Lys(29) and/or Lys(33) rather than the more common Lys(48)/Lys(63). We find that AMPK and other AMPK-related kinases are also polyubiquitinated in cells. We identified non-USP9X-binding mutants of NUAK1 and MARK4 and find that these are hyper-ubiquitinated and not phosphorylated at their T-loop residue targeted by LKB1 when expressed in cells, suggesting that polyubiquitination may inhibit these enzymes. The results of the present study demonstrate that NUAK1 and MARK4 are substrates of USP9X and provide the first evidence that AMPK family kinases are regulated by unusual Lys(29)/Lys(33)-linked polyubiquitin chains.     

Pyruvoyl-dependent histidine decarboxylases. Preparation and amino acid sequences of the beta chains of histidine decarboxylase from Clostridium perfringens and Lactobacillus buchneri

Histidine decarboxylase (HisDCase) from Lactobacillus buchneri was purified to homogeneity. Its subunit structure, (alpha beta)6, and enzymatic properties resemble closely those of the immunologically cross-reactive HisDCase of Lactobacillus 30a (Recsei, P. A., and Snell, E. E. (1984) Annu. Rev. Biochem. 53, 357-387). The complete amino acid sequences of the beta chains of the HisDCase from L. buchneri (81 residues) and Clostridium perfringens (86 residues) were then determined to be a and b, respectively. (a) SEFDKKLNTLGVDRISVSPYKKWSRGYMEPGNIGNGYVSGLKVDAG VVDKTDDMVLDGIGSYDRAETKNAYIGQINMTTAS. (b) TLSEGIHKNIKNIKVRAP KIDKTAISPYDRYCDGYGMPGAYGDGYVSVLKVSVGTVKK TDDILLDGIVSYDRAEINDAYVGQINMLTAS. SEFDKKLNTLGVDRISVSPYKKWSRGYMEPGNIGNGYVSGLKVDAGVV. Although these sequences differ substantially near the NH2-terminal ends, there is striking homology near the COOH termini and also near the NH2 terminus of the two alpha chains (pyruvoyl-Phe-X-Gly-Val-, where X is Ser or Cys). If the four known pyruvoyl-dependent HisDCases arise from inactive proenzymes by the mechanism previously demonstrated for the HisDCase of Lactobacillus 30a (Recsei, P. A., Huynh, Q. K. and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 973-977), then each of these proenzymes has the sequence -Thr-Ala-Ser-Ser-Phe- at the activation site (where -Ser- becomes the COOH terminus of the beta chain and -Ser- becomes the pyruvoyl group blocking the NH2 terminus of the alpha chain), and the sequences around this activation site are highly conserved in all four enzymes. These facts support the assumptions that the four enzymes have evolved from a common ancestral protein, are formed from inactive pyruvate-free proenzymes by similar mechanisms, and have similar catalytic mechanisms.     

Electron-spin resonance of nitrosyl haemoglobins: normal alpha and beta chains and mutants Hb M Iwate and Hb Zürich.

At 77 K the electron spin resonance (ESR) spectra of the NO derivatives of the mutant haemoglobins Hb M Iwate and Hb Zurich as well as of the isolated chains of normal haemoglobin were studied. Two types of ESR spectra differing in the g-value and the hyperfine splitting at gzz were observed. The type II spectrum is characterized by a hyperfine structure at gzz = 2.005 with a splitting constant of deltaH = 23 G (14NO) or 32 G (15NO), respectively. In the type I spectrum the splitting constant of the hyperfine structure at gzz = 2.009 amounts to deltaH = 18 G (14NO) or 23 G (15NO), respectively. In some cases this hyperfine structure is coincident with another one at gxx = 2.064 with nearly identical splitting constant. In addition, the type I spectrum is characterized by an increased ESR absorption at gxx = 2.064. At neutral pH the NO derivatives of the isolated chains as well as of the mutant haemoglobins give rise to a type II spectrum. In correspondence with previous results gained with normal NO haemoglobin, the ESR spectra of the NO-alpha chains and NO-Hb Zurich show a transition to type I in the acid region. This transition is favoured by binding of 2,3-bisphosphoglycerate. On the other hand, the ESR spectra of the NO-beta chains and NO-Hb M Iwate are of the type II also at acid pH. The NO-beta chains show a transition of the ESR spectrum from type II to type I only at alkaline pH. These results indicate that in the tetrameric NO haemoglobin only the alpha chains are responsible for the transition of the ESR spectrum from type II to type I in the acid region. The two types of ESR spectra are interpreted in terms of two kinds of haem-NO complexes differing in the iron-NO and iron-imidazole distances. The type II spectrum is attributed to a complex with a relatively short iron-imidazole distance which is responsible for a weakened sigma-bond in trans position. The type I spectrum arises then from a complex with a larger iron-imidazole bond leading to an approach of the NO molecule to the iron. The influence of the protein conformation upon the iron-imidazole bond length is discussed with regard to the ESR spectra of the mutant NO haemoglobins and considering the influence of agents modifying the protein structure.     

Properties of hemoglobin G. Ferrara (beta57(E1) Asn replaced by Lys).

Hemoglobin G. Ferrara is an abnormal human hemoglobin in which an asparagine residue is replaced by a lysyl residue at position beta57 (beta57 Asn replaced by Lys). Oxygen equilibria show that cooperativity and alkaline Bohr effect are maintained to normal levels while the acid Bohr effect appears increased; in addition, a smaller effect of diphosphoglycerate is also observed. Flash photolysis experiments performed as a function of protein concentration show that the fraction of quickly reacting form is always higher than that of human hemoglobin A. This fact, together with the increase of the oxygen affinity observed at acid pH values, may be related to an enhanced dissociation of the molecule into dimers. Several attempts to isolate the native chains by treatment of the protein with p-chloromercuribenzoate were unsuccessful due to the great instability of the isolated variant beta-chains, which precipitated completely during incubation with p-chloromercuribenzoate. Therefore, although the substitution is on the surface of the molecule, there are several properties of hemoglobin G. beta Ferrara which are clearly different from hemoglobin A.     

Structure and function of the tryptophan synthase alpha(2)beta(2) complex. Roles of beta subunit histidine 86

To probe the structural and functional roles of active-site residues in the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium, we have determined the effects of mutation of His(86) in the beta subunit. His(86) is located adjacent to beta subunit Lys(87), which forms an internal aldimine with the pyridoxal phosphate and catalyzes the abstraction of the alpha-proton of L-serine. The replacement of His(86) by leucine (H86L) weakened pyridoxal phosphate binding approximately 20-fold and abolished the circular dichroism signals of the bound coenzyme and of a reaction intermediate. Correlation of these results with previous crystal structures indicates that beta-His(86) plays a structural role in binding pyridoxal phosphate and in stabilizing the correct orientation of pyridoxal phosphate in the active site of the beta subunit. The H86L mutation also altered the pH profiles of absorbance and fluorescence signals and shifted the pH optimum for the synthesis of L-tryptophan from pH 7.5 to 8.8. We propose that the interaction of His(86) with the phosphate of pyridoxal phosphate and with Lys(87) lowers the pK(a) of Lys(87) in the wild-type alpha(2)beta(2) complex and thereby facilitates catalysis by Lys(87) in the physiological pH range.     

Inactivation of chloroplast H(+)-ATPase by modification of Lys beta 359, Lys alpha 176 and Lys alpha 266.

Treatment of isolated, latent chloroplast ATPase with pyridoxal-5-phosphate (pyridoxal-P) in presence of Mg2+ causes inhibition of dithiothreitol-activated plus heat-activated ATP hydrolysis. The amount of [3H]pyridoxal-P bound to chloroplast coupling factor 1 (CF1) was estimated to run up to 6 +/- 1 pyridoxal-P/enzyme, almost equally distributed between the alpha- and beta-subunits. Inactivation, however, is complete after binding of 1.5-2 pyridoxal-P/CF1, suggesting that two covalently modified lysines prevent the activation of the enzyme. ADP as well as ATP in presence of Mg2+ protects the enzyme against inactivation and concomittantly prevents incorporation of a part of the 3H-labeled pyridoxal-P into beta- and alpha-subunits. Phosphate prevents labeling of the alpha-subunit, but has only a minor effect on protection against inactivation. The data indicate a binding site at the interface between the alpha- and beta-subunits. Cleavage of the pyridoxal-P-labeled subunits with cyanogen bromide followed by sequence analysis of the labeled peptides led to the detection of Lys beta 359, Lys alpha 176 and Lys alpha 266, which are closely related to proposed nucleotide-binding regions of the alpha- and beta-subunits.     

Two mutations in recombinant Hb beta F41(C7)Y, K82 (EF6)D show additive effects in decreasing oxygen affinity.

Based on the properties of two low oxygen affinity mutated hemoglobins (Hb), we have engineered a double mutant Hb (rHb beta YD) in which the beta F41Y substitution is associated with K82D. Functional studies have shown that the Hb alpha 2 beta 2(C7)F41Y exhibits a decreased oxygen affinity relative to Hb A, without a significantly increased autooxidation rate. The oxygen affinity of the natural mutant beta K82D (Hb Providence-Asp) is decreased due to the replacement of two positive charges by two negative ones at the main DPG-binding site. The functional properties of both single mutants are interesting in the view of obtaining an Hb-based blood substitute, which requires: (1) cooperative oxygen binding with an overall affinity near 30 mm Hg at half saturation, at 37 degrees C, and in the absence of 2,3 diphosphoglycerate (DPG), and (2) a slow rate of autooxidation in order to limit metHb formation. It was expected that the two mutations were at a sufficient distance (20 A) that their respective effects could combine to form low oxygen affinity tetramers. The double mutant does display additive effects resulting in a fourfold decrease in oxygen affinity; it can insure, in the absence of DPG, an oxygen delivery to the tissues similar to that of a red cell suspension in vivo at 37 degrees C. Nevertheless, the rate of autooxidation, 3.5-fold larger than that of Hb A, remains a problem.     

Hemoglobin Athens-Georgia, or alpha 2 beta 2 40(C6)Arg replaced by Lys, a hemoglobin variant with an increased oxygen affinity.

A new hemoglobin variant, termed hemoglobin Athens-Georgia, has been found in a 23-year-old Caucasian student and three members of her family. The electrophoretic mobility of this variant at pH 9.0 is slightly less than that of hemoglobin-A. Arginyl residue in position 40 of the beta chain, corresponding to position 6 of the C helix, has been replaced by a lysyl residue. This amino acid substitution is at the alpha1-beta2 contact and slightly affects the oxygen binding properties of the hemoglobin molecule. Hemoglobin Athens-Georgia has an increased affinity for oxygen, a normal heme-heme interaction and a normal Bohr effect. Hematological abnormalities are not associated with this variant.     

Involvement of peptide histidine isoleucine (PHI) in prolactin secretion induced by serotonin in rats

The possible role of hypothalamic peptide histidine isoleucine (PHI) in prolactin (PRL) secretion induced by serotoninergic mechanisms was investigated in male rats using a passive immunization technique. Intracerebroventricular injection of serotonin (5HT, 10 micrograms/rat) raised plasma PRL levels both in urethane-anesthetized rats and in conscious rats pretreated with normal rabbit serum (0.5 ml/rat, iv, 30 min before). Plasma PRL responses to 5HT were blunted in these animals when they were pretreated with rabbit antiserum specific for PHI (0.5 ml/rat, iv, 30 min before) (mean +/- SE peak plasma PRL: anesthetized rats 271.3 +/- 38.3 ng/ml vs 150.0 +/- 12.6 ng/ml, p less than 0.01, conscious rats 54.3 +/- 6.8 ng/ml vs 30.7 +/- 4.1 ng/ml, p less than 0.025). These results suggest that hypothalamic PHI is involved, at least in part, in PRL secretion induced by central serotoninergic stimulation in the rat.     

Electron paramagnetic resonance of Hb St Louis beta28 (B10) Leu replaced by Gln.

Hemoglobin St Louis beta28 (B10) Leu replaced by Gln is a new mutant which occurs as a natural valency hybrid (alpha2beta+2), or hemoglobin M (Cohen-Solal, M., Seligmann, M., Thillet, J. and Rosa, J. (1973) FEBS Lett. 33, 37-41). The electron paramagnetic resonance (EPR) spectrum of native Hb St Louis at pH 6.2 shows a mixture of three species. Two are high spin, one with tetragonal symmetry, like Hb+ A, the other with rhombic distortion. The third is a low-spin form corresponding to a hemichrome with the distal (E7) histidine as the sixth ligand of the ferric iron. The hemichrome is also found in red blood cells. After oxidation to the alpha+2beta+2 form, three EPR species are seen. Surprisingly, there remains only one high-spin signal, with almost tetragonal symmetry. Besides the low-spin hemichrome, a hydroxy signal is observed even at pH 6.2. These observations imply interactions between the alpha and beta hemes.     

Molecular cloning and characterization of a high affinity dopamine receptor (D1 beta) and its pseudogene.

We have cloned a novel human intronless gene encoding a G-protein-coupled receptor of the dopamine receptor family. Expression of this receptor in Cos-7 cells led to the high affinity binding of a number of dopamine D1 antagonists, with a binding profile similar to that of the previously described dopamine D1 receptor. In contrast, the agonist binding profile of this new receptor did not exactly match any previously defined dopamine D1 receptor and was notable for its unusually high affinity for dopamine. This new receptor caused a 13-fold increase in adenylylcyclase activity in transfected Cos-7 cells, following addition of dopamine. Messenger RNA encoding this new receptor appears to be widely distributed in the human brain, including cortical regions, choroid plexus, hippocampus, and brain stem. This new receptor appears to be identical to the recently described dopamine D5 receptor. A second closely related gene, GL39, was isolated and shown to represent a pseudogene, the first to be described in the G-protein-coupled receptor superfamily. This pseudogene exhibits 94% nucleotide sequence homology to the GL30 sequence and may have arisen from a gene duplication event followed by a mutation approximately 8 million years ago, prior to the emergence of man. This recently evolved pseudogene is transcribed in the human brain with a tissue distribution similar to that for its closely related functional gene.     

Structure of deoxy hemoglobin C (beta six Glu replaced by Lys) in two crystal forms

Five crystal forms of the abnormal human hemoglobin Hb³ C (beta six Glu → Lys) have been grown. Two of them are grown with liganded Hb C, three with deoxy Hb C. The structures of two of the deoxy crystal forms were determined by the method of molecular replacement, using deoxy Hb A as the model structure. Fourier maps were calculated for each Hb C structure, using data to a resolution of 5 Å in one case and 4 Å in the second case. The structural differences between each deoxy Hb C structure and the deoxy Hb A model are found mostly at the molecular surface. Energetically favorable interactions involving the variant residue, beta six lysine, occur in both Hb C crystal forms, and could explain the lowered solubility and enhanced tendency of deoxy Hb C to crystallize in vivo.