Mouse haemoglobin Beta chains. Sequence data on embryonic y chain and genetic linkage of the Y-chain locus to the adult beta-chain locus Hbb.

Embryonic haemoglobin y-chain types were determined by starch-gel electrophoresis for inbred Mus musculus strains Sec/ReJ and Au/SsJ (with Hbbs and Hbbp adult beta-chain alleles respectively) and for random-bred HA/ICR (Swiss) mice, which had been selected to be homozygous for the adult Hbbd beta-chain allele. The strain with the Hbbs allele had y1 embryonic beta chain and the strain with the Hbbd allele had y2. The strain with the Hbbp allele also had y2 chain. A breeding study showed that the maximum recombination frequency between the Hbb locus and the y-chain locus is 5.4% at the 95% confidence level. Embryonic y2 chain was sequenced for 39 positions from the N-terminus with an Edman-Begg sequenator. Some tryptic-peptide composition data were obtained on both y1 and y2 chains. No amino acid substitutions between y1 and y2 chains were found in the 40 positions for which there are common data on the two chains. y2 and adult beta chains are different in 12 of the first 21 positions, but show considerable similarity thereafter. It is suggested that homologous but unequal crossing-over between y-chain and adult beta-chain loci could have given rise to the adult beta-chain allele Hbbd, which produces structurally different major and minor beta chains from two closely linked genes.     

Evolutionary potential: A mathematical hypothesis of mouse hemoglobin beta chain evolution

Summary This paper examines the possibility that the linkage arrangements and regulatory properties of genes may be influenced by selection. A mathematical hypothesis is developed in order to show how selective properties of hemoglobin beta chains could have influenced the linkage and regulation of their structural genes. The hypothesis is applied to the case of mouse hemoglobin beta chains. In most mice, closely-linked pairs of loci (doublets) code for two structurally divergent beta chains in unequal amounts. Some mouse strains have singlet alleles, however, coding for another beta chain variant. With the mathematical hypothesis, one can show that selectively determined evolutionary potentials may have favored changes in proportions of major and minor chains produced by a doublet allele. In the extreme case, zero production of the minor chain may give a selective advantage, leading to a ringlet; conversely, selection may favor linking another gene to the singlet locus to give a doublet. A specific prediction of the model is the stable maintenance under certain conditions of multiple alleles at regulatory loci. The concept of evolutionary potential thus suggests that selection could have influenced the evolution of genotypic fitnesses, in addition to causing changes in gene frequencies as in standard population genetics models.     

Assignment of the alpha and beta chains of human propionyl-CoA carboxylase to genetic complementation groups.

Propionicacidemia is a metabolic disorder resulting from a deficiency of propionyl-CoA carboxylase activity. The enzyme is composed of two polypeptides: a 72,000-dalton alpha chain which contains the biotin ligand and a 56,000-dalton beta chain. It has been suggested that the two major complementation groups in this disorder, pccA and pccBC (with subgroups pccB and pccC), correspond to the genes encoding these two chains. To correlate gene product with complementation groups, 15 mutant and four normal human fibroblast strains were analyzed by [35S]methionine and [3H]biotin labeling. Immunoprecipitation and gel electrophoresis of the polypeptides revealed that alpha chains are synthesized by mutants of pccBC and both subgroups but not in four out of five pccA mutants. On the other hand, beta chains were detected only in pccB mutants. We suggest that pccA encodes the alpha chain of PCC while pccBC encodes the beta chain, and furthermore predict that the beta chain is unstable in the absence of the alpha chain.     

Perissodactyla: the primary structure of hemoglobins from the lowland tapir (Tapirus terrestris): glutamic acid in position 2 of the beta chains

The hemoglobins from a lowland tapir (Tapirus terrestris) were analysed and the complete primary structure is described. The globin chains were separated on CM cellulose column in 8M urea and the amino-acid sequences were determined in the liquid phase sequenator. The results show that globin consists of two alpha chains (alpha I and alpha II) and beta major and beta minor components. The alpha chains differ only at one position: alpha I contains aspartic acid and alpha II glycine. The beta chains are heterogeneous: aspartic and glutamic acid were found at position beta 21 and beta 73 of the beta major components and asparagine and serine at position beta 139. In the beta minor components four positions were found with more than one amino acid, namely beta 2, beta 4, beta 6 and beta 56. The sequences are compared with those of man, horse and rhinoceros. Four residues of horse methemoglobin, which are involved in the alpha 1 beta 1 contacts are substituted in tapir hemoglobins. In the alpha chains: alpha 107(G14)Ser----Val, alpha 111-(G18) Val----Leu, alpha 115(GH3) Asn----Asp or Gly; in the beta chains: beta 116(G18) Arg----Gln. The amino acid at beta 2 of the major components is glutamic acid while glutamine and histidine are found in the minor components. Although glutamic acid, a binding site for ATP, does not interact with 2,3-bisphosphoglycerate, glutamine and histidine in the minor components are responsible for the slight effect of 2,3-bisphosphoglycerate on tapir hemoglobin.     

Allelic constitution of the hemoglobin beta chain in wild populations of the house mouse,Mus musculus

We studied the allelic frequency of the hemoglobin beta chain (Hbb) of wild mice, Mus musculus, collected from 46 localities, mostly in Asia and surrounding areas. The wild populations in the northern part of China, Korea, and the central part of Japan exhibited an almost monomorphic distribution of Hbbp. In the southern part of Asia, the frequency of Hbbp decreased and Hbbd was predominant. Although Hbbs and Hbbd are generally found in Europe, the Hbbp allele was present in Southeastern Europe (Bulgaria). In the light of these results, the Hbbp allele might have originated in mice of northern Asia.     

Differential control of the synthesis of two hemoglobin beta chains in normal mice

A fetal-to-adult switch in the proportion of the mouse minor hemoglobin is described. Although mice have no fetal hemoglobin per se, the timing of this switch in the mouse suggests that the mechanism of its control may directly parallel that of the human switch from fetal to adult hemoglobin expression. The mouse minor hemoglobin is expressed only in strains with the "diffuse" allele for the beta chain complex locus. Fetal liver cells of these mice synthesize a much greater proportion of the betaminor globin chain that do adult hematopoietic cells. Consequently, circulating fetal erythrocytes carry a high level of the minor hemoglobin containing it. By the time of birth, a lowered proportion of betaminor is synthesized in the liver. This low proportion continues to be expressed during early erythroid maturation in the adult. The fetal-to-adult switch is the first indication that in normal mice, the two beta chain loci can be expressed noncoordinately. The similarity between the patterns of the decline of the minor hemoglobin in mice and of the disappearance of fetal hemoglobin in humans suggests that the minor hemoglobin in the "diffuse" mouse may function to some degree as a fetal hemoglobin in the period between the disappearance of the embryonic hemoglobins and the time of birth.     

The primary structure of three hemoglobin chains from the indigo snake (Drymarchon corais erebennus,Serpentes): first evidence for alphaD chains and two beta chain types in snakes

The hemoglobin of the indigo snake (Drymarchon corais erebennus, Colubrinae) consists of two components, HbA and HbD, in the ratio of 1:1. They differ in both their alpha and beta chains. The amino acid sequences of both a chains (alphaA and alphaD) and one beta chain (betaI) were determined. The presence of an alphaD chain in a snake hemoglobin is described for the first time. A comparison of all snake beta chain sequences revealed the existence of two paralogous beta chain types in snakes as well, which are designated as betaI and betaII type. For the discussion of the physiological properties of Drymarchon hemoglobin, the sequences were compared with those of the human alpha and beta chains and those of the closely related water snake Liophis milians where functional data are available. Among the heme contacts, the substitution alphaD58(E7)His-->Gln is unusual but most likely without any effect. The residues responsible for the main part of the Bohr effect are the same as in mammalian hemoglobins. In each of the three globin chains only two residues at positions involved in the alpha1/beta2 interface contacts, most important for the stability and the properties of the hemoglobin molecule, are substituted with regard to human hemoglobin. On the contrary, nine, eleven, and six alpha1/beta1 contact residues are replaced in the alphaA, alphaD, betaI chains, respectively.     

Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin

The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.     

Amino acid sequences of the alpha and beta chains of adult hemoglobin of the brown lemur, Lemur fulvus fulvus.

Globin prepared from hemoglobin of the brown lemur (Lemur fulvus fulvus) was separated into alpha and beta chains by chromatography on a CM 52 column. The S-aminoethylated alpha and beta chains were each digested with trypsin and resulting peptides were isolated. The amino acid sequences of the tryptic peptides were established. The ordering of these peptides in the alpha and beta chains was deduced from the homology of their amino acid sequences with that of human adult hemoglobin. The primary structure of brown lemur hemoglobin thus obtained differs from that of human hemoglobin in 15 amino acids in the alpha chain and 26 in the beta chain.     

Domain IV of mouse laminin beta1 and beta2 chains.

Domain IV, consisting of about 230 residues, represents a particular protein module so far found only in laminin beta1 and beta2 chains. Both domains were obtained by recombinant production in mammalian cells. They showed a globular structure, as expected from electron microscopic examination of laminins. Fragment beta1IV was obtained as a monomer and a disulfide-bonded dimer, and both were modified to approximately 50% by a single chondroitin sulfate chain attached to Ser721 of an SGD consensus sequence. Dimerization is caused by an odd number of cysteines, with three of them having a partial thiol character. Whether both modifications also occur in tissue forms of laminin remains to be established. Fragment beta2IV was only obtained as a monomer, as it lacked one crucial cysteine and the SGD sequence. It required, however, the presence of two adjacent LE modules for proper folding. Polyclonal antibodies raised against both fragments showed no cross-reaction with each other and allowed establishment of beta chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 5-25-fold lower content of beta2 compared with beta1 chains in various tissue extracts of adult mice. Tissues derived from beta2-deficient mice failed to react with the beta2-specific antibodies but showed a twofold higher content of beta1 than heterozygotes. The antibodies to beta2 showed broader tissue staining than reported previously, including in particular a distinct reaction with the extrasynaptic endomysium of skeletal muscle. Immunogold staining localized both beta chains primarily to basement membranes of kidney, muscle and various other tissues.     

The third molecule associated with interleukin 2 receptor alpha and beta chain.

It is known that the affinity cross-linking study of the human high-affinity Interleukin 2 (IL-2) receptor reveals triplet bands consisting of 70 kDa alpha chain(Tac)-IL-2 and the 90/80 kDa doublet. We found the cell lines lacking the lower band of the doublet in spite of the expression of both alpha and beta chains. No IL-2 binding was detectable in the presence of anti-Tac antibody in these cells. Immunoprecipitation from the cell extract of [125 I] IL-2-cross-linked T cells with anti-beta chain polyclonal IgG detected the upper band, but not lower band of the doublet. These data suggest that the lower band of the doublet represents an unknown IL-2-binding protein (p65) distinct from the beta chain and this molecule may be involved in the intermediate-affinity IL-2 binding together with the beta chain.     

Genetic variation in mouse beta globin cysteine content modifies glutathione metabolism: implications for the use of mouse models

Allelic variation in the mouse beta globin gene complex (Hbb) produces structurally different beta globins in different mouse strains. Like humans, mice with HbbS alleles produce a single beta globin with one reactive cysteine (beta Cys93). In contrast, mice with HbbD alleles produce two structurally different beta globins, each containing an additional cysteine (beta Cys13). beta Cys93 forms mixed disulfides with glutathione and plays a pivotal role in the activities of hemoglobin, glutathione, and nitric oxide. Similar roles for mouse beta Cys13 have not been described. We used capillary electrophoresis to compare reduced glutathione (GSH), glutathione disulfide (GSSG), and S-glutathionyl hemoglobin levels in erythrocytes from inbred C57BL/6J (homozygous HbbS/S) and 129S1/SvImJ (homozygous HbbD/D) mice and their homozygous and heterozygous B6129S/F2J hybrid offspring. S-glutathionyl hemoglobin was nearly undetectable in inbred or hybrid mice with only monocysteinyl beta globins (HbbS/S) but represented up to 10% of total hemoglobin in mice with polycysteinyl beta globins (HbbS/D or HbbD/D). The stepwise increase in beta globin sulfhydryl group concentration in HbbS/S, HbbS/D, and HbbD/D F2 mice was associated with increasing hemoglobin-bound glutathione and decreasing free glutathione (GSH + GSSG) concentrations. Total erythrocyte glutathione (GSH + GSSG + hemoglobin-bound) was not significantly different between groups. In vitro studies showed that beta Cys13 in mouse HbbD beta globins was more susceptible to disulfide exchange with GSSG than beta Cys93. We conclude that reactive beta globin sulfhydryl group concentration is genetically determined in mice, and that polycysteinyl beta globins markedly influence intraerythrocyte glutathione distribution between free and hemoglobin-bound compartments. Although Hbb heterozygosity and polycysteinyl beta globins are common in wild mouse populations, all common human beta globins contain only one reactive cysteine, and homozygosity is the norm. These fundamental differences in mouse and human beta globin genetics have important implications for the study of mouse biology and for the use of some mouse strains as models for humans.     

Oxygen equilibria of hybrid-heme hemoglobins containing proto- and mesoheme groups. On the nonequivalence of alpha and beta chains.

Hybrid-heme hemoglobins, alpha(meso)2beta(proto)2 and alpha(proto)2beta(meso)2, were prepared, and the O2 equilibria of their alpha and beta chains were measured separately at the isosbestic points of the partner chains at different pH values and in the presence and absence of inositol hexaphosphate. The Adair equation was extended to distinguish between the O2 saturations of the alpha and beta chains, and the seven equilibrium parameters were obtained by curve fitting to those equations. The results showed that the beta chains have an affinity slightly higher than the alpha chains in the binding of the first O2 molecule. For the second O2 molecule, the molecular species that has been oxygenated on the alpha chain has a higher affinity than that carrying O2 on the beta chain. The slopes of the Hill plots were higher for the alpha chain. The O2 saturation curves for the alpha and beta chains were calculated from the parameters averaged for the hybrids alpha(meso)2beta(proto)2 and alpha(proto)2beta(meso)2 in order to cancel the effects of the heme replacement. The curves showed that the difference in O2 saturation between the two kinds of chains depends on the conditions and on the degree of O2 saturation. It was concluded that the functional difference between the chains is small enough so that it is not required to modify the models already accepted for the cooperativity of hemoglobin.     

Light chains of sea urchin kinesin identified by immunoadsorption

Previous studies with monoclonal antibodies indicate that sea urchin kinesin contains two heavy chains arranged in parallel such that their N-terminal ends fold into globular mechanochemical heads attached to a thin stalk ending in a bipartite tail [Scholey et al., 1989]. In the present, complementary study, we have used the monoclonal antikinesin, SUK4, to probe the quaternary structure of sea urchin (Strongylocentrotus purpuratus) kinesin. Kinesin prepared from sea urchin cytosol sedimented at 9.6 S on sucrose density gradients and consisted of 130-kd heavy chains plus an 84-kd/78 kd doublet (1 mol heavy chain: 1 mol doublet determined by gel densitometry). Low levels of 110-kd and 90-kd polypeptides were sometimes present as well. The 84-kd/78 kd polypeptides are thought to be light chains because they were precipitated from the kinesin preparation at a stoichiometry of one mol doublet per 1 mol heavy chain using SUK4-Sepharose immunoaffinity resins. The 110-kd and 90-kd peptides, by contrast, were removed using this immunoadsorption method. SUK4-Sepharose immunoaffinity chromatography was also used to purify the 130-kd heavy chain and 84-kd/78-kd doublet (1 mol heavy chain: 1 mol doublet) directly from sea urchin egg cytosolic extracts, and from a MAP (microtubule-associated protein) fraction eluted by ATP from microtubules prepared in the presence of AMPPNP but not from microtubules prepared in ATP. The finding that sea urchin kinesin contains equimolar quantities of heavy and light chains, together with the aforementioned data on kinesin morphology, suggests that native sea urchin kinesin is a tetramer assembled from two light chains and two heavy chains.     

Amino-acid sequences of the alpha and beta chains of adult hemoglobins of the Grand Galago, Galago crassicaudatus

The adult Grand Galago (Galago crassicaudatus) was found to have two hemoglobin components (Hb I and Hb II) which were separated by carboxymethyl cellulose column chromatography. The alpha and beta chains of each component were isolated. The tryptic peptides of the alpha and beta chains were each isolated and sequenced by the conventional method. The alignment of these peptides in each chain was deduced from the homology of their sequences with that of human adult hemoglobin. The alpha chains from Hb I and Hb II were considered to be identical. On the other hand, there was only one amino-acid difference between the two beta chains at the 125th residue from the N-terminus.     

Amino acid sequences of the alpha and beta chains of adult hemoglobin of the slender loris, Loris tardigradus.

alpha and beta chains from adult hemoglobin of the slender loris (Loris tardigradus) were isolated by Amberlite CG-50 column chromatography. After S-aminoethylation, both chains were digested with trypsin and the amino acid sequences of the tryptic peptides obtained were analyzed. Further, the order of these tryptic peptides in each chain was deduced from their homology with the primary structures of alpha and beta chains of human adult hemoglobin. Comparing the primary structures of the alpha and beta chains of adult hemoglobin of the slender loris thus obtained with those of adult hemoglobin of the slow loris, 4 amino acid substitutions in the alpha chains and 2 in the beta chains were recognized.     

Amino acid sequence of the beta chain of human fibrinogen.

The beta chain of human fibrinogen contains 461 amino acid residues, 15 of which are methionines. The calculated molecular weight, independent of a single carbohydrate cluster, is 52 230. In this regard, we have isolated and characterized all 16 cyanogen bromide fragments. In one case (CNI), we have concentrated on a disputed portion of a previously reported fragment. The arrangement of the cyanogen bromide peptides was deduced by the use of overlap fragments obtained from the tryptic digestion of modified and unmodified beta-chains and from digestions with staphylococcal protease, as well as by considerations involving the plasmic digestion products of fibrinogen. In one case two adjacent fragments were aligned by homology with the corresponding segments of the gamma chain. The homology of the beta chain with the gamma chain is especially strong over the course of the carboxy-terminal two-thirds of the sequence. Neither of these chains appears to be homologous with the alpha chain in these regions. With a few minor exceptions, the sequence reported in this article is in agreement with data reported by other groups in Stockholm and Munich.     

Analysis of murine S-glutathionyl hemoglobins and beta globin haplotype by dynamic capillary isoelectric focusing

Genetic variation in the number and reactivity of beta globin sulfhydryl groups causes variation in erythrocyte redox status in mouse populations. These experiments demonstrate the use of capillary isoelectric focusing for measuring endogenous S-glutathionyl hemoglobin and identifying mouse beta globin (Hbb) haplotype in inbred and outbred mouse strains with mono-cysteinyl or di-cysteinyl beta globins. Hbb haplotype can be readily determined in all strains based on characteristic differences in peak profiles or on peak mobility shift induced by thiol exchange with glutathione disulfide in vitro. This method could prove useful for in vivo study of factors that influence thiol protein modification.     

Posttranslational modifications and beta/gamma chain associations of human laminin alpha1 and laminin alpha5 chains: purification of laminin-3 from placenta

Laminins assemble into trimers composed of alpha, beta, and gamma chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the laminin isoforms formed by laminin alpha1 and alpha5 chains. Comparative pulse-chase experiments and deglycosylation studies in JAR cells established that the M(r) 360,000 laminin alpha1 chain is glycosylated into a mature M(r) 400,000 band while the M(r) 370,000 laminin alpha5 chain is glycosylated into a M(r) 390,000 form that upon secretion is further processed into a M(r) 380,000 form. Hence, despite the shorter peptide length of alpha1 chain in comparison with the alpha5 chain, secreted alpha1 assumes a larger size in SDS-PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR laminins identified laminin alpha1 and laminin alpha5 chains in laminin-1 and laminin-10. In placenta laminin alpha1 chain (M(r) 400,000) and laminin alpha5 chain (M(r) 380, 000/370,000 doublet) were found in laminin-1/-3 and laminin-10/-11. Immunohistochemically we could establish that the laminin alpha1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin beta2 chains. Surprisingly, a fraction of the laminin alpha1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin beta1-beta3 chains, possibly pointing to an unexpected complexity in the chain composition of alpha1-containing laminin isoforms.