Hb natal or alpha 2(minus Tyr-Arg) beta 2: a high oxygen affinity alpha chain variant with a deleted carboxy-terminus resulting from a TAC----TAA (Tyr----terminating codon) mutation in codon alpha 140

The discovery is reported of a fast-moving alpha chain variant (Hb Natal) which is characterized by a shortened alpha polypeptide chain because of the deletion of the Tyr-Arg carboxy-terminal residues. Through amplification of appropriate segments of DNA and hybridization with synthetic oligonucleotide probes, it was possible to detect a C----A mutation in codon 140 of the alpha 2 globin gene, which causes a change in the codon for tyrosine to a terminating codon. Hb Natal or alpha 2 (minus Tyr-Arg) beta 2 has a high affinity for oxygen without a Bohr effect and heme-heme interaction. These results provide direct evidence for the importance of the tyrosine residue at alpha 140 in the oxygenation-deoxygenation process.     

A new hemoglobin variant altering the alpha 1 beta 2 contact: Hb Chemilly alpha 2 beta 2 99(G1)Asp leads to Val

The contribution of hepatic glycogen to lipogenesis was studied in isolated, intact rat hepatocytes. To establish its importance as a substrate for lipogenesis, the glycogen of isolated hepatocytes was prelabelled with 14C from glucose. Evidence is presented that neither glucose nor glycogen constitute major sources of carbon for de novo synthesis of fatty acids and that less than 1% of glycogen is converted into fatty acids.     

Hemoglobin Fort Gordon or alpha2beta2145 Tyr replaced by Asp, a new high-oxygen-affinity hemoglobin variant.

Hemoglobin Fort Gordon, alpha2beta2145 Tyr replaced by Asp (HC2), has been observed in a 20-year-old black male with compensatory erythrocytosis. The variant was readily identified by electrophoresis and chromatography, and comprised about 30% of the red cell hemoglobin. The substitution was identified through analyses of tryptic peptides of various digests of the isolated beta chain. The oxygen affinity of whole blood was increased; two components were observed one of which had a greatly increased affinity for oxygen and a markedly reduced subunit cooperativity. It appears that the Tyr replaced by Asp substitution resembles the Tyr replaced by His substitution in hemoglobin Bethesda (Bunn, H. F. et al. (1972) J. Clin. Invest. 51, 2299-2309; Olson, J. S. and Gibson, G. H. (1972) J Biol. Chem. 247, 3662-3670; Adamson et al. (1972) J. Clin. Invest. 51, 2883-2888) in that both inhibit the quarternary change of the oxy to the deoxy conformation, resulting in greatly altered functional properties. Studies of a few members of the family were negative.     

Characterization of a new electrophoretically silent hemoglobin variant. Hb saale OR alpha 2beta 2 84(EF8)Thr --> Ala

A new abnormal hemoglobin was detected in a young German anemic patient by cation-exchange high performance liquid chromatography (HPLC). Using a combination of electrospray mass spectrometry, HPLC, direct sequencing, and family screening with polymerase chain reaction/restriction digestion approach, we have characterized this hemoglobin variant as resulting from a Thr --> Ala replacement at beta84(EF8). It could be separated neither by electrophoresis nor by isoelectric focusing. Hb Saale is slightly unstable, exhibiting a moderate tendency to auto-oxidize. Functional properties and the heterotropic interactions are similar to those of Hb A.     

Hb J-Antakya or alpha 2 beta (2)65(E9)Lys----Met in a Turkish family and Hb complutense or alpha 2 beta (2)127(H5)Gln----Glu in a Spanish family; correction of a previously published identification

Almost 10 years ago we reported in this journal the characterization of Hb Hacettepe or alpha 2 beta (2)127(H5)Gln----Glu. Unfortunately, we have to conclude that the original characterization of this Turkish variant was in error. The corrected data are presented in this short communication. The variant (alpha 2 beta (2)65(E9)Lys----Met) was (re)named Hb J-Antakya, after the city where the family resides. An abnormal Hb, observed in a Spanish family and named Hb Complutense, had the beta 127 Gln----Glu substitution, erroneously assigned to the Turkish variant.     

Hemoglobin Roseau-Pointe a Pitre alpha 2 beta 2(90) (F6) Glu----Gly: a new hemoglobin variant with slight instability and low oxygen affinity

A Dominican neonate carrying a new abnormal hemoglobin, hemoglobin Roseau Pointe-à-Pitre alpha 2 beta 2(90)(F6) Glu----Gly, was detected in Guadeloupe during application of a cord blood screening program. This variant behaved in isoelectrofocusing as an Hb D, and displayed instability and low whole blood oxygen affinity. In the affected family it was present, either isolated, or in association with a beta+ thalassemia trait.     

A new abnormal human hemoglobin: Hb Prato (alpha 2 31 (B12) Arg leads to Ser beta 2).

An abnormal human hemoglobin was found in a hemolysate from a 5-year-old healthy child living in Prato (Tuscany, Italy). Strutctural studies demonstrated a previously unreported amino acid substitution, alpha 31 (B12) Arg leads to Ser (this is an alpha 1 beta 1 contact). The new variant has been named Hb Prato. It was unstable in isopropanol and heat-denaturation tests, but has normal functional properties, with respect to whole blood studies. Family studies indicated that the variant had been inherited from the mother, a 39-year-old woman of Sicilian extraction. Hb Prato occurs at 20 and 28% in hemolysates from the boy and woman, respectively.     

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.     

Carbohydrate-carbohydrate binding of ganglioside to integrin alpha(5) modulates alpha(5)beta(1) function

Gangliosides GT1b and GD3, components of keratinocyte membranes, inhibit keratinocyte adhesion to fibronectin. Although ganglioside sialylation is known to be important, the mechanism of inhibition is unknown. Using purified insect recombinant alpha(5) and beta(1) proteins and alpha(5)beta(1) integrin from lysed keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3 inhibit the binding of alpha(5)beta(1) to fibronectin. Co-immunoprecipitation of GT1b and alpha(5)beta(1) from SCC12 cells and direct binding of GT1b and GD3 to affinity-purified alpha(5)beta(1) from SCC12 cells and insect recombinant alpha(5)beta(1), particularly the alpha(5) subunit, further suggest interaction between ganglioside and alpha(5)beta(1). The carbohydrate moieties of integrin appear to be critical since gangliosides are unable to bind deglycosylated forms of alpha(5)beta(1) from SCC12 and insect cells or poorly glycosylated recombinant alpha(5)beta(1) from Escherichia coli cells. The GT1b-alpha(5)beta(1) interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in alpha(5)beta(1). The preferential binding of GT1b to high mannose rather than reduced mannose ovalbumin further implicates the binding of GT1b to mannose structures. These data provide evidence that highly sialylated gangliosides regulate alpha(5)beta(1)-mediated adhesion of epithelial cells to fibronectin through carbohydrate-carbohydrate interactions between GT1b and the alpha(5) subunit of alpha(5)beta(1) integrin.     

The Hb A variant (beta73 Asp-->Leu) disrupts Hb S polymerization by a novel mechanism

Polymerization of a 1:1 mixture of hemoglobin S (Hb S) and the artificial mutant HbAbeta73Leu produces a dramatic morphological change in the polymer domains in 1.0 M phosphate buffer that are a characteristic feature of polymer formation. Instead of feathery domains with quasi 2-fold symmetry that characterize polymerization of Hb S and all previously known mixtures such as Hb A/S and Hb F/S mixtures, these domains are compact structures of quasi-spherical symmetry. Solubility of Hb S/Abeta73Leu mixtures was similar to that of Hb S/F mixtures. Kinetics of polymerization indicated that homogeneous nucleation rates of Hb S/Abeta73Leu mixtures were the same as those of Hb S/F mixtures, while exponential polymer growth (B) of Hb S/Abeta73Leu mixtures were about three times slower than those of Hb S/F mixtures. Differential interference contrast (DIC) image analysis also showed that fibers in the mixture appear to elongate between three and five times more slowly than in equivalent Hb S/F mixtures by direct measurements of exponential growth of mass of polymer in a domain. We propose that these results of Hb S/Abeta73Leu mixtures arise from a non-productive binding of the hybrid species of this mixture to the end of the growing polymer. This "cap" prohibits growth of polymers, but by nature is temporary, so that the net effect is a lowered growth rate of polymers. Such a cap is consistent with known features of the structure of the Hb S polymer. Domains would be more spherulitic because slower growth provides more opportunity for fiber bending to spread domains from their initial 2-fold symmetry. Moreover, since monomer depletion proceeds more slowly in this mixture, more homogeneous nucleation events occur, and the resulting gel has a far more granular character than normally seen in mixtures of non-polymerizing hemoglobins with Hb S. This mixture is likely to be less stiff than polymerized mixtures of other hybrids such as Hb S with HbF, potentially providing a novel approach to therapy.     

Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins.

The alpha(1)beta(1) and alpha(2)beta(1) integrins are cell surface collagen receptors. Cells expressing the alpha(1)beta(1) integrin preferentially adhere to collagen IV, whereas cells expressing the alpha(2)beta(1) integrin preferentially adhere to collagen I. Recombinant alpha(1) and alpha(2) integrin I domains exhibit the same collagen type preferences as the intact integrins. In addition, the alpha(2) integrin I domain binds echovirus 1; the alpha(1) I domain does not. To identify the structural components of the I domains responsible for the varying ligand specificities, we have engineered several alpha(1)/alpha(2) integrin I domain chimeras and evaluated their virus and collagen binding activities. Initially, large secondary structural components of the alpha(2) I domain were replaced with corresponding regions of the alpha(1) I domain. Following analysis in echovirus 1 and collagen binding assays, chimeras with successively smaller regions of alpha(1) I were constructed and analyzed. The chimeras were analyzed by ELISA with several different alpha(2) integrin monoclonal antibodies to assess their proper folding. Three different regions of the alpha(1) I domain, when present in the alpha(2) I domain, conferred enhanced collagen IV binding activity upon the alpha(2) I domain. These include the alpha3 and alpha5 helices and a portion of the alpha6 helix. Echovirus 1 binding was lost in a chimera containing the alphaC-alpha6 loop; higher resolution mapping identified Asn(289) as playing a critical role in echovirus 1 binding. Asn(289) had not been implicated in previous echovirus 1 binding studies. Taken together, these data reveal the existence of multiple determinants of ligand binding specificities within the alpha(1) and alpha(2) integrin I domains.     

Hb Doha or alpha 2 beta 2[X-N-Met-1(NA1)Val----Glu]; a new beta-chain abnormal hemoglobin observed in a Qatari female

Structural analysis of a fast-moving hemoglobin variant, present in three members of a Qatari family, identified a Val----Glu substitution at position 1 (NA1) of the beta-chain. The introduction of this glutamic acid residue prevents the removal of the initiator methionine, thus extending the N-terminus by one residue to Met-Glu-His-Leu-Thr-. The methionine residue is blocked by an as yet not completely identified molecule. The presence of the variant in a heterozygote does not have clinical consequences.     

Hemoglobin Fannin-Lubbock [alpha2 beta 2 119 (GH2) Gly replaced by Asp]. A new hemoglobin variant at the alpha1 beta 1 contact.

Hemoglobin Fannin-Lubbock was found in a 9-year-old Mexican-American female. The abnormal hemoglobin was detected as a fast-moving variant by electrophoresis on cellulose acetate at pH 8.4. Structural analysis indicated a substitution in the beta-chain of aspartic acid for glycine at position 119, a position involved in the alpha1beta1 contact of the hemoglobin tetramer. This contact between unlike chains is larger and undergoes a smaller shift during the process of oxygenation and deoxygenation that the alpha1beta2 contact (Perutz, M.F., Muirhead, H., Cox, J.M. and Goaman, L.C.G. (1968) Nature 219, 131-139). Mutations in this contact tend to cause slight or no changes in functional behavior. Apart from a mild anemia, the propositus did not exhibit any obvious clinical symptoms.