Genomic organization of the human interleukin-12 receptor β2-chain gene

 The interleukin-12 receptor (IL-12R) is composed of two subunits, referred to as β1 and β2. Both chains are necessary for high-affinity IL-12 binding and signalling, although only the IL-12Rβ2 chain contains the intracellular tyrosine residues responsible for STAT4 activation. This study presents the intron-exon organization of the human IL-12Rβ2-chain gene. Polymerase chain reaction (PCR) primers designed across the cDNA (U46198) were used to trace introns, by comparing PCR product sizes obtained using cDNA and genomic DNA as templates. PCR products spanning introns were sequenced to determine the exact splice sites and flanking regions. The coding region of the gene was found to consist of 15 exons and 14 introns. All intron-exon boundaries are consistent with the consensus sequence for splice junctions (5′ GT/AG 3′). Comparison of the intron-exon organization with the human GCSFR gene indicated a remarkably well conserved genomic organization between these two class I cytokine receptors. Interestingly, we identified an alternatively spliced mRNA, encoding a putative, truncated protein, lacking all signalling potential. Received: 21 July 1999 / Revised: 2 September 1999     

Primary structure of the major β-chain of rat haemoglobins

The amino acid sequence of the major β-chain, ^IIβ, from rat haemoglobins was established with an automated sequencer. Amino acid heterogeneities were found that appear to result from allelic variation at particular residues. We applied several new or unusual techniques in determining the sequence: (1) reaction of the polypeptide with dansylaziridine for detection of cysteine; (2) blockage of the N-terminal residue and the ε-amino group of lysine residues with 1-fluoro-2-nitro-4-trimethylammoniobenzene iodide and subsequent identification of the modified lysine phenylthiohydantoin by absorbance at 420nm; (3) identification of histidine phenylthiohydantoin by its blue fluorescence under long-wave u.v. light; (4) cleavage of the chain into two or three fragments and subsequent sequencing without purification [a detailed statement giving the major phenylthiohydantoins assigned at each step for each sequence run before their alignment in individual sequences has been deposited as Supplementary Publication SUP 50084 (10 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5]; (5) separation of fragments produced by CNBr cleavage by cation-exchange chromatography; (6) peptide sequencing after attachment of the peptide to cytochrome c. The amino acid sequence was confirmed by amino acid compositions of the complete chain, of CNBr fragments 1 and 3, and of 11 purified tryptic peptides.     

Biochemical characterization of the β-chain variant haptoglobin Marburg

Summary -chains were isolated from two individuals heterozygous for the -chain mutant haptoglobin Marburg. Total amino acid composition and tryptic peptides were compared with -chains from common haptoglobin types. ^Mb chain preparations are characterized by the presence of -chains with an atypical electrophoretic migration rate and by at least three, possibly four additional peptides in their tryptic digests. It is probable that haptoglobin Marburg is the result of an mutational event other than a single base substitution.Supported by US-PHS grant AM 11796 and by US-PHS grant HD 03321 and aided by a grant from the Deutsche Forschungsgemeinschaft.     

Experiments with β-chain variants of the hemoglobin of Mus musculus

Starch gel electrophoretic and ultracentrifuge methods failed to demonstrate any differences between the hemoglobins of mice of the Shanghai and HBBP/Cag strains and crosses among these strains. The apparent identity of these hemoglobins is thought to stem from the contribution of Asian mice to the British mouse fancy from which the laboratory strains having Hbb-p in part descerd. Maleate buffer of pH 7 or above can be used to prevent the formation of disulfide-bridged dimers of mouse hemoglobins. However, the minor electrophoretic bands of Hbb-p and Hbb-d react with approximately twice as much maleate as the major bands of each of these hemoglobins, although the minor bands like the major contain only one free cysteine group per chain. This can be explained by the alkylation of the -amino of lysine residue 76, but some evidence for the alkylation of histidine in the minor band of Hbb-p is also presented.     

Evidence of homology between the β-chain of human haptoglobin and the chymotrypsin family of serine proteases

Amino acid sequences from the β-chain of human haptoglobin are compared with those sequences known for the serine proteases of the chymotrypsin family. In a comparison of some 171 residues of the haptoglobin β-chain (approximately 60% of the protein molecule), approximately 30% of these are identical to residues occurring in sequences of either bovine trypsin, bovine chymotrypsin A, bovine chymotrypsin B, porcine elastase, or bovine thrombin B-chain, and an additional 10% are chemically similar. A combined comparison of the haptoglobin β-chain with the above five serine proteases gave an identity of 56% and a chemical similarity of 11%. Similarity of primary structure is also striking around two of the five half-cystinyl residues so far characterized in long lengths of sequence. These data provide substantial evidence that the β-chain of haptoglobin is homologous to the chymotrypsin family of serine proteases. Proposals are also presented to explain the occurrence of internal homology in the N-terminal region of the β-chain.     

Characterization of the MHC class II ��-chain gene in ducks

In humans, classical MHC class II molecules include DQ, DR, and DP, which are similar in structure but consist of distinct α- and β-chains. The genes encoding these molecules are all located in the MHC class II gene region. In non-mammalian vertebrates such as chickens, only a single class II α-chain gene corresponding to the human DRA has been identified. Here, we report a characterization of the duck MHC class II α-chain (Anpl-DRA) encoding gene, which contains four exons encoding a typical signal peptide, a peptide-binding α1 domain, an immunoglobulin-like α2 domain, and Tm/Cyt, respectively. This gene is present in the duck genome as a single copy and is highly expressed in the spleen. Sequencing of cDNA and genomic DNA of the Anpl-DRA of different duck individuals/strains revealed low levels of genetic polymorphism, especially in the same strain, although most duck individuals have two different alleles. Otherwise, we found that the duck gene is located next to class II β genes, which is the same as in humans but different from the situation in chickens.     

Comparison of the TCR ζ-chain with the FcR γ-chain in chimeric TCR constructs for T cell activation and apoptosis

A promising strategy for cancer treatment is adoptive gene therapy/immunotherapy by genetically modifying T cells with a chimeric T cell receptor (cTCR). When transduced T cells (T-bodies) specifically bind to tumor antigens through cTCR, they will become cytotoxic T lymphocytes (CTL) and lyse the tumor cells in a non-major histocompatibility complex (MHC)-restricted manner. Both the FcR gamma-chain and the TCR zeta-chain have been used to construct such cTCR, and both have shown specific cytolytic functions against tumor cells. However, most researchers believe that the zeta-chain generates stronger cytolytic activities against tumor than the gamma-chain and therefore would be a better candidate for cTCR construction. On the other hand, because of the lack of costimulation signaling in such constructs, the T-body might cause activation-induced T cell death (AICD) when bound to tumor antigens. Therefore, one can argue that the gamma-chain might generate less AICD than the zeta-chain because the gamma-chain has only one immunoreceptor tyrosine-based activation motif (ITAM), and the cytolytic activities can be therefore recycled. Two cTCR, GAHgamma and GAHzeta, were constructed and evaluated for cytokine production, specific cytolytic function and AICD in T-bodies after exposure to tumor cells. Using EGP-2-positive LS174T colorectal carcinoma cells as targets, there was no substantial difference observed between a gamma-chain or zeta-chain as the T-body signaling moiety in terms of specific cytolytic functions and induced cytokine production. This paper also demonstrates that, in the absence of a costimulation system, tumor antigen may not trigger apoptosis of T cells transduced with a cTCR carrying either an FcR gamma-chain or a TCR zeta-chain. These observations challenge current ideas about the role of ITAM in T cell activation.     

Primary structure of the hemoglobin α-chain of rose-ringed parakeet (Psittacula krameri)

The structure of the hernoglobin α-chain of Rose-ringed Parakeet was determined by sequence degradations of the intact subunit, the CNBr fragments, and peptides obtained by digestion with staphylococcal Glu-specific protease and trypsin. Using this analysis, the complete α-chain structure of 21 avian species is known, permitting comparisons of the protein structure and of avian relationships. The structure exhibits differences from previously established avian α-chains at a total of 61 positions, five of which have residues unique to those of the parakeet (Ser-12, Gly-65, Ser-67, Ala-121, and Leu-134). The analysis defines hemoglobin variation within an additional avian order (Psittaciformes), demonstrates distant patterns for evaluation of relationships within other avian orders, and lends support to taxonomic conclusions from molecular data.     

Hemoglobin binding with haptoglobin: Delineation of the haptoglobin binding site on the α-chain of human hemoglobin

Previous studies from this laboratory employing a comprehensive synthetic overlapping peptide strategy showed that the -chain of human hemoglobin (Hb) contains a single haptoglobin (HP) binding region residing within residues 121–135. The present study describes a precise delineation of this Hp-binding site on the -chain. Two overlapping peptides (111–125 and 121–135) spanning this region and a panel of five peptides decreasing at the C-terminal from residue 135 by decrements of two residues (119–135, 119–133, 119–131, 119–129, and 119–127) were synthesized, purified, and characterized. Quantitative radiometric titration of¹²⁵I-labeled human HP (type 2-1) with adsorbents of each of these synthetic peptides showed that the peptide 119–127 retained a Hp-binding activity equivalent to that of peptide 121–135. This finding indicated that Lys-127 marked the C-terminal boundary of the binding site. Another panel of eight peptides was then synthesized, which had their C-terminus fixed at Lys-127 and increased at the N-terminus by one-residue increments from residue 122 up to residue 115 (122–127, 121–127, 120–127, 119–127, 118–127, 117–127, 116–127, and 115–127). The binding of¹²⁵I-Hp to adsorbents of these peptides demonstrated that the N-terminal boundary of the site did not extend beyond Valine 121. It is, therefore, concluded that the Hp-binding site on the -chain of human Hb comprises residues 121–127.     

The effects of stabilizing mutations in the central part of the α-chain of tropomyosin on the structural and functional properties of αβ-tropomyosin heterodimers

The effects of the D137L/G126R double mutation in the central part of the tropomyosin α-chain via the simultaneous replacement of two highly conserved non-canonical residues, viz., Asp137 and Gly126, by canonical residues Leu and Arg, respectively, on the properties of the αβ-tropomyosin heterodimer have been studied. It has been shown using circular dichroism that this mutation substantially increases the thermal stability of αβ-tropomyosin heterodimers, which, nevertheless, remains lower than that of αα-tropomyosin homodimers with these mutations in both α-chains. The stability of tropomyosin complexes with F-actin has also been studied by measuring the temperature dependences of their dissociation, which is detected by a decrease in light scattering. It has been revealed that αβ-tropomyosin heterodimers carrying the D137L/G126R mutation in the α-chain dissociate from the surface of actin filaments at a higher temperature than ββ-homodimers but at a lower temperature than αα-homodimers with these mutations in both α-chains. It has also been shown using the in vitro motility assay that D137L/G126R substitution in the α-chain increases the sliding velocity of regulated actin filaments in the case of αα-homodimers, while it noticeably decreases the velocity in the case of αβ-tropomyosin heterodimers. Thus, we can conclude that mutations in one of the chains of the tropomyosin dimeric molecule may have different effects on the properties of tropomyosin homodimers and heterodimers.     

Application of DFT methods to the study of the coordination environment of the VO2+ ion in V?proteins

Density functional theory (DFT) methods were used to simulate the environment of vanadium in several V proteins, such as vanadyl-substituted carboxypeptidase (sites A and B), vanadyl-substituted chloroplast F(1)-ATPase (CF(1); site 3), the reduced inactive form of vanadium bromoperoxidase (VBrPO; low- and high-pH sites), and vanadyl-substituted imidazole glycerol phosphate dehydratase (IGPD; sites α, β, and γ). Structural, electron paramagnetic resonance, and electron spin echo envelope modulation parameters were calculated and compared with the experimental values. All the simulations were performed in water within the framework of the polarizable continuum model. The angular dependence of [Formula: see text] and [Formula: see text] on the dihedral angle θ between the V=O and N-C bonds and on the angle φ between the V=O and V-N bonds, where N is the coordinated aromatic nitrogen atom, was also found. From the results it emerges that it is possible to model the active site of a vanadium protein through DFT methods and determine its structure through the comparison between the calculated and experimental spectroscopic parameters. The calculations confirm that the donor sets of sites B and A of vanadyl-substituted carboxypeptidase are [[Formula: see text], H(2)O, H(2)O, H(2)O] and [N(His)(||), N(His)(⊥), [Formula: see text], H(2)O], and that the donor set of site 3 of CF(1)-ATPase is [[Formula: see text], OH(Thr), H(2)O, H(2)O, [Formula: see text]]. For VBrPO, the coordination modes [N(His)(||), N(His)(∠), OH(Ser), H(2)O, H(2)O(ax)] for the low-pH site and [N(His)(||), N(His)(∠), OH(Ser), OH(-), H(2)O(ax)] or [N(His)(||), N(His)(∠), [Formula: see text], H(2)O] for the high-pH site, with an imidazole ring of histidine strongly displaced from the equatorial plane, can be proposed. Finally, for sites α, β, and γ of IGPD, the subsequent deprotonation of one, two, and three imidazole rings of histidine and the participation of a carboxylate group of a glutamate residue ([N(His)(||), [Formula: see text], H(2)O, H(2)O], [N(His)(||), N(His)(||), [Formula: see text], H(2)O], and [N(His)(||), N(His)(||), [Formula: see text], OH(-), [Formula: see text]], respectively) seems to be the most plausible hypothesis.     

Elucidation of the origin of multiple conformations of the human α3-chain type VI collagen C-terminal Kunitz domain: The reorientation of the Trp21 ring

Summary The human 3-chain type VI collagen C-terminal Kunitz domain fragment (3(VI)) has been studied by two-dimensional ¹H–¹H and ¹H–¹³C NMR spectroscopy at 303 K. It is shown that the secondary structure of the protein is strikingly similar to that of BPTI, and that a number of unusual H chemical shifts, which are highly conserved in Kunitz-domain proteins, are also observed for 3(VI). Further-more a series of exchange cross peaks observed in ¹H–¹H spectra shows that a large number of protons in the central -sheet exist in two different chemical environments, corresponding to two unequally populated conformations that are slowly exchanging on the NMR time scale. Several protons, including Ser⁴⁷⁽⁵³⁾ H, Arg³²⁽³⁸⁾ H², and Gln⁴⁸⁽⁵⁴⁾ H², all located in the vicinity of the Trp²¹⁽²⁷⁾ ring in the crystal structure of 3(VI) [Arnoux, B. et al. (1995) J. Mol. Biol., 246, 609–617], have very different chemical shifts in the two conformations, the most affected being Gln⁴⁸⁽⁵⁴⁾ H² (=1.53 ppm), which is placed directly above the Trp²¹⁽²⁷⁾ ring in the crystal structure of 3(VI). It is concluded that the origin of the multiple conformations of the central -sheet is a reorientation of the Trp²¹⁽²⁷⁾ ring. From the intensities of corresponding signals in the two conformations, the population of the minor conformation was found to be 6.4±0.2% of that of the major conformation, while a rate constant k_M=1.01±0.05 s^-1 for the major to minor interconversion was obtained from a series of NOESY spectra with different mixing times. In addition, it is shown that Cys¹⁴⁽²⁰⁾-Cys³⁸⁽⁴⁴⁾ disulfide bond isomerization, previously observed in BPTI [Otting, G. et al. (1993) Biochemistry, 32, 3570–3582], is also likely to occur in 3(VI).     

Characterization and strain distribution of four alleles at the hemoglobin α-chain structural locus in the mouse

In a survey of mice from 40 inbred strains, largely previously untested, four alleles were distinguished at the Hba locus, determining structure of adult mouse hemoglobin chain. This finding supports and extends previous sequence studies by others. Methods are given by which each phenotype was characterized by its solubility profile at varying pH and by its chromatography pattern. Concordance was complete between histidine-positive T-4 (defining Hba ^c) and high-intermediate solubility profile. In three inbred strains, a distinct new low-solubility profile, not associated with his-positive T-4, was recognized, and mice with this phenotype were classified as Hba ^d. Implications of observed widely distributed four-allele polymorphism of mouse hemoglobin -chain structure are discussed.This investigation was supported in part by NIH research grant CA-01074 from the National Cancer Institute and in part by the Virginia and D. K. Ludwig Foundation. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

A novel I247T missense mutation in the haptoglobin 2 β-chain decreases the expression of the protein and is associated with ahaptoglobinemia

We have identified a novel base substitution at codon 247 in the -chain of the haptoglobin 2 (Hp²) allele in a Ghanaian with the Hp0 (ahaptoglobinemic) phenotype. The heterozygous TC substitution caused reduced expression of the protein when the mutant was transfected into COS7 cells. The base substitution resulted in a missense change of the non-polar amino acid isoleucine to the polar amino acid threonine at a position in the -chain that is highly conserved among several species. We had previously identified a mutation in the Hp gene promoter region for the same individual, which gives her genotype as –61CHp²/–61CHp²(I247T). Since the –61C mutation also leads to low Hp expression, the genotype represents the first and most definitive ahaptoglobinemic case reported in Africa.     

Analysis of the disulfide bond arrangement of the HIV-1 envelope protein CON-S gp140 ΔCFI shows variability in the V1 and V2 regions

Disulfide bonding of cysteines is one of the most important protein modifications, and it plays a key role in establishing/maintaining protein structures in biologically active forms. Therefore, the determination of disulfide bond arrangement is one important aspect to understanding the chemical structure of a protein and defining its functional domains. Herein, aiming to understand how the HIV-1 envelope protein's structure influences its immunogenicity, we used an MS-based approach, liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance (LC/ESI-FTICR) mass spectrometry, to determine the disulfide linkages on an oligomeric form of the group M consensus HIV-1 envelope protein (Env), CON-S gp140 ΔCFI. This protein has marked improvement in its immunogenicity compared to monomeric gp120 and wild-type forms of gp140 Envs. Our results demonstrate that the disulfide connectivity in the N-terminal region of CON-S gp140 ΔCFI is different from the disulfide bonding previously reported in the monomeric form of gp120 HIV-1 Env. Additionally, heterogeneity of the disulfide bonding was detected in this region. These data suggest that the V1/V2 region does not have a single, conserved disulfide bonding pattern and that variability could impact immunogenicity of expressed Envs.     

Analysis of sheep T-cell receptor β-chain heterogeneity

 We analyzed nucleotide and deduced amino acid sequence heterogeneity of sheep T-cell receptor β-chain cDNAs isolated from an anchored-polymerase chain reaction library. Evaluation of 34 individual rearrangements has defined 18 new β-chain variable region sequences which have been clustered into 13 families. Presumptive allelic polymorphisms of four of these variable regions have been defined, as well as ten distinct β-chain joining region sequences. The present analysis indicates that sheep T-cell receptor β-chains are composed of characteristic leader, variable, joining, and constant region sequences, and that imprecise joining and N-region addition contribute significantly to diversity in the third hypervariable region. Thus, it appears that sheep, like all other mammals studied to date, employ somatic rearrangement of multiple germline genes to create β-chain heterogeneity. These findings have allowed us to estimate the diversity of the sheep T-cell receptor β-chain variable region repertoire, and they provide information that will permit the evaluation of the role that specific T-cell populations play in naturally occurring and experimental diseases of sheep. Received: 20 October 1997 / Revised: 20 April 1998