The complete amino acid sequence of the murine transplantation antigen H-2Db as deduced by molecular cloning

A mouse cDNA library derived from the EL4 cell line (b-haplotype) was screened with a probe containing a small part of the H-2K^b coding region. One of the clones isolated, pH203, encodes a protein whose deduced amino acid sequence is identical with the known sequence of H-2D^b in 141 of 141 positions available for comparison. The clone, therefore, is believed to code for the H-2D^b transplantation antigen. The cDNA insert of pH203 contains the coding region for residues 82 through the carboxy-terminus of H-2D^b, and includes 476 nucleotides of the 3-untranslated sequence. Comparison between the H-2D^b cDNA clone and a previously isolated H-2K^b cDNA clone shows homologies of 83% and 91% at the amino acid and nucleotide levels, respectively. Analysis of DNA sequences at the 3-coding and untranslated regions suggests that the mRNAs of H-2K^b and H-2D^b are spliced differently at their 3-coding ends.     

Class I MHC molecules rather than other mouse genes dictate influenza epitope recognition by cytotoxic T cells

Influenza nucleoprotein (NP) is an important target antigen for influenza A virus cross-reactive cytotoxic T cells (Tc). Here we examine the NP epitope recognized by cloned and polyclonal BALB/c Tc and the genetics of this recognition pattern. We can define NP residues 147–161 as the epitope seen in conjunction with K ^d , the only H-2^d class I responder allele for NP restriction. H-2 ^d /H-2 ^b F₁ mice (C57BL × DBA/2) primed by influenza infection lyse only H-2^d target cells treated with peptide 147–161 while H-2^b targets are recognized only after treatment with NP residues 365–379 (previously found to be recognized by D^b restricted Tc cells). Tc cell recognition of NP peptide 147–161 is entirely dictated by expression of K ^d and not by other B10 or OH background genes of congenic mice. Restriction of a unique NP sequence by each responder class I major histocompatibility complex (MHC) allele suggests that antigen and class I MHC interact for Tc recognition.     

Phylogenetic conservation of a class III major histocompatibility complex antigen, factor B. Isolation and nucleotide sequencing of mouse factor B cDNA clones

The complement protein factor B is a novel serine protease which is encoded within the major histocompatibility complex in man, guinea pig, and mouse. To determine the structure of mouse factor B, cDNA clones were isolated from mouse strains of two different major histocompatibility complex haplotypes, H-2k and H-2d, and clones of 0.9 and 1.5 kilobases, respectively, were sequenced. The H-2d clone includes the H-2k clone sequence and spans 94% of the Bb-coding sequence. No differences in sequence or in restriction enzyme sites were observed between the H-2k and H-2d clones. The H-2d clone displays 83% nucleotide homology and 83% (derived) amino acid homology with that of human factor B; there are no insertions or deletions. Comparison of the mouse and human factor B sequence reveals extensive regional homology at the catalytic residues and in the NH2-terminal portion of the Bb fragment.     

Characterization and sequencing of cDNA clone encoding the phloem protein PP2 of Cucurbita pepo

Direct N-terminal amino acid sequencing of the phloem protein 2 (PP2) from 3-month old Cucurbita pepo L. (pumpkin), purified by SDS-PAGE and blotted onto PVDF membrane, showed that the protein had a blocked N-terminus. However, after in situ cleavage of the polypeptide in a gel slice by cyanogen bromide, 75 residues of sequence on two cyanogen bromide fragments were determined. An oligonucle-otide probe based on this amino acid sequence was used to screen a cDNA library, constructed from mRNA of 3–5-day old seedling hypocotyls, in ZAP II. A cDNA clone (p11A) predicted an amino acid sequence of 218 residues, in full agreement with the sequences determined for two CNBr fragments of PP2, and suggests that the N-terminus of the protein is a blocked methionine residue which is cleaved off by CNBr. Two additional cDNA clones were sequenced but no heterogeneity in the PP2 sequence was found. The deduced amino acid sequence of C. pepo differs in nine residues from the recently published sequence of Cucurbita maxima (Bostwick et al., Plant Cell 4 (1992) 1539–1548). Southern blot showed that PP2 is encoded by a gene family with a relatively large number of members (estimated as 7–15 per haploid genome).     

Biochemical studies on the H-2K mutant B6.C-H-2 bm10

The H-2K glycoprotein from the MHC mutant bm10 was analyzed biochemically to determine where primary structural differences distinguished it from the parental standard molecule, K^b. Comparative peptide maps showed differences in two peptides known to be part of the parental CNBr fragment spanning amino acids 139 to 228. Partial sequence analyses of CNBr fragments and tryptic peptides identified two tightly clustered amino acid substitutions at amino acids 165 (Val to Met) and 173 (Lys to unknown). The substitutions in bm10 represent the most carboxy-terminal substitutions characterized in the K^b molecules of the spontaneous, histogenically active H-2 mutants.     

Somatic cell variants of H-2k: b: A point mutation in the first extracellular domain results in altered immune recognition

A cell-surface-associated variant H-2K product was expressed by an Abelson virus-induced pre-B-cell line after chemical mutagenesis with ethyl methane sulfonate. The variant cell line (R8.313) was previously demonstrated to have altered allodeterminants in K^b as demonstrated by both K^b-specific monoclonal antibody binding and alloreactive cytotoxic T lymphocyte (CTL) cytolysis. The mutant H-2K ^b gene from R8.313 was cloned and characterized in detail. DNA sequence analysis of the region of the gene corresponding to the three extracellular domains identified a single point mutation resulting in a leucine-to-phenylalanine substitution at amino acid residue 82. The site of mutation within the 1 domain was confirmed by oligonucleotide hybridization analysis. Mouse L-cell fibroblasts transfected with the mutant gene were recognized with the same monoclonal antibody binding and CTL lytic pattern as the R8.313 cell line, confirming that the altered phenotype of the mutant cell line was due to a point mutation in the H-2K ^b gene. These data further extend the hypothesis that the region of amino acid residues 70–90 in the 1 domain is important in the formation of both antibody and CTL-defined recognition structures on major histocompatibility complex class I molecules.     

Biochemical evidence for structurally distinct H-2Dd antigens differing in serological properties

H-2D^d antigens, as defined by the private H-2.4 determinant, exist as two immunochemically distinct populations in H-2^a and H-2^dm2 splenocytes and in the transformed cell line, RADA1(H-2 ^a). The two populations are distinguishable by the anti-H-2.28 serum, k/r anti-h2, which is directed, in part, against the H-2.28 family of public determinants encoded by the D end of the b haplotype. Sequential precipitates of lentil-lectin-purified glycoprotein extracts metabolically labeled with radioactive amino acids reveal that approximately one-quarter to one-third of the H-2D^d antigens, designated H-2D^d (b28 ⁺), react with this antiserum, whereas two-thirds to three-quarters, designated H-2D^d(b28^–), do not. Paired-label tryptic peptide maps in this and a previous study indicate that H-2D^d(b28⁺) and H-2D^d(b28 ^–) are closely related structurally and are more likely to represent modified forms of the same gene product rather than products of different genes, although the existence of closely related genetic loci is not rigorously excluded. Together, H-2D^d(b28⁺) and H-2D^d(b28^–) have a radioactivity level seven times higher than H-2L^d, which also reacts with the anti-H-2.28 serum but which lacks the H-2.4 determinant. As yet unresolved, however, is the question of whether the observed quantitative differences between these three antigens reflect actual molar differences at the cellular level, or whether the variation is the result of metabolic or compositional factors. In any case, a complex serological and structural relationship is found to exist between antigens encoded by the D/L end of the MHC.     

The 24kDa outer envelope membrane protein from spinach chloroplasts: molecular cloning,in vivo expression and import pathway of a protein with unusual properties

The 24 kDa outer envelope membrane protein of spinach chloroplasts (omp24) represents a major constituent of this membrane. Sequences of tryptic and endoprotease Glu-C peptides derived from omp24 allowed the design of oligonucleotides which were used to generate a DNA fragment by polymerase chain reaction using spinach cDNA as template. This fragment served as a probe to screen a cDNA library for a full-length clone of the omp24 coding sequence. The protein predicted from the complete sequence only has 148 amino acids and a molecular mass of 16294 Da. It is an acidic protein (calculated isoelectric point 4.8) with a high content of proline residues. Expression of the coding sequence in Escherichia coli and characterization of the purified recombinant protein produced revealed that the overestimation of its molecular mass by SDS-PAGE (ca. 25 kDa) is due to its abnormal amino acid composition. Despite its rather low hydrophobicity (polarity index 49%), omp24 appears to be deeply embedded in the outer membrane. Insertion of omp24 into the membrane proceeds almost independently of surface receptors or targeting sequence but, in contrast to other known outer envelope membrane proteins, is stimulated by ATP.Abbreviations CNBr cyanogen bromide - IP isoelectric point - NCS N-chlorosuccinimide - NTA nitrilotriacetic acid - omp24 outer envelope membrane protein of spinach chloroplasts - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SV8 protease Staphylococcus aureus V8 protease (Endoprotease Glu-C) - TPT chloroplast triose phosphate/phosphate translocator     

Fine specificity and T-cell receptor β-chain gene rearrangements of five H-2Db-specific cytotoxic T-cell clones

A panel of cytotoxic T lymphocyte clones that recognize H-2^b target cells has been established. Six different clones were distinguished according to the following criteria. First, the fine specificity of the clones was determined by testing proliferation and cytotoxicity on target cells of recombinant mice. Clone 221 recognized H-2K^b, and five other clones recognized H-2D^b. Clone 433 distinguished itself from the other five D^b-specific clones by cross-reacting with an antigen on H-2^k cells. Second, the presence of an idiotypic determinant as defined by the 3179 clone-specific monoclonal antibodies was investigated in cytotoxicity inhibition experiments. One of the D^b-specific clones, 653, was inhibited by these antibodies and was therefore clearly different from the other D^b-specific clones. The third criterion involved the rearrangement pattern of the DNA coding for the chain of the T-cell receptor. Southern blot analysis showed that each clone had a unique pattern. Interestingly, clone 653 , which expresses the same idiotypic determinant as clone 3F9, had deleted the C 1 gene cluster, whereas this gene is functionally expressed in clone 3179.Abbreviations used in this paper C constant gene segment - Con A concanavalin A - CTLs cytotoxic T lymphocytes - D diversity gene segment - ³H-dThd tritiated thymidine - J joining gene segment - kb kilobase pairs - LPS lipopolysaccharide - MHC major histocompatibility complex - MLC mixed lymphocyte culture - SDS sodium dodecyl sulfate - V ariable gene segment     

Structural and immunological reactivity of the principal neutralizing determinant V3 of glycoprotein gp 120 of HIV-1

The variable domain V3 in the outer glycoprotein gp 120 of HIV-1 is a highly important region with respect to immune response during the course of viral infection. Neutralizing antibodies are produced against this domain; in addition, it has been shown to be a functionally active epitope for T helper and cytotoxic T cells. The high degree of amino acid variability in individual HIV-isolates, however, limits the use of the V3-domain in approaches to vaccine development. In order to characterize the residues important for antibody interaction and binding to MHC class I proteins, we constructed a consensus sequence of the V3-domain with broad reactivity [1] and used synthetic peptides derived from this consensus with individual residues altered to alanine. These peptides were used as antigens in ELISA tests to define the amino acids which are important for binding to human and rabbit/anti-peptide immunoglobulins. In addition, we used these alanine-derived peptides in interaction studies with human HLA-A2.1 and mouse H-2D^d by testing their capacity to stabilize the respective MHC class I protein complexes on the surface of mutant cell lines T2 and RMA-S transfected with D^d gene. The experimental tests allowed us to define individual residues involved in antibody and MHC-protein interaction, respectively. In a further approach, we used those results to design interaction models with HLA-A2.1 and H-2D^d. Therefore, a structural model for H-2D^d was built that exhibits an overall similar conformation to the parental crystal structure of HLA-A2.1. The resulting interaction models show V3-peptide bound in an extended β-conformation with a bulge in its centre for both H-2D^d and HLA-A2.1 complexes. The N- and C-termini of V3 peptide reside in conserved pockets within both MHC-proteins. Anchoring residues could be determined that are crucial for the binding of the respective MHC class I haplotype. The cross-reactivity of V3-peptide in enhancing the expression of two different MHC class I molecules (H-2D^d and HLA-A2.1) is shown to be based on similar peptide binding that induces an almost identical peptide conformation.     

Structural characterization of antigens encoded by rabbitRLA-11 histocompatibility genes

Products of rabbitRLA-11 histocompatibility genes were isolated from rabbit lymphoid tumor cells (RL-5) and characterized. The tumor cells were grown in culture with radioactive amino acids and were lysed by treatment with the detergent NP-40. Glycoprotein molecules were isolated by affinity chromatography using immobilized lentil lectin. Chromatography on purified sheep anti-rabbit beta-2-microglobulin yielded a 43,000 dalton glycoprotein fraction, designated RLA-11gp, which was noncovalently associated with beta-2-microglobulin. N-terminal amino acid sequence analysis using radiochemical methods allowed assignment of 28 of the N-terminal 35 residues. The data revealed 89% homology of the RLA-11gp N-terminus with that of the human HLA-B7 and 82% with the mouse H-2K^b histocompatibility antigens. Comparison of the RLA-11 gp N-terminal sequence data obtained in this work to sequence data reported for major histocompatibility complex antigens of other species revealed no amino acid substitutions unique to the rabbit antigens.     

Mass spectrometric analysis of rabbit and bovine trypsin-solubilized cytochrome b5

The sequence and blocking group of the amino-terminal 15 amino acids of rabbit trypsin-solubilized cytochrome b₅ were determined by liquid secondary ion mass spectrometry (LSIMS) and tandem mass spectrometry (MS/MS). The molecular weights of peptides generated from aStaphylococcus aureus V8 protease digest of this protein were determined by LSIMS analysis and the two peptides containing the blocked amino-terminus were sequenced by tandem mass spectrometry to yield the sequence; N-acetyl-Ala-Ala-Glu-Ser-Asp-Lys-Asp-Val-Lys-Tyr-Tyr-Thr-Leu-Glu-Glu. Comparison of this sequence with a recently reported cDNA sequence (Dariushet al., 1988) indicates that Gln at position 3 is selectively deamidated, although no other discrepancies were found. Intact rabbit and bovine trypsin-solubilized cytochrome b₅ were also analyzed by LSIMS on a high-field mass spectrometer equipped with a diode array detector. Mass measurement of the unresolved protonated molecular ion peak tops gave average molecular weights of 9462.2±2 and 9502.3±2 for bovine and rabbit trypsin-solubilized cytochrome b₅, respectively. In both cases, these molecular weights correspond to a cytochrome b₅ fragment consisting of amino acids Asp(7)-Arg(88). The average molecular weight for the rabbit amino-terminal-blocked form of trypsin-solubilized cytochrome b₅ was found to be 10,144.5±2, which was consistent with the molecular weight predicted for the extended N-acetylated form (residues 1–88) of M_r 10,146.1.     

Characterization of a weak allele of the GL1 gene of Arabidopsis thaliana

A genomic clone containing the gl1–2 allele has been isolated and sequenced. The predicted amino acid sequence of the gl1–2 protein is identical to that of the GL1-Col allele up to position 201. At this point in the coding region of gl1–2 there is a deletion relative to the wild-type sequence that results in an in-frame stop codon at position 202. This deletion removes 27 amino acid residues, including a highly negatively charged region, from the predicted gl1–2 polypeptide. The loss of this negatively charged carboxy-terminal region from the gl1–2 product is most likely the cause of the partial loss of gene activity which results in a reduction in leaf trichome initiation.     

Evolutionary conservation of protein regions in the protonmotive cytochromeb and their possible roles in redox catalysis

Summary The amino acid sequences of the protonmotive cytochromeb from seven representative and phylogenetically diverse species have been compared to identify protein regions or segments that are conserved during evolution. The sequences analyzed included both prokaryotic and eukaryotic examples as well as mitochondrial cytochromeb and chloroplastb ₆ proteins. The principal conclusion from these analyses is that there are five protein regions-each comprising about 20 amino acid residues—that are consistently conserved during evolution. These domains are evident despite the low density of invariant residues. The two most highly conserved regions, spanning approximately consensus residues 130–150 and 270–290, are located in extramembrane loops and are hypothesized to constitute part of the Q_o reaction center. The intramembrane, hydrophobic protein regions containing the heme-ligating histidines are also conserved during evolution. It was found, however, that the conservation of the protein segments extramembrane to the histidine residues ligating the low potential b566 heme group showed a higher degree of sequence conservation. The location of these conserved regions suggests that these extramembrane segments are also involved in forming the Q_o reaction center. A protein segment putatively constituting a portion of the Q_i reaction center, located approximately in the region spanned by consensus residues 20–40, is conserved in species as divergent as mouse andRhodobacter. This region of the protein shows substantially less sequence conservation in the chloroplast cytochromeb ₆. The catalytic role of these conserved regions is strongly supported by locations of residues that are altered in mutants resistant to inhibitors of cytochromeb electron transport.     

Positive selection of Tcrb-V10b+ T cells

The Tcrb-V10b⁺ T cell population has been examined with a newly established antibody, KT10b, specific for Tcrb-V10b but not Tcrb-V10a. H-2E⁺ mice have higher levels of Tcrb-V10b⁺ T cells (4.3%–11.%) than H-2E^– mice (2.2%–4.9%). This difference appears to be determined by levels of Tcrb-V10b⁺ T cells in the CD4 population. F₁ mice between H-2E⁺ and H-2E^– mice dominantly express higher levels of Tcrb-V10b⁺ T cells. [NOD (E–) x (NOD x A (E⁺))F₁] backcross mice show positive selection of Tcrb-V10b⁺ CD4⁺ T cells by H-2E. On the other hand other backcross analyses reveal positive selection of Tcrb-V10b⁺ CD8⁺ T cells by certain major histocompatibility class I molecules. Involvement of non-H-2 antigens in these positive selections remain to be determined. Address correspondence and offprint requests to: K. Tomonari.     

Covalent Structure of Botulinum Neurotoxin Type B; Location of Sulfhydryl Groups and Disulfide Bridge and Identification of C-Termini of Light and Heavy Chains

Botulinum neurotoxin (NT) serotype B, produced by Clostridium botulinum (proteolytic strain), is a 150-kDa single-chain polypeptide of 1291 amino acids, of which 10 are Cys residues [Whelan et al. (1992), Appl. Environ. Microbiol. 58, 2345–2354] The posttranslational modifications of the gene product were found to consist of excision of only the initiating Met residue, limited proteolysis (nicking) of the 1290-residue-long protein between Lys 440 and Ala 441, and formation of at least one disulflde bridge. The dichain (nicked) protein, in a mixture with the precursor single-chain (unnicked) molecules, was found to have a 50-kDa light chain (Pro 1 through Lys 440) and a 100-kDa heavy chain (Ala 441 through Glu 1290). The limited in vivo nicking of the single-chain NT to the dichain form, by protease endogenous to the bacteria, and the nonfacile in vitro cleavage by trypsin of the Lys 440–Ala 441 bond appear to be due to the adjacent Ala 441–Pro 442 imide bond's probable cis configuration in a mixed population of molecules with cis and trans configurations. The two chains were found connected by an interchain disulfide formed by Cys 436 and Cys 445. Six other Cys residues, at positions 70, 195, 308, 777, 954, and 1277, were found in sulfhydryl form. In addition, a Cys at position 1220 or 1257 appeared to be in sulfhydryl form, hence our experimental results could not unambiguously identify presence of an intrachain disulfide bridge near the C-terminus of the NT. A total of 384 amino acid residues, including the 6 Cys residues at positions 70, 195, 308, 436, 445, and 1277, were identified by direct protein-chemical analysis; thus 29.7% of the protein's entire amino acid sequence predicted from the nucleotide sequence was confirmed. The 6 amino acids, residues 945–950, did not match with the sequence predicted in 1992, but did match with a later report of 1995. The above determinations were made by a combination of chemical (CNBr and acidic cleavage at Asp–Pro) and enzymatic (trypsin, clostripain, and pepsin) cleavages of the NT, and NT carboxymethylated with iodoacetamide (with or without ¹⁴C label), separation and isolation of the fragments by SDS–PAGE (followed by electroblotting onto PVDF membrane), and/or reversed-phase HPLC, and analyses of the fragments for the N-terminal amino acid sequences by Edman degradation and amino acid compositions.     

Purification, characterization, and relation to bikunin of rat urinary trypsin inhibitors

Two forms of urinary trypsin inhibitor (UTI-1 and UTI-2) were purified from pooled urine of normal male rats to apparent homogeneity by salting out, affinity chromatography, gel filtration, and reverse-phase HPLC. UTIs-1 and 2 were shown to be thermostable glycoproteins with the respective molecular weights of 22,000 and 18,000 estimated by SDS-PAGE. These inhibitors combined with bovine trypsin in a 1:1 molar ratio: the K _d values were 2.5 × 10^–10 and 2.3 × 10^–10 M, respectively. Amino acid composition and sequence analysis indicated that UTI-1 corresponded to rat bikunin of which the amino acid sequence was deduced from a rat liver cDNA clone encoding ₁-microglobulin [Lindqvist et al. (1992), Biochim. Biophys. Acta 1130, 63–67] except that the protein sequence seemed to lack C-terminal serine, and UTI-2 corresponded to UTI-1 lacking N-terminal 21 amino acid residues.     

Identification of critical amino acids involved in α1-β interaction in voltage-dependent Ca channels

In voltage-dependent Ca²⁺ channels, the α₁ and β subunits interact via two cytoplasmic regions defined as the Alpha Interaction Domain (AID) and Beta Interaction Domain (BID). Several novel amino acids for that interaction have now been mapped in both domains by point mutations. It was found that three of the nine amino acids in AID and four of the eight BID amino acids tested were essential for the interaction. Whereas the important AID amino acids were clustered around five residues, the important BID residues were more widely distributed within a larger 16 amino acid sequence. The affinity of the AID_A GST fusion protein for the four interacting β_1b BID mutants was not significantly altered compared with the wild-type β_1b despite the close localization of mutated residues to disruptive BID amino acids. Expression of these interactive β mutants with the full-length α_1A subunit only slightly modified the stimulation efficiency when compared with the wild-type β_1b subunit. Our data suggest that non-disruptive BID sequence alterations do not dramatically affect the β subunit-induced current stimulation.     

A barley cDNA clone encoding a type III chlorophyll a/b-binding polypeptide of the light-harvesting complex II

The nucleotide sequence of a leaf cDNA clone encoding a Type III chlorophyll a/b-binding (CAB) protein of light-harvesting complex II (LHCII) in barley is reported. Sequence comparisons and results from in vitro import into chloroplasts demonstrate that the cDNA clone encodes a functional transit peptide of 45 amino acid residues and a mature polypeptide of 223 residues with a predicted molecular mass of 24.3 kDa. After insertion into thylakoids, the mature protein is resistant to protease attack. Hybridization analysis using a gene-specific probe shows that the gene is expressed in dark-grown seedlings and that the amount of mRNA increases during illumination.     

Immunoselection of structural H-2Kb variants: Use of cloned cytolytic T cells to select for loss of a CTL-defined allodeterminant

Functional studies concerning the unique interaction between class I H-2 allodeterminants and cytolytic T lymphocyte (CTL) antigen receptors have benefitted from the development of H-2K^b mutant mouse strains and somatic H-2 variants selected with monoclonal antibody. Here, we describe the development of a novel approach to immunoselection of somatic H-2K^b variants employing a K^b-specific CTL clone as the negative selective agent. The rationale for this method is that the use of an alloreactive CTL clone as the selective agent should enable us to detect the emergence of structural K^b variants based on their loss of the relevant CTL-defined allodeterminant. Thus, these structural variants are well suited to an in-depth analysis of the functional relationship between H-2 antigens and receptor recognition by CTL. Using this approach, we successfully isolated two types of structural K^b variants, as well as numerous Kb-loss variants. The functional studies described in this paper indicate that these structural variants exhibit alterations in expression of both CTL-defined and serologically defined H-2K^b allodeterminants. The structural characterization of such variants should enable us to identify the precise amino acid residues responsible for the creation of the relevant CTL-defined K^b allodeterminants.