Structure of the apical membrane antigen I (AMA-1) of Plasmodium chabaudi.
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Amino Acid Sequence↗
Biomedical subjects
A M Lew
Publications and source records attributed to A M Lew.
A protective monoclonal antibody recognizes a linear epitope in the precursor to the major merozoite antigens of Plasmodium chabaudi adami.
The monoclonal antibody 5C10/66 was shown to afford strong protection in mice against fulminating Plasmodium chabaudi adami infection. This was remarkable, as immunity to this organism is regarded to be mainly T-cell mediated. This antibody identified a 250-kDa molecule in schizonts and an 83-kDa fragment in merozoites. A cDNA clone selected by 5C10/66 was the homologue of the Plasmodium falciparum precursor to the major merozoite surface antigen (PMMSA). Comparison with the P. falciparum sequence showed that the P. chabaudi adami clone encoded the middle portion of the gene and that it can also be divided into variable and conserved blocks. Screening of a set of all possible octamer peptides predicted by the cDNA clone revealed that the core epitope of 5C10/66 was Glu-Thr-Thr-Glu-Thr. This region resides in a variable block of PMMSA.
Amino Acid Sequence↗
Antifolate drug selection results in duplication and rearrangement of chromosome 7 in Plasmodium chabaudi.
We selected lines of Plasmodium chabaudi that are resistant to high levels of the antifolate drug pyrimethamine and have shown that rearrangement and duplication of a portion of chromosome 7 has occurred in the resistant lines. This chromosomal duplication results in an increase in the chromosome number from 14 to 15: two derived chromosomes (450 kilobases and 1.1 megabases) were smaller than the original chromosome 7 (1.3 megabases), so that essentially only a 200-kilobase region was duplicated. This region contained the DHFR-TS gene and the closely linked Hsp70 gene. We have macrorestriction mapped chromosome 7 from the pyrimethamine-susceptible line (DS) and also the duplicated chromosome 7s in the resistant line. From these maps, we have proposed a process for the karyotype changes. Sequencing of the DHFR gene from the parent and derived chromosomes showed that there were no mutations in the coding sequence. As a result of the duplication of the DHFR-TS gene, there is at least a twofold increase in expression of the DHFR-TS gene, and this may explain the ability of the pyrimethamine-resistant lines to grow in increased amounts of the drug.
Amino Acid Sequence↗
Thymus-dependent and thymus-independent developmental pathways for peripheral T cell receptor-gamma delta-bearing lymphocytes.
To elucidate the developmental pathways of T cells that bear TCR gamma delta, we have analyzed the kinetics of expression and biochemical characteristics of gamma delta receptors in the thymus and spleen of normal and athymic (nude) mice, as well as nude mice engrafted with neonatal thymuses. TCR gamma delta-bearing thymocytes and splenocytes have a CD4-8- phenotype, and both populations express products of the C gamma 1 locus. TCR gamma delta-bearing cells develop in the thymus before their appearance in the spleen. Young nude mice have no detectable TCR gamma delta-bearing cells in their spleens. When young nude mice are given thymus grafts, TCR gamma delta-bearing cells of host origin first develop in the engrafted thymus, followed by their appearance in the spleen. In the absence of a thymus graft, the spleens of old nude mice eventually develop small numbers of TCR gamma delta + cells, as well as TCR alpha beta + cells. These results demonstrate that there is a major thymic-dependent pathway for TCR gamma delta expression, as well as a minor thymic-independent pathway seen in older nude mice. The development of TCR gamma delta + cells in the thymus before their appearance in the spleen, both in normal ontogeny as well as in the thymus-engrafted nude mouse model, suggests that thymic TCR gamma delta + cells are precursors of the thymus-dependent population of peripheral TCR gamma delta + cells.
Aging↗
The biosynthesis and assembly of T cell receptor alpha- and beta-chains with the CD3 complex.
The biosynthesis, processing, and assembly of the TCR alpha- and beta-chains with each other and with the CD3 complex were investigated on both cell surface positive (TCR+CD3-) and negative (TCR-CD3-) cell lines. The results indicate that 1) in cell surface TCR-CD3- cell lines (MOLT 3, CCRF-CEM), TCR-beta, but not alpha-chains are present intracellularly. TCR-beta-CD3 complexes are readily found in these cell lines, but no evidence for final processing or cell surface expression of such incomplete TCR-CD3 complexes is observed. 2) In the cell surface TCR+CD3+ cell line HPB-ALL, both alpha- and beta-chains are present intracellularly. Whereas non-glycosylated forms of TCR-beta chain can be detected, only more mature forms of TCR alpha-chains are detected indicating that the alpha-chains are more rapidly glycosylated than the beta-chains. 3) The large majority of the intracellular alpha- and beta-chains is not disulfide linked and a small fraction of these is associated with CD3. 4) Only small amounts of the total intracellular TCR chains are found as CD3-associated disulfide-linked alpha beta-heterodimers. 5) Final processing of TCR chains for cell surface expression takes place after formation of these TCR-alpha beta-CD3 complexes. Thus, both the TCR alpha- and beta-chains are over-produced and only relatively small amounts of these chains form CD3-associated heterodimers that are processed for cell surface expression. Analogous results were obtained with a non-leukemic CTL clone. Based on these observations, a model for the biosynthesis and assembly of the TCR-CD3 complex is presented.
Antigens, Differentiation, T-Lymphocyte↗
Signals controlling alternative splicing of major histocompatibility complex H-2 class I pre-mRNA.
The use of alternative splice acceptor sites during the removal of intron 7 in pre-mRNA splicing produces two forms of H-2Kb protein: the predominant form, derived from a transcript that has spliced at the upstream splice acceptor site for exon 8 (long exon 8), and a Kb molecule derived from a transcript that has spliced at the downstream acceptor site for exon 8 (short exon 8). We have identified a potential lariat branch point adenosine for the upstream acceptor splice site. This adenosine is found 28 bp from the splice junction and is contained in the sequence AGTGATGG. D-region genes, which use only the downstream splice site, have the sequence AGTGGTGG. We have used in vitro mutagenesis to change this A of the H-2Kb gene to G and have made the reciprocal change in H-2Dd. Elimination of this adenosine in H-2Kb alters the pattern of pre-mRNA splicing and results in a predominance of the Kb molecules with short exon 8 encoded sequences. However, the addition of an adenosine in H-2Dd is not sufficient to direct splicing to the upstream site.
Animals↗
H-2K molecules have two different C-termini, one of which is K-region specific.
Amino acid sequences encoded by exon 8 of the H-2K and H-2D/L genes appear to be locus specific. The majority of H-2Kb molecules contain 10 amino acids that are derived from exon 8. In contrast, the H-2Db, -Dd and -Ld molecules contain only one amino acid which is encoded by exon 8, even though the genetic information exists to encode 10 amino acids analogous to those encoded by the majority of H-2Kb transcripts. We have produced a rabbit anti-peptide serum reactive with the exon 8 encoded sequence of H-2Kb (alpha K-C) that specifically immunoprecipitates a molecule of 45 K mol. wt from spleen cell lysates of b, d, p, q and k haplotype mice. Further analysis by Western blots indicated that virtually all mouse strains express a 45 K protein reactive with alpha K-C. In sequential immunoprecipitations of spleen cell lysates from b, q, p and d haplotype mice using alpha K-C followed by H-2K or H-2D private specificity alloantisera, the anti-peptide serum removed nearly all of the molecules reactive with the anti-H-2K alloantisera (except in the k haplotype) and none of the molecules reactive with the anti-H-2D serum. In addition, no D-region molecules possessing a long C-terminal sequence were detected with an antiserum directed against a representative D-region long C-terminal peptide. We conclude that even though the genetic information for an extended exon 8 exists in K, D and L locus genes, apparently only K-region molecules are expressed with such a C-terminus. Furthermore, in most haplotypes the great majority of H-2K molecules are produced using long exon 8; however, H-2Kk is produced mostly from short exon 8. The absence or presence of key adenosine residues is predicted to be responsible for the variability in class I exon 8 splicing.
Animals↗
Comparison of antibody avidity and titre elicited by peptide as a protein conjugate or as expressed in vaccinia.
The immunogenicity of a malaria peptide presented in various forms was tested in terms of the quality and quantity of the antibody response in rabbits. Peptide conjugated to a protein carrier, diphtheria toxoid (DT), required strong adjuvants (e.g. muramyl dipeptide, MDP and Freund's adjuvant, FCA) to elicit high levels of antibody with high avidity. Alum was a poor adjuvant, producing the lowest titre and avidity of antibody compared with all the other groups. Peptide expressed in vaccinia (and without carrier) produced intermediate levels of antibody but the avidity of the antibodies produced were comparable to that produced by peptide conjugates given with muramyl dipeptide.
Acetylmuramyl-Alanyl-Isoglutamine↗
Expression of the human T cell receptor as defined by anti-isotypic antibodies.
Anti-isotypic reagents against the human T cell receptor (TcR) were made by immunizing rabbits with peptides which corresponded to sites within the constant region of the alpha- and beta-chains. These antibodies were shown to immunoprecipitate a heterodimer of 80,000 to 90,000 m.w. that could be reduced to chains of 44,000 to 50,000 and 37,000 to 40,000 m.w. In addition, an anti-peptide serum against CD3 delta-chain was made. The anti-alpha peptide serum reacted with all human TcR (from phytohemagglutinin-stimulated lymphocytes, cytotoxic T cell clones, and the T cell leukemias: HPB-ALL, Jurkat, JA3, and JM), and the anti-beta peptide serum reacted with only human TcR of the C beta 2 isotype (from a cytotoxic T cell clone which had a C beta 2 transcript, HPB-ALL, and a proportion of phytohemagglutinin-stimulated lymphocytes, but not with Jurkat, JA3, and JM). A comparison of the detergents NP-40 and digitonin revealed that digitonin was more efficient at keeping the TcR/CD3 complex intact, but was less efficient at solubilizing the total amount of TcR or the total amount of CD3. With these reagents and the use of digitonin, it was shown that all of the alpha, beta, and CD3 moieties on the surface of a T cell leukemia HPB-ALL occur as a bound TcR/CD3 complex. The proportion of C beta 1 to C beta 2 isotype expressed on the surface of phytohemagglutinin-stimulated peripheral blood lymphocytes was 0.8, indicating approximately equal use of the two beta-chain isotypes.
Animals↗
Analysis of T-cell receptor gamma chain expression in the thymus.
While much is known about the structure and function of the T-cell receptor (TCR) alpha beta heterodimer, information has just begun to emerge about the gamma protein, the product of a third rearranging T-cell receptor gene. We describe the use of antiserum to a carboxy-terminal peptide common to several of the murine gamma chain constant regions and a monoclonal antibody to the murine T3 complex to identify products of this T-cell receptor gene family in a subpopulation of Lyt2-, L3T4- thymocytes. The gamma chain has a molecular weight of 35kD, is disulfide bonded to a 45kD partner termed delta (delta) and is associated with the T3 complex. The cells that bear this second T-cell receptor appear to represent a distinct lineage differentiating within the thymus.
Animals↗
Multiple class I molecules generated from single genes by alternative splicing of pre-mRNAs.
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Animals↗
Thy-1+ dendritic epidermal cells express T3 antigen and the T-cell receptor gamma chain.
The murine epidermis is a heterogeneous epithelium composed of keratinocytes, melanocytes, Langerhans cells, and a recently described subpopulation (2-3%) of bone-marrow-derived leukocytes with a dendritic morphology and the cell surface phenotype Thy-1+, L3T4-, Lyt-2-. Previous studies have demonstrated that cell lines derived from freshly explanted Thy-1+ dendritic epidermal cells (DEC) have abundant mRNA for rearranged T-cell receptor (TCR) gamma-chain genes. Analysis of Thy-1+ DEC in situ, freshly isolated cell suspensions of Thy-1+ DEC, and long-term Thy-1+ DEC lines demonstrated that 100% of the Thy-1+ DEC reacted with a monoclonal antibody to the epsilon chain of the murine T3 complex and that 40-60% of resident Thy-1+ DEC were also reactive with an antiserum to the TCR gamma chain. Two Thy-1+ DEC lines expressed a disulfide-linked 70-kDa molecule that could be precipitated with an anti-gamma-chain antiserum and could be coprecipitated with an antiserum to the T3 delta chain; the molecule appeared as a single 34-kDa band under reducing conditions. The phenotype of Thy-1+ DEC (T3+, L3T4-, Lyt-2-, TCR gamma chain+) thus resembles that of the recently described subpopulation of murine and human lymphocytes that have been identified in the thymus, peripheral blood, and fetal blood.
Animals↗
Evidence of a second polymorphic ELA class I (ELA-B) locus and gene order for three loci of the equine major histocompatibility complex.
Two antisera, B-442 and R-2046, were produced by immunizing offspring with purified peripheral blood lymphocytes from a parent matched for the ELA-A specificity carried on the unshared haplotype. Absorption analysis demonstrated that these antisera contained at least two families of cytotoxic antibodies, one directed against antigens present on T and B cells, and a second directed preferentially against antigens present on surface Ig positive cells. Immunoprecipitation studies using these antisera demonstrated that both antisera contain antibodies specific for glycoproteins with molecular weights characteristic of class I and class II MHC antigens. In lymphocyte typing tests of unfractionated lymphocytes, only the class I activity was readily detectable since the class II activity killed less than 25% of the cells. Family studies demonstrated that these antisera recognize products of genes linked to the ELA system. Based on two recombinants in an extended family it became apparent that the specificities detected by B-442 and R-2046 are not products of the ELA-A locus, but rather they are products of at least one other locus, defined in this paper as ELA-B. In this family a third recombinant was found between the A blood group system and the ELA-A locus. Based on these three recombinants, the most probable linear relationship of the following genes is: A blood group system/ELA-A/ELA-B.
Animals↗
Characterization of T cell receptor gamma chain expression in a subset of murine thymocytes.
While much information exists about the structure and function of the clonally distributed T cell receptor (TCR) alpha beta heterodimer, little is known about the gamma protein, the product of a third rearranging TCR gene. An antiserum to a carboxyl-terminal peptide common to several of the murine gamma chain constant regions and a monoclonal antibody to the murine T3 complex were used to identify products of this TCR gene family in a subpopulation of Lyt2-, L3T4- thymocytes. This subpopulation does not express TCR alpha or full-length TCR beta messenger RNA. The gamma chain is a 35-kilodalton (kD) protein that is disulfide-bonded to a 45-kD partner and is associated with the T3 complex. Analysis of the glycosylation pattern of this thymic gamma chain revealed that the major variable region gamma (V gamma) gene transcribed in activated peripheral T cells is absent from this subpopulation. The cells that bear this second T cell receptor may therefore represent a distinct lineage differentiating within the thymus.
Amino Acid Sequence↗
Nucleotide sequence and comparative analysis of H-2D class 1 H-2 gene.
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Animals↗
The gene encoding the equine soluble class I molecule is linked to the horse MHC.
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Animals↗
A soluble class I molecule analogous to mouse Q10 in the horse and related species.
Horse serum is shown to contain a soluble class I molecule analogous to the secreted Q10 molecule in the mouse. This molecule has several similarities to the recently described mouse Q10 molecule: it is smaller than membrane-bound equine class I molecules; it occurs in a high molecular mass complex of 200-300 kd in serum; and the serum levels of the equine molecule are similar to that of the Q10 molecule (about 30 micrograms/ml). A soluble molecule is also detected in the sera of species related to the horse; it has in fact been found in all the wild members of the order Perissodactyla so far tested. However, it was not detected in the serum of members of the orders Carnivora, Sirenia, Proboscidea, Artiodactyla, and Primates that were tested, nor in the serum of members of the order Rodentia other than in that of the genus Mus.
Animals↗
Alternative protein products with different carboxyl termini from a single class I gene, H-2Kb.
In addition to mRNA encoding the canonical form of the murine class I antigen H-2Kb (348 amino acid residues), mRNA that would encode a shortened form of H-2Kb (missing 9 amino acids from the C-terminus) has been identified in C57BL/6 spleen cells by RNase-protection studies. The alternative transcripts of H-2Kb arise through the use of different AG acceptor splice sites for exon VIII. The existence of a shortened H-2Kb protein was demonstrated by sequential immunoprecipitation. Lysates of spleen cells that had been labeled with [35S]methionine and [3H]histidine were precleared with rabbit anti-peptide serum reactive with the C-terminus of the canonical H-2Kb. The shortened form of H-2Kb was immunoprecipitated from this lysate with H-2Kb alloantiserum. Both forms of H-2Kb were isolated by NaDodSO4/PAGE. Tryptic peptide mapping confirmed that these molecules differed only at their C-terminus. The shortened form of H-2Kb is also found in a B-cell line (R8) but not in three cloned T-cell lines or in a T-cell lymphoma (EL4), suggesting that regulatory events are involved in producing the two forms of H-2Kb. Putative lariat branch points involved in these alternative splicing events for the 3' coding region of H-2 class I pre-mRNAs are proposed.
Animals↗