RESUMO
Immunization of mice with tumors genetically engineered to express the B7 costimulatory molecules amplifies the antitumor immune response mediated by CD8+ cytolytic T lymphocytes (CTL). In this report, we examined the effect of B7-CD28 costimulation on the hierarchy of tumor epitopes. Using a combination of affinity chromatography/reversed-phase high performance liquid chromatography and CTL cloning, we show that major histocompatibility complex (MHC) class I molecules from EL4 lymphoma cells can present at least six distinct CTL epitopes presented by MHC class I molecules. Nevertheless, mice immunized with wild-type B7-negative EL4 cells develop CTL only to one immunodominant epitope. In contrast, immunization with B7-transduced EL4 cells led to not only the amplification of the CTL response to this immunodominant epitope, but also to the recognition of five otherwise silent subdominant epitopes. The adoptive transfer of a CTL clone against such a subdominant epitope cured mice bearing EL4 lymphoma growing as an ascites tumor. The fact that CTL response can be spread to normally silent epitopes as a result of B7-CD28 costimulation suggests a novel approach to manipulate the hierarchy of CTL epitopes and offers an opportunity to explore novel targets for T cell-mediated cancer therapy.
Assuntos
Antígenos de Neoplasias/imunologia , Antígeno B7-1/imunologia , Antígenos CD28/imunologia , Citotoxicidade Imunológica , Epitopos/análise , Antígenos de Histocompatibilidade Classe I/imunologia , Linfoma/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Linhagem Celular , Epitopos/imunologia , Feminino , Imunoterapia Adotiva , Cinética , Linfoma/patologia , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Antibody-blocking studies have demonstrated the role of CD6 in thymocyte-thymic epithelial (TE) cell adhesion. Here we report that CD6 expressed by COS cells mediates adhesion to TE cells and that this interaction is specifically blocked with an anti-CD6 monoclonal antibody (mAb) or with a mAb (J4-81) that recognized a TE cell antigen. We isolated and expressed a cDNA clone encoding this antigen and show that COS cells transfected with this cDNA bind a CD6 immunoglobulin fusion protein (CD6-Rg). This antigen, which we named ALCAM (activated leukocyte-cell adhesion molecule) because of its expression on activated leukocytes, appears to be the human homologue of the chicken neural adhesion molecule BEN/SC-1/DM-GRASP. The gene was mapped to human chromosome 3q13.1-q13.2 by fluorescence in situ hybridization of cDNA probes to metaphase chromosomes. We prepared an ALCAM-Rg fusion protein and showed that it binds to COS cell transfectants expressing CD6, demonstrating that ALCAM is a CD6 ligand. The observations that ALCAM is also expressed by activated leukocytes and that both ALCAM and CD6 are expressed in the brain suggest that ALCAM-CD6 interactions may play a role in the binding of T and B cells to activated leukocytes, as well as in interactions between cells of the nervous system.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Cromossomos Humanos Par 3 , Glicoproteínas/biossíntese , Molécula de Adesão de Leucócito Ativado , Sequência de Aminoácidos , Animais , Adesão Celular , Moléculas de Adesão Celular/biossíntese , Linhagem Celular , Chlorocebus aethiops , Bandeamento Cromossômico , Mapeamento Cromossômico , Sequência Consenso , Células Epiteliais , Epitélio/fisiologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Cariotipagem , Ligantes , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Homologia de Sequência de Aminoácidos , Linfócitos T/citologia , Linfócitos T/imunologia , Timo/citologia , Timo/fisiologia , Transfecção , Células Tumorais CultivadasRESUMO
The transforming growth factor-beta 1 (TGF beta 1) and -beta 2 (414) precursors both contain three predicted sites of N-linked glycosylation within their pro regions. These are located at amino acid residues 72, 140, and 241 for the TGF beta 2 (414) precursor and at residues 82, 136, and 176 for the TGF beta 1 precursor; both proteins contain mannose-6-phosphate (M-6-P) residues. The major sites of M-6-P addition are at Asn (82) and Asn (136), the first two sites of glycosylation, for the TGF beta 1 precursor. We now show that the major site of M-6-P addition within the TGF beta 2 (414) precursor is at Asn241, the third glycosylation site. To determine the importance of N-linked glycosylation to the secretion of TGF beta 1 and -beta 2, site-directed mutagenesis was used to change the Asn residues to Ser residues; the resulting DNAs were transfected into COS cells, and their supernatants were assayed for TGF beta activity. Substitution of Asn (241) of the TGF beta 2 (414) precursor resulted in an 82% decrease in secreted TGF beta 2 bioactivity. Mutation at Asn72 resulted in a 44% decrease, while mutation at Asn140 was without effect. Elimination of all three glycosylation sites resulted in undetectable levels of TGF beta 2. These results were compared with similar mutations made in the cDNA encoding the TGF beta 1 precursor. Mutagenesis of the two M-6-P-containing sites (Asn82 and Asn136) resulted in an 83% decrease in secreted TGF beta 1; replacement of Asn82 and Asn136 with Ser individually resulted in 85% and 42% decreases in activity, respectively. Substitution of Asn176 with Ser was without effect, while substitution of all three sites of glycosylation resulted in undetectable levels of TGF beta 1 activity, similar to the results obtained with TGF beta 2. The nine Cys residues within the mature region of TGF beta 1 were mutated to serine, and their effects on TGF beta 1 secretion were evaluated. Mutation of most Cys residues resulted in undetectable levels of TGF beta 1 protein or activity in conditioned medium. Mutation of Cys (355) led to the secretion of inactive TGF beta 1 monomers, suggesting that this residue is either directly involved in dimer formation or required for correct interchain disulfide bond formation.
Assuntos
Cisteína , Mutagênese Sítio-Dirigida , Precursores de Proteínas/genética , Proteínas Recombinantes/biossíntese , Fator de Crescimento Transformador beta/genética , Sequência de Aminoácidos , Animais , Células CHO , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Glicosilação , Manosefosfatos/análise , Proteínas Recombinantes/farmacologia , Mapeamento por Restrição , Transfecção , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Kedarcidin, a new chromoprotein antitumor antibiotic, was isolated from the culture broth of a novel actinomycete strain L585-6 (ATCC 53650). The antibiotic was recovered from the culture filtrate by adsorption to QAE ion exchanger and purified by successive application of gel filtration and ion exchange chromatography with Sephadex G-50 and DEAE-Sephadex, respectively. Kedarcidin is an acidic complex (pI 3.65) with an apparent molecular weight of 12,400. The complex consists of a highly unstable, solvent extractable chromophore and a water soluble peptide. The apoprotein is a single chain polypeptide of 114 residues.
Assuntos
Antibacterianos , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/isolamento & purificação , Peptídeos , Actinomycetales/metabolismo , Alcinos/química , Alcinos/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Cromatografia Líquida de Alta Pressão , Cicloparafinas/química , Cicloparafinas/isolamento & purificação , Enedi-Inos , Peptídeos e Proteínas de Sinalização Intercelular , Focalização Isoelétrica , Dados de Sequência Molecular , Naftalenos/química , Naftalenos/isolamento & purificação , Proteínas/química , Proteínas/isolamento & purificaçãoRESUMO
Three cysteine residues are located in the pro region of the transforming growth factor beta 1 (TGF-beta 1) precursor at amino acid positions 33, 223, and 225. Previous studies (Gentry, L. E., Lioubin, M. N., Purchio, A. F., and Marquardt, H. (1988) Mol. Cell. Biol. 8, 4162-4168) with purified recombinant TGF-beta 1 (rTGF-beta 1) precursor produced by Chinese hamster ovary (CHO) cells revealed that Cys-33 can form a disulfide bond with at least 1 cysteine residue in mature TGF-beta 1, contributing to the formation of a 90-110-kDa protein. We now show that Cys-223 and Cys-225 form interchain disulfide bonds. Site-directed mutagenesis was used to change these Cys codons to Ser codons, and mutant constructs were transfected into COS cells. Analysis of recombinant proteins by immunoblotting showed that by substituting Cys-33 the 90-110-kDa protein is not formed, and thus, more mature dimer (24 kDa) is obtained, corresponding to a 3- to 5-fold increase in biological activity. Substitution of Cys-223 and/or Cys-225 resulted in near wild-type levels of mature TGF-beta 1. Furthermore, cells transfected with plasmid coding for Ser at positions 223 and 225 expressed only monomeric precursor proteins and released bioactive TGF-beta 1 that did not require acid activation, suggesting that dimerization of the precursor pro region may be necessary for latency.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Cisteína , DNA de Cadeia Simples/isolamento & purificação , Mutação , Precursores de Proteínas , Proteínas/genética , RNA Bacteriano/isolamento & purificação , Fator de Crescimento Transformador beta , Fatores de Crescimento Transformadores/genética , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Linhagem Celular , Códon/genética , DNA de Cadeia Simples/metabolismo , Genes , Dados de Sequência Molecular , Biossíntese de Proteínas , Proteínas/isolamento & purificação , RNA Bacteriano/metabolismo , TransfecçãoRESUMO
The class Ib antigen HLA-G is expressed as a membrane-bound protein like classical class Ia molecules (M.HLA-G) but, unlike typical class I, is also expressed as a soluble protein (S.HLA-G) with a unique C terminus. Our results show that, similar to classical class I proteins, the membrane-bound form of HLA-G associated with TAP, as evidenced by the ability to immunoprecipitate HLA-G class I heavy chain with TAP antisera. In contrast, the soluble G protein did not appear to associate with TAP in the same manner, since similar immunoprecipitation experiments failed to detect soluble G complex. A detailed analysis of peptides bound to the soluble and membrane HLA-G proteins expressed in the B lymphoblastoid cell line 721.221 showed that, like class Ia complexes, both HLA-G proteins consist of heavy and light chains complexed with nonameric peptides in a 1:1:1 ratio. The two proteins bind essentially the same set of peptides, which are derived from a variety of intracellular proteins and define a peptide motif for HLA-G. The peptides contain Leu at the C terminus and Pro or small hydrophobic amino acids in position 3 followed by Pro or Gly in position 4. The complexity of the bound peptides is lower than that found for some class Ia complexes, but is more similar to class Ia than to the limited repertoire of some murine class Ib molecules.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antígenos HLA/química , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Transportadores de Cassetes de Ligação de ATP/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Linhagem Celular , Antígenos HLA/imunologia , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica/fisiologia , TransfecçãoRESUMO
Hsc70, the constitutive form of the heat shock protein 70 family of proteins, is involved in a number of biological activities which include protein folding and molecular chaperoning. Previously, we had shown that the immunosuppressant 15-deoxyspergualin (DSG) specifically interacted with Hsc70, as well as the Hsp90 family of proteins. Although the exact binding site on Hsc70 for protein substrates is unknown, a recent study shows that the extreme C-terminal four amino acids 647EEVD650 play a role in regulating AT-Pase activity, substrate binding, and interaction with HDJ-1. These four amino acids are also found at the C-terminus of Hsp90 and may be involved in similar functions. In this study, we show that DSG binds specifically to this EEVD regulatory domain. Binding of DSG to Hsc70 did not affect its ability to bind peptides. These results suggest that in addition to the ATP binding domain, there are two additional substrate binding domains on Hsc70. DSG should provide a tool for understanding the role of the EEVD motif in biological processes.
Assuntos
Guanidinas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Imunossupressores/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/efeitos dos fármacos , Ligação Competitiva , Bovinos , Reagentes de Ligações Cruzadas/metabolismo , Etildimetilaminopropil Carbodi-Imida/metabolismo , Guanidinas/farmacologia , Imunossupressores/farmacologia , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica/efeitos dos fármacosRESUMO
A murine monoclonal antibody, ME20, with high selectivity for melanomas, has been utilized to isolate a unique membrane-bound (designated ME20-M) and secreted (designated ME20-S) antigen from H3606 human melanoma cells. ME20-M was purified from the cell lysate and ME20-S from the conditioned medium of H3606 cells by immunoaffinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weights were 105,000 and 76,000, respectively. Analyses of ME20-M and ME20-S by amino acid sequencing identified the processing sites. Signal peptide cleavage occurs at Thr-24 of pro-ME20 antigen, yielding ME20-M (25 to 661). In addition, proteolytic processing of the precursor at Val-467 yields ME20-S (25 to 467). We report the characterization of Asn-linked glycosylation sites in ME20-M and ME20-S to determine the involvement of oligosaccharides in the proteolytic processing of pro-ME20 antigen. Tryptic peptide maps of ME20-M and ME20-S were prepared and the glycosylation sites identified by sequence analyses. Oligosaccharides were enzymatically released and characterized by high-performance anion-exchange chromatography. We found high-mannose-type structures at Asn-57, Asn-82, and Asn-87 of ME20-M, whereas ME20-S contained 73% complex-type and 27% high-mannose-type oligosaccharides at the same sites. To assess the role of oligosaccharides in the processing of the ME20 antigen, we tested the effect of the oligosaccharide processing modifier deoxymannojirimycin, a compound that inhibits synthesis of hybrid- and complex-type oligosaccharides. Deoxymannojirimycin had no effect on the synthesis and relative rate of synthesis of ME20-M, but markedly reduced the synthesis of ME20-S without affecting the rate of secretion. The reported results suggest that carbohydrate maturation of the ME20 antigen may be important for processing and secretion.
Assuntos
Antígenos de Neoplasias/metabolismo , Melanoma/imunologia , 1-Desoxinojirimicina/farmacologia , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/isolamento & purificação , Asparagina/metabolismo , Sítios de Ligação , Metabolismo dos Carboidratos , Membrana Celular/imunologia , Meios de Cultivo Condicionados , Glicosilação , Humanos , Camundongos , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Tripsina , Células Tumorais CultivadasRESUMO
Activated leukocyte cell adhesion molecule (ALCAM) was recently identified as a ligand for CD6, a signaling receptor expressed on T cells, a subset of B cells, and some cells in the brain. Receptor-ligand binding assays, antibody blocking experiments, and examination of the tissue distribution of these two cell surface proteins suggest that CD6-ALCAM interactions play an important role in mediating the binding of thymocytes to thymic epithelial cells and of T cells to activated leukocytes. Presently, the details of CD6-ALCAM interactions and of signaling through CD6 are unknown. A series of truncated human ALCAM and CD6 immunoglobulin fusion proteins were produced and tested in different binding assays to analyze ALCAM-CD6 interactions in more detail. In this study, we report that the amino-terminal Ig-like domain of human ALCAM specifically binds to the third membrane-proximal scavenger receptor cysteine-rich (SRCR) domain of human CD6. Using thrombin-cleaved Ig fusion proteins containing single or multiple ALCAM or CD6 domains, we were able to determine that the stoichiometry of the interaction between the amino-terminal ALCAM domains and the membrane-proximal CD6 SRCR domain is 1:1. These results provide the first example of an Ig-like domain mediating an interaction with an SRCR domain.