RESUMO
B and T lymphocyte attenuator (BTLA) is an immunoglobulin superfamily member surface protein expressed on B and T cells. Its ligand, herpesvirus entry mediator (HVEM), is believed to act as a monomeric agonist that signals via the CRD1 of HVEM to inhibit lymphocyte activation: HVEM is also the receptor for lymphotoxin-α and LIGHT, which both bind in the CRD2 and CRD3 domains of the HVEM molecule, and for CD160 which competes with BTLA. We have shown that recombinant HVEM and a panel of different monoclonal antibodies specifically bind murine BTLA on both B and T cells and that some antibodies inhibit anti-CD3ε-induced T cell proliferation in vitro, but only when constrained appropriately with a putatively cross-linking reagent. The antibodies had no significant effect on in vitro T cell proliferation in a mixed lymphocyte reaction (MLR) assay nor on in vitro DO11.10 antigen-induced T cell proliferation. None of these antibodies, nor HVEM-Fc, had any significant effect on in vitro B cell proliferation induced by anti-immunoglobulin M antibodies (±anti-CD40) or lipopolysaccharide. We further elucidated the requirements for inhibition of in vitro T cell proliferation using a beads-based system to demonstrate that the antibodies that inhibited T cell proliferation in vitro were required to be presented to the T cell in a cis, and not trans, format relative to the anti-CD3ε stimulus. We also found that antibodies that inhibited T cell proliferation in vitro had no significant effect on the antibody captured interleukin-2 associated with the in vivo activation of DO11.10 T cells transferred to syngeneic recipient BALB/c mice. These data suggest that there may be specific structural requirements for the BTLA molecule to exert its effect on lymphocyte activation and proliferation.
Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Ativação Linfocitária , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/imunologia , Linfócitos T/imunologia , Animais , Complexo CD3/imunologia , Antígenos CD40/imunologia , Reagentes de Ligações Cruzadas/química , Imunoglobulina M/imunologia , Interleucina-2/sangue , Interleucina-2/imunologia , Linfotoxina-alfa/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Membro 14 de Receptores do Fator de Necrose Tumoral/imunologiaRESUMO
Pleiotrophin (PTN) is a secreted growth factor that induces neurite outgrowth and is mitogenic for fibroblasts, epithelial, and endothelial cells. During tumor growth PTN can serve as an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN, ALK is highly expressed during perinatal development of the nervous system and down-modulated in the adult. Here we show in cell-free assays as well as in radioligand receptor binding studies in intact cells that PTN binds to the ALK ECD with an apparent Kd of 32 +/- 9 pm. This receptor binding is inhibited by an excess of PTN, by the ALK ECD, and by anti-PTN and anti-ECD antibodies. PTN added to ALK-expressing cells induces phosphorylation of both ALK and of the downstream effector molecules IRS-1, Shc, phospholipase C-gamma, and phosphatidylinositol 3-kinase. Furthermore, the growth stimulatory effect of PTN on different cell lines in culture coincides with the endogenous expression of ALK mRNA, and the effect of PTN is enhanced by ALK overexpression. From this we conclude that ALK is a receptor that transduces PTN-mediated signals and propose that the PTN-ALK axis can play a significant role during development and during disease processes.
Assuntos
Proteínas de Transporte/metabolismo , Citocinas/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Neoplasias das Glândulas Suprarrenais , Quinase do Linfoma Anaplásico , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/enzimologia , Divisão Celular , Sistema Livre de Células , Clonagem Molecular , Biblioteca Gênica , Substâncias de Crescimento/metabolismo , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , Receptores Proteína Tirosina Quinases , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
We have developed specific antibodies against fragments of anaplastic lymphoma kinase (ALK) in order to develop tools for characterizing the expression and biological function of this orphan receptor. The first fragment consisted of residues 280 to 480 of the murine extracellular domain, was expressed in Escherichia coli (E. coli), purified in the presence of urea from the pellet of mechanically lysed cells and injected into rabbits as an unfolded protein in urea. The second fragment consisted of residues 1519 to 1619 of the murine sequence, corresponding to the C-terminal side of the kinase domain. It was expressed in E. coli as a soluble glutathione-S-transferase fusion protein, purified from the supernatant of broken cells and injected into rabbits as a folded protein. Both antisera were purified using antigen affinity chromatography, with the polyclonal antibodies eluted stepwise using three different buffers, 0.1 M glycine, pH 2.9, followed by 7 M urea, pH 4, followed by 6 M guanidine-HCl (GdnHCl), pH 4. Antisera prepared against either antigen contained antibodies that eluted in each of the three pools, indicating that solvents more chaotropic than acid were required to elute antibody populations that were tightly bound to the antigen column. All three antibody pools were reactive towards their respective antigens upon Western blot analysis. Purified polyclonal antibodies (pAbs) to both fragments also recognized the full-length protein expressed in Chinese hamster ovary cells. In every case, the pAbs eluting in GdnHCl were the most sensitive for detecting full-length ALK.
Assuntos
Anticorpos/isolamento & purificação , Fragmentos de Peptídeos/imunologia , Proteínas Tirosina Quinases/química , Quinase do Linfoma Anaplásico , Animais , Anticorpos/imunologia , Western Blotting , Células CHO , Cricetinae , Eletroforese em Gel de Poliacrilamida , Fragmentos de Peptídeos/genética , Receptores Proteína Tirosina Quinases , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , SolventesRESUMO
Antibodies are routinely purified by acid/salt elution from antigen affinity columns. The antibodies recovered with this procedure are active, but the recovery of protein is often low. We investigated the effect of acid and other denaturing or chaotropic solvents on the conformation of monoclonal antibodies (mAbs) made against the extracellular region of Her2 receptor (sHer2) derived from Chinese hamster ovary cells. The mAb remain almost completely folded in the 0.1 M glycine, pH 2.9, commonly used for elution, with the beta-sheet secondary structure intact, and only very small changes detected in the environment of the tryptophans. In 7 M urea, 50 mM NaAc pH 4.0, the antibody was partially unfolded, with the Trp environment further perturbed and some of the beta-sheet structure converted to disordered structure. In 6 M guanidine HCl, 50 mM NaAc, pH 4.0, the antibody is completely unfolded, with no secondary or tertiary structure present. The antibodies exposed to glycine or urea were refolded by dialysis into phosphate-buffered saline (PBS), while the guanidine HCl-denatured antibodies were refolded by dialysis into 7 M urea, pH 4.0, followed by dialysis into PBS. The refolded antibodies were capable of forming antigen-antibody complexes which could be isolated by gel filtration chromatography. Two different mAbs were subjected to immunoaffinity chromatography on sHer2-Sepharose. mAb86 was eluted by 0.1 M Gly, pH 2.9, while mAb52 was eluted with the 7 M urea, 50 mM NaAc, pH 4.0. The isolated antibodies were refolded by dialysis into PBS, analyzed for their ability to recognize native sHer2 by immunoprecipitation, and denatured sHer2 by Western blot analysis. Both preparations recognized the native protein, but precipitated slightly different forms of sHer2, indicating that they might recognize different epitopes. The mAb52 is a more sensitive reagent for Western blot analysis. Thus, this procedure can be used to recover antibodies which would not be recovered with glycine as the only eluate. It is also possible that the antibodies can be fractionated by the different eluants into populations which can be used for different applications.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Animais , Anticorpos Monoclonais/metabolismo , Complexo Antígeno-Anticorpo/metabolismo , Células CHO , Cromatografia de Afinidade , Cricetinae , Dobramento de Proteína , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Solventes/farmacologiaRESUMO
In the previous paper we described the effect of several different solvents on the structure of antibodies and demonstrated that 0.1 M glycine, pH 2.9, 7 M urea, pH 4.0, and 6 M guanidine-HCl, pH 4.0, unfold the antibodies to different degrees. Antibodies can be refolded from all of these solvents by dialysis. Polyclonal antibodies (pAbs) are a mixture of antibodies which recognize and bind different epitopes on the same antigen, with the strength of the antigen-antibody binding varying with each subpopulation. When rabbit antisera to the extracellular domain of Her2 receptor (sHer2), derived from Chinese hamster ovary cells, was applied to an antigen column, bound pAbs were recovered with a step-wise elution of 0.1 M glycine, pH 2.9 (44% of the total recovered pAb), 7 M urea, pH 4.0 (29%), and 6 M guanidine-HCl, pH 4.0 (27%), with baseline resolution between them. Fluorescence spectra of the pAbs confirmed that the 0. 1 M glycine pH 2.9 sample had near-native structure, the pAbs in 7 M urea, pH 4.0, were partially unfolded, and the pAbs in the 6 M guanidine-HCl, pH 4.0, were totally unfolded. The glycine- or urea-eluted sample was refolded by dialysis into PBS, while the guanidine-HCl-eluted sample was first dialyzed into the 7 M urea pH 4.0 buffer and then into PBS. The refolded material from glycine or urea had native-like spectra, while the spectrum of the protein refolded from 6 M guanidine-HCl was slightly perturbed. All three of these subpopulations of pAbs formed antigen-antibody complexes which could be isolated by gel-filtration chromatography, precipitated sHer2 during immunoprecipitation, and recognized sHer2 in Western blots. The guanidine-HCl-eluted material was most sensitive for Western blotting. Identical results were obtained with pAbs applied either in the batch mode or to the top of the column, indicating that antibody aggregation which may occur when applied from the top of the column is not responsible for the distribution of pAbs into different subpopulations. These results indicate that the sequential use of these three increasingly chaotropic solvents to elute antibodies results in both increased recovery of antibodies and fractionation of pAbs into subpopulations with potentially different antigen binding characteristics.