ABSTRACT
A mammalian expression vector with features optimized for simple expression and purification of secreted proteins has been developed. This vector was constructed to facilitate X-ray crystallographic studies of cysteine-rich glycoproteins that are difficult to express by other means. Proteins expressed with this vector possess an N-terminal human growth hormone domain and an octahistidine tag separated from the desired polypeptide sequences by a tobacco etch virus protease recognition site. Advantages of this vector are high levels of expression, simple detection and purification of expressed proteins, and reliable cleavage of the fusion protein. Cotransfection of this vector with a dihydrofolate reductase gene allows amplification of expression levels with methotrexate. Over one dozen cysteine-rich secreted proteins have been expressed in sufficient quantity for structural studies using this vector; the structure of at least one of these proteins has been determined.
Subject(s)
Cloning, Molecular/methods , Genetic Vectors , Amino Acid Sequence , Animals , Base Sequence , CHO Cells , Cricetinae , Crystallography, X-Ray , DNA , Glycosylation , Human Growth Hormone/chemistry , Human Growth Hormone/genetics , Human Growth Hormone/metabolism , Humans , Molecular Sequence Data , Protein Conformation , Proteins/metabolismABSTRACT
Engagement of TCR by its ligand, the MHC/peptide complex, causes T cell activation. T cells respond positively to stimulation with agonists, and are inhibited by antagonist MHC/peptide ligands. Failure to induce proper conformational changes in the TCR or fast TCR/MHC dissociation are the leading models proposed to explain anergy induction by antagonist ligands. In this study, we demonstrate that presentation of between 1 and 10 complexes of agonist/MHC II by unfixed APC induces T cell anergy that persists up to 7 days and has characteristics similar to anergy induced by antagonist ligand or TCR occupancy without costimulation. Furthermore, anergy-inducing doses of hemagglutinin 306-318 peptide led to the engagement of less than 1000 TCR/CD3 complexes. Thus, engagement of a subthreshold number of TCR by either a low density of agonist/MHC or a 2-3 orders of magnitude higher density of antagonist/MHC causes anergy. Moreover, we show that anergy induced by low agonist concentrations is inhibited in the presence of IL-2 or cyclosporin A, suggesting involvement of the calcineurin signaling pathway.
Subject(s)
Clonal Anergy/immunology , Receptors, Antigen, T-Cell/agonists , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Clonal Anergy/drug effects , Clone Cells , Cyclosporine/pharmacology , Cytokines/biosynthesis , Dose-Response Relationship, Immunologic , Down-Regulation/immunology , HLA-DR1 Antigen/analysis , Hemagglutinin Glycoproteins, Influenza Virus , Hemagglutinins, Viral/analysis , Hemagglutinins, Viral/immunology , Hemagglutinins, Viral/pharmacology , Humans , Immunosuppressive Agents/pharmacology , Interleukin-2/pharmacology , Ligands , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Macromolecular Substances , Orthomyxoviridae/immunology , Peptide Fragments/analysis , Peptide Fragments/immunology , Peptide Fragments/pharmacology , Receptors, Antigen, T-Cell/antagonists & inhibitors , Receptors, Antigen, T-Cell/biosynthesis , Receptors, Interleukin-2/biosynthesis , T-Lymphocytes/chemistry , Time Factors , Up-Regulation/immunologyABSTRACT
NuMA is a nuclear protein during interphase but redistributes to the spindle poles early in mitosis. To investigate its role during spindle formation, we tested spindle assembly in frog egg extracts from which NuMA was immunodepleted. Immunodepletion revealed that NuMA forms a complex with cytoplasmic dynein and dynactin. The depleted extracts failed to assemble normal mitotic spindles, producing, instead, chromatin-associated irregular arrays of microtubules lacking characteristic spindle poles. A subdomain of the NuMA tail was shown to induce microtubule aster formation by mediating microtubule bundling. Our findings suggest that NuMA forms bifunctional complexes with cytoplasmic dynein and dynactin that can tether microtubules at the spindle poles and that are essential for mitotic spindle pole assembly and stabilization.
