ABSTRACT
HLA-DR-positive NK cells, found in both healthy individuals and patients with different inflammatory diseases, are characterized as activated cells. However, data on their capacity for IFNγ production or cytotoxic response vary between studies. Thus, more precise investigation is needed of the mechanisms related to the induction of HLA-DR expression in NK cells, their associations with NK cell differentiation stage, and functional or metabolic state. In this work, HLA-DR-expressing NK cell subsets were investigated using transcriptomic analysis, metabolic activity assays, and analysis of intercellular signaling cascades. We demonstrated that HLA-DR+CD56bright NK cells were characterized by a proliferative phenotype, while HLA-DR+CD56dim NK cells exhibited features of adaptive cells and loss of inhibitory receptors with increased expression of MHC class II trans-activator CIITA. The activated state of HLA-DR-expressing NK cells was confirmed by higher levels of ATP and mitochondrial mass observed in this subset compared to HLA-DR- cells, both ex vivo and after stimulation in culture. We showed that HLA-DR expression in NK cells in vitro can be induced both through stimulation by exogenous IL-2 and IL-21, as well as through auto-stimulation by NK-cell-produced IFNγ. At the intracellular level, HLA-DR expression depended on the activation of STAT3- and ERK1/2-mediated pathways, with subsequent activation of isoform 3 of the transcription factor CIITA. The obtained results broaden the knowledge about HLA-DR-positive NK cell appearance, diversity, and functions, which might be useful in terms of understanding the role of this subset in innate immunity and assessing their possible implications in NK cell-based therapy.
Subject(s)
Cell Differentiation , HLA-DR Antigens , Interferon-gamma , Killer Cells, Natural , Killer Cells, Natural/metabolism , Killer Cells, Natural/immunology , Humans , HLA-DR Antigens/metabolism , HLA-DR Antigens/genetics , Interferon-gamma/metabolism , CD56 Antigen/metabolism , Lymphocyte Activation/immunology , Interleukin-2/metabolism , Interleukin-2/pharmacology , Cells, Cultured , Nuclear Proteins , Trans-ActivatorsABSTRACT
T cell maturation and activation depend upon T cell receptor (TCR) interactions with a wide variety of antigenic peptides displayed in a given major histocompatibility complex (MHC) context. Complementarity-determining region 3 (CDR3) is the most variable part of the TCRα and -ß chains, which govern interactions with peptide-MHC complexes. However, it remains unclear how the CDR3 landscape is shaped by individual MHC context during thymic selection of naïve T cells. We established two mouse strains carrying distinct allelic variants of H2-A and analyzed thymic and peripheral production and TCR repertoires of naïve conventional CD4+ T (Tconv) and naïve regulatory CD4+ T (Treg) cells. Compared with tuberculosis-resistant C57BL/6 (H2-Ab) mice, the tuberculosis-susceptible H2-Aj mice had fewer CD4+ T cells of both subsets in the thymus. In the periphery, this deficiency was only apparent for Tconv and was compensated for by peripheral reconstitution for Treg We show that H2-Aj favors selection of a narrower and more convergent repertoire with more hydrophobic and strongly interacting amino acid residues in the middle of CDR3α and CDR3ß, suggesting more stringent selection against a narrower peptide-MHC-II context. H2-Aj and H2-Ab mice have prominent reciprocal differences in CDR3α and CDR3ß features, probably reflecting distinct modes of TCR fitting to MHC-II variants. These data reveal the mechanics and extent of how MHC-II shapes the naïve CD4+ T cell CDR3 landscape, which essentially defines adaptive response to infections and self-antigens.
Subject(s)
Complementarity Determining Regions/immunology , Histocompatibility Antigens Class II/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes, Regulatory/immunology , Tuberculosis/genetics , Alleles , Animals , CD4-Positive T-Lymphocytes/immunology , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Histocompatibility Antigens Class II/genetics , Humans , Mice , Mice, Inbred C57BL , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/genetics , Spleen/immunology , T-Lymphocytes, Regulatory/chemistry , Tuberculosis/immunologyABSTRACT
The diversity, architecture, and dynamics of the TCR repertoire largely determine our ability to effectively withstand infections and malignancies with minimal mistargeting of immune responses. In this study, we have employed deep TCRß repertoire sequencing with normalization based on unique molecular identifiers to explore the long-term dynamics of T cell immunity. We demonstrate remarkable stability of repertoire, where approximately half of all T cells in peripheral blood are represented by clones that persist and generally preserve their frequencies for 3 y. We further characterize the extremes of lifelong TCR repertoire evolution, analyzing samples ranging from umbilical cord blood to centenarian peripheral blood. We show that the fetal TCR repertoire, albeit structurally maintained within regulated borders due to the lower numbers of randomly added nucleotides, is not limited with respect to observed functional diversity. We reveal decreased efficiency of nonsense-mediated mRNA decay in umbilical cord blood, which may reflect specific regulatory mechanisms in development. Furthermore, we demonstrate that human TCR repertoires are functionally more similar at birth but diverge during life, and we track the lifelong behavior of CMV- and EBV-specific T cell clonotypes. Finally, we reveal gender differences in dynamics of TCR diversity constriction, which come to naught in the oldest age. Based on our data, we propose a more general explanation for the previous observations on the relationships between longevity and immunity.
Subject(s)
Aging , Fetal Blood/cytology , Fetal Blood/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocytes/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Clone Cells , Female , Humans , Immunodominant Epitopes , Longevity , Male , Middle Aged , Molecular Dynamics Simulation , Receptors, Antigen, T-Cell, alpha-beta/immunology , Software , T-Lymphocytes/physiology , Time Factors , Young AdultABSTRACT
Deep profiling of antibody and T cell-receptor repertoires by means of high-throughput sequencing has become an attractive approach for adaptive immunity studies, but its power is substantially compromised by the accumulation of PCR and sequencing errors. Here we report MIGEC (molecular identifier groups-based error correction), a strategy for high-throughput sequencing data analysis. MIGEC allows for nearly absolute error correction while fully preserving the natural diversity of complex immune repertoires.
Subject(s)
DNA Fingerprinting/methods , High-Throughput Nucleotide Sequencing/standards , Receptors, Antigen, T-Cell/genetics , Research Design , DNA Fingerprinting/standards , Polymerase Chain Reaction/standardsABSTRACT
Emerging high-throughput sequencing methods for the analyses of complex structure of TCR and BCR repertoires give a powerful impulse to adaptive immunity studies. However, there are still essential technical obstacles for performing a truly quantitative analysis. Specifically, it remains challenging to obtain comprehensive information on the clonal composition of small lymphocyte populations, such as Ag-specific, functional, or tissue-resident cell subsets isolated by sorting, microdissection, or fine needle aspirates. In this study, we report a robust approach based on unique molecular identifiers that allows profiling Ag receptors for several hundred to thousand lymphocytes while preserving qualitative and quantitative information on clonal composition of the sample. We also describe several general features regarding the data analysis with unique molecular identifiers that are critical for accurate counting of starting molecules in high-throughput sequencing applications.
Subject(s)
Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Lymphocyte Count , Lymphocytes/metabolism , Computational Biology/methods , DNA, Complementary , Gene Expression Profiling/methods , Humans , Lymphocytes/immunology , Male , Middle Aged , Position-Specific Scoring Matrices , Receptors, Antigen, B-Cell/chemistry , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/geneticsABSTRACT
The decrease of TCR diversity with aging has never been studied by direct methods. In this study, we combined high-throughput Illumina sequencing with unique cDNA molecular identifier technology to achieve deep and precisely normalized profiling of TCR ß repertoires in 39 healthy donors aged 6-90 y. We demonstrate that TCR ß diversity per 10(6) T cells decreases roughly linearly with age, with significant reduction already apparent by age 40. The percentage of naive T cells showed a strong correlation with measured TCR diversity and decreased linearly up to age 70. Remarkably, the oldest group (average age 82 y) was characterized by a higher percentage of naive CD4(+) T cells, lower abundance of expanded clones, and increased TCR diversity compared with the previous age group (average age 62 y), suggesting the influence of age selection and association of these three related parameters with longevity. Interestingly, cross-analysis of individual TCR ß repertoires revealed a set >10,000 of the most representative public TCR ß clonotypes, whose abundance among the top 100,000 clones correlated with TCR diversity and decreased with aging.
Subject(s)
Aging/immunology , Genetic Variation/immunology , High-Throughput Nucleotide Sequencing/methods , Receptors, Antigen, T-Cell, alpha-beta/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Aging/genetics , Amino Acid Sequence , Base Sequence , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Child , Complementarity Determining Regions/genetics , Female , Flow Cytometry , Humans , Male , Middle Aged , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Young AdultABSTRACT
Mycobacterium tuberculosis (Mtb) remains a major threat worldwide, although only a fraction of infected individuals develops tuberculosis (TB). TB susceptibility is shaped by multiple genetic factors, and we performed comparative immunological analysis of two mouse strains to uncover relevant mechanisms underlying susceptibility and resistance. C57BL/6 mice are relatively TB-resistant, whereas I/St mice are prone to develop severe TB, partly due to the MHC-II allelic variant that shapes suboptimal CD4+ T cell receptor repertoire. We investigated the repertoires of lung-infiltrating helper T cells and B cells at the progressed stage in both strains. We found that lung CD4+ T cell repertoires of infected C57BL/6 but not I/St mice contained convergent TCR clusters with functionally confirmed Mtb specificity. Transcriptomic analysis revealed a more prominent Th1 signature in C57BL/6, and expression of pro-inflammatory IL-16 in I/St lung-infiltrating helper T cells. The two strains also showed distinct Th2 signatures. Furthermore, the humoral response of I/St mice was delayed, less focused, and dominated by IgG/IgM isotypes, whereas C57BL/6 mice generated more Mtb antigen-focused IgA response. We conclude that the inability of I/St mice to produce a timely and efficient anti-Mtb adaptive immune responses arises from a suboptimal helper T cell landscape that also impacts the humoral response, leading to diffuse inflammation and severe disease.
Subject(s)
Adaptive Immunity , Genetic Predisposition to Disease , Mice, Inbred C57BL , Mycobacterium tuberculosis , Tuberculosis , Animals , Mice , Mycobacterium tuberculosis/immunology , Adaptive Immunity/genetics , Tuberculosis/immunology , Tuberculosis/genetics , Lung/immunology , Lung/pathology , B-Lymphocytes/immunology , Disease Models, Animal , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunologyABSTRACT
The development of magnesium calcium phosphate bone cements (MCPCs) has garnered substantial attention. MCPCs are bioactive and biodegradable and have appropriate mechanical and antimicrobial properties for use in reconstructive surgery. In this study, the cement powders based on a (Ca + Mg)/P = 2 system doped with Zn2+ at 0.5 and 1.0 wt.% were obtained and investigated. After mixing with a cement liquid, the structural and phase composition, morphology, chemical structure, setting time, compressive strength, degradation behavior, solubility, antibacterial activities, and in vitro behavior of the cement materials were examined. A high compressive strength of 48 ± 5 MPa (mean ± SD) was achieved for the cement made from Zn2+ 1.0 wt.%-substituted powders. Zn2+ introduction led to antibacterial activity against Staphylococcus aureus and Escherichia coli strains, with an inhibition zone diameter of up to 8 mm. Biological assays confirmed that the developed cement is cytocompatible and promising as a potential bone substitute in reconstructive surgery.
ABSTRACT
Autoimmunity is intrinsically driven by memory T and B cell clones inappropriately targeted at self-antigens. Selective depletion or suppression of self-reactive T cells remains a holy grail of autoimmune therapy, but disease-associated T cell receptors (TCRs) and cognate antigenic epitopes remained elusive. A TRBV9-containing CD8+ TCR motif was recently associated with the pathogenesis of ankylosing spondylitis, psoriatic arthritis and acute anterior uveitis, and cognate HLA-B*27-presented epitopes were identified. Following successful testing in nonhuman primate models, here we report human TRBV9+ T cell elimination in ankylosing spondylitis. The patient achieved remission within 3 months and ceased anti-TNF therapy after 5 years of continuous use. Complete remission has now persisted for 4 years, with three doses of anti-TRBV9 administered per year. We also observed a profound improvement in spinal mobility metrics and the Bath Ankylosing Spondylitis Metrology Index (BASMI). This represents a possibly curative therapy of an autoimmune disease via selective depletion of a TRBV-defined group of T cells. The anti-TRBV9 therapy could potentially be applicable to other HLA-B*27-associated spondyloarthropathies. Such targeted elimination of the underlying cause of the disease without systemic immunosuppression could offer a new generation of safe and efficient therapies for autoimmunity.
Subject(s)
Spondylitis, Ankylosing , Humans , Epitopes , HLA-B Antigens , Immunotherapy , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/therapeutic use , Spondylitis, Ankylosing/drug therapy , T-Lymphocytes , Tumor Necrosis Factor Inhibitors/therapeutic useABSTRACT
AIM: This study is aimed at investigation of electrophysiological effects of α1-adrenoreceptor (α1-AR) stimulation in the rat superior vena cava (SVC) myocardium, which is one of the sources of proarrhythmic activity. METHODS: α1-ARs agonists (phenylephrine-PHE or norepinephrine in presence of atenolol-NE + ATL) were applied to SVC and atrial tissue preparations or isolated cardiomyocytes, which were examined using optical mapping, glass microelectrodes or whole-cell patch clamp. α1-ARs distribution was evaluated using immunofluorescence. Kir2.X mRNA and protein level were estimated using RT-PCR and Western blotting. RESULTS: PHE or NE + ATL application caused a significant suppression of the conduction velocity (CV) of excitation and inexcitability in SVC, an increase in the duration of electrically evoked action potentials (APs), a decrease in the maximum upstroke velocity (dV/dtmax ) and depolarization of the resting membrane potential (RMP) in SVC to a greater extent than in atria. The effects induced by α1-ARs activation in SVC were attenuated by protein kinase C inhibition (PKC). The whole-cell patch clamp revealed PHE-induced suppression of outward component of IK1 inward rectifier current in isolated SVC, but not atrial myocytes. These effects can be mediated by α1A subtype of α-ARs found in abundance in rat SVC. The basal IK1 level in SVC was much lower than in atria as a result of the weaker expression of Kir2.2 channels. CONCLUSION: Therefore, the reduced density of IK1 in rat SVC cardiomyocytes and sensitivity of this current to α1A-AR stimulation via PKC-dependent pathways might lead to proarrhythmic conduction in SVC myocardium by inducing RMP depolarization, AP prolongation, CV and dV/dtmax decrease.
Subject(s)
Potassium , Receptors, Adrenergic, alpha-1 , Vena Cava, Superior , Action Potentials , Animals , Heart Atria , Myocardium , RatsABSTRACT
The interplay between T- and B-cell compartments during naïve, effector and memory T cell maturation is critical for a balanced immune response. Primary B-cell immunodeficiency arising from X-linked agammaglobulinemia (XLA) offers a model to explore B cell impact on T cell subsets, starting from the thymic selection. Here we investigated characteristics of naïve and effector T cell subsets in XLA patients, revealing prominent alterations in the corresponding T-cell receptor (TCR) repertoires. We observed immunosenescence in terms of decreased diversity of naïve CD4+ and CD8+ TCR repertoires in XLA donors. The most substantial alterations were found within naïve CD4+ subsets, and we have investigated these in greater detail. In particular, increased clonality and convergence, along with shorter CDR3 regions, suggested narrower focused antigen-specific maturation of thymus-derived naïve Treg (CD4+CD45RA+CD27+CD25+) in the absence of B cells - normally presenting diverse self and commensal antigens. The naïve Treg proportion among naïve CD4 T cells was decreased in XLA patients, supporting the concept of impaired thymic naïve Treg selection. Furthermore, the naïve Treg subset showed prominent differences at the transcriptome level, including increased expression of genes specific for antigen-presenting and myeloid cells. Altogether, our findings suggest active B cell involvement in CD4 T cell subsets maturation, including B cell-dependent expansion of the naïve Treg TCR repertoire that enables better control of self-reactive T cells.
Subject(s)
Agammaglobulinemia/immunology , Genetic Diseases, X-Linked/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Adolescent , Adult , Agammaglobulinemia/genetics , Aged , Aged, 80 and over , B-Lymphocytes/immunology , Case-Control Studies , Complementarity Determining Regions/genetics , Genes, T-Cell Receptor beta , Genetic Diseases, X-Linked/genetics , Humans , Immunosenescence/genetics , Immunosenescence/immunology , Male , Memory T Cells/immunology , Middle Aged , Models, Immunological , Transcriptome , Young AdultABSTRACT
Photosensitizers are chromophores that generate reactive oxygen species (ROS) upon light irradiation. They are used for inactivation of specific proteins by chromophore-assisted light inactivation (CALI) and for light-induced cell killing in photodynamic therapy. Here we report a genetically encoded photosensitizer, which we call KillerRed, developed from the hydrozoan chromoprotein anm2CP, a homolog of green fluorescent protein (GFP). KillerRed generates ROS upon irradiation with green light. Whereas known photosensitizers must be added to living systems exogenously, KillerRed is fully genetically encoded. We demonstrate the utility of KillerRed for light-induced killing of Escherichia coli and eukaryotic cells and for inactivating fusions to beta-galactosidase and phospholipase Cdelta1 pleckstrin homology domain.
Subject(s)
Escherichia coli/radiation effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Kidney/cytology , Kidney/radiation effects , Photosensitizing Agents/metabolism , Base Sequence , Cell Line , Cell Proliferation/radiation effects , Cell Survival/radiation effects , Escherichia coli/physiology , Humans , Light , Molecular Sequence Data , Protein EngineeringABSTRACT
Age-related changes can significantly alter the state of adaptive immune system and often lead to attenuated response to novel pathogens and vaccination. In present study we employed 5'RACE UMI-based full length and nearly error-free immunoglobulin profiling to compare plasma cell antibody repertoires in young (19-26 years) and middle-age (45-58 years) individuals vaccinated with a live yellow fever vaccine, modeling a newly encountered pathogen. Our analysis has revealed age-related differences in the responding antibody repertoire ranging from distinct IGH CDR3 repertoire properties to differences in somatic hypermutation intensity and efficiency and antibody lineage tree structure. Overall, our findings suggest that younger individuals respond with a more diverse antibody repertoire and employ a more efficient somatic hypermutation process than elder individuals in response to a newly encountered pathogen.
Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Immunity, Active , Receptors, Antigen, B-Cell/metabolism , Yellow Fever Vaccine/immunology , Adult , Animals , Antibodies, Viral/immunology , Female , Humans , Immunity, Active/genetics , Immunoglobulin Constant Regions/genetics , Immunoglobulin Heavy Chains/genetics , Male , Middle Aged , Receptors, Antigen, B-Cell/genetics , Somatic Hypermutation, Immunoglobulin , Vaccination , Yellow Fever/prevention & control , Young AdultABSTRACT
The relationship between maternal and child immunity has been actively studied in the context of complications during pregnancy, autoimmune diseases, and haploidentical transplantation of hematopoietic stem cells and solid organs. Here, we have for the first time used high-throughput Illumina HiSeq sequencing to perform deep quantitative profiling of T cell receptor (TCR) repertoires for peripheral blood samples of three mothers and their six children. Advanced technology allowed accurate identification of 5 × 10(5) to 2 × 10(6) TCR beta clonotypes per individual. We performed comparative analysis of these TCR repertoires with the aim of revealing characteristic features that distinguish related mother-child pairs, such as relative TCR beta variable segment usage frequency and relative overlap of TCR beta complementarity-determining region 3 (CDR3) repertoires. We show that thymic selection essentially and similarly shapes the initial output of the TCR recombination machinery in both related and unrelated pairs, with minor effect from inherited differences. The achieved depth of TCR profiling also allowed us to test the hypothesis that mature T cells transferred across the placenta during pregnancy can expand and persist as functional microchimeric clones in their new host, using characteristic TCR beta CDR3 variants as clonal identifiers.
Subject(s)
Biotechnology/methods , Cell Physiological Phenomena , Proteins/metabolism , Cell Line , HeLa Cells , HumansABSTRACT
The far-red fluorescent protein mKate (lambda(ex), 588 nm; lambda(em), 635 nm; chromophore-forming triad Met(63)-Tyr(64)-Gly(65)), originating from wild-type red fluorescent progenitor eqFP578 (sea anemone Entacmaea quadricolor), is monomeric and characterized by the pronounced pH dependence of fluorescence, relatively high brightness, and high photostability. The protein has been crystallized at a pH ranging from 2 to 9 in three space groups, and four structures have been determined by x-ray crystallography at the resolution of 1.75-2.6 A. The pH-dependent fluorescence of mKate has been shown to be due to reversible cis-trans isomerization of the chromophore phenolic ring. In the non-fluorescent state at pH 2.0, the chromophore of mKate is in the trans-isomeric form. The weakly fluorescent state of the protein at pH 4.2 is characterized by a mixture of trans and cis isomers. The chromophore in a highly fluorescent state at pH 7.0/9.0 adopts the cis form. Three key residues, Ser(143), Leu(174), and Arg(197) residing in the vicinity of the chromophore, have been identified as being primarily responsible for the far-red shift in the spectra. A group of residues consisting of Val(93), Arg(122), Glu(155), Arg(157), Asp(159), His(169), Ile(171), Asn(173), Val(192), Tyr(194), and Val(216), are most likely responsible for the observed monomeric state of the protein in solution.
Subject(s)
Crystallography, X-Ray/methods , Luminescent Proteins/chemistry , Amino Acid Sequence , Animals , Electrons , Hydrogen-Ion Concentration , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Protein Isoforms , Sea Anemones , Sequence Homology, Amino Acid , Red Fluorescent ProteinABSTRACT
Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Luminescent Agents/chemistry , Luminescent Proteins/chemistry , Proteins/analysis , Fluorescence , HeLa Cells , Humans , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Red Fluorescent ProteinABSTRACT
For deep imaging of animal tissues, the optical window favorable for light penetration is in near-infrared wavelengths, which requires proteins with emission spectra in the far-red wavelengths. Here we report a far-red fluorescent protein, named Katushka, which is seven- to tenfold brighter compared to the spectrally close HcRed or mPlum, and is characterized by fast maturation as well as a high pH-stability and photostability. These unique characteristics make Katushka the protein of choice for visualization in living tissues. We demonstrate superiority of Katushka for whole-body imaging by direct comparison with other red and far-red fluorescent proteins. We also describe a monomeric version of Katushka, named mKate, which is characterized by high brightness and photostability, and should be an excellent fluorescent label for protein tagging in the far-red part of the spectrum.
Subject(s)
Biotechnology/methods , Fluorescence Resonance Energy Transfer/methods , Fluorescent Dyes , Luminescent Proteins , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Animals , Biotechnology/instrumentation , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Fluorescence Resonance Energy Transfer/instrumentation , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Luminescent Proteins/analysis , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Microscopy, Confocal/instrumentation , Microscopy, Fluorescence/instrumentation , Transgenes , Xenopus laevis , Red Fluorescent ProteinABSTRACT
Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.