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1.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207579

RESUMO

Biomanufacturing processes may be optimized by storing cell culture media at room temperature, but this is currently limited by their instability and change in color upon long-term storage. This study demonstrates that one of the critical contributing factors toward media browning is tryptophan. LC-MS technology was utilized to identify tryptophan degradation products, which are likely formed primarily from oxidation reactions. Several of the identified compounds were shown to contribute significantly to color in solutions but also to exhibit toxicity against CHO cells. A cell-culture-compatible antioxidant, a-ketoglutaric acid, was found to be an efficient cell culture media additive for stabilizing components against degradation, inhibiting the browning of media formulations, and decreasing ammonia production, thus providing a viable method for developing room-temperature stable cell culture media.


Assuntos
Meios de Cultura/química , Triptofano/metabolismo , Animais , Células CHO , Cricetulus , Oxirredução , Triptofano/análise
2.
Biotechnol Bioeng ; 118(9): 3395-3408, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33738790

RESUMO

Increasing demands for protein-based therapeutics such as monoclonal antibodies, fusion proteins, bispecific molecules, and antibody fragments require researchers to constantly find innovative solutions. To increase yields and decrease costs of next generation bioprocesses, highly concentrated cell culture media formulations are developed but often limited by the low solubility of amino acids such as tyrosine, cystine, leucine, and isoleucine, in particular at physiological pH. This study sought to investigate highly soluble and bioavailable derivatives of leucine and isoleucine that are applicable for fed-batch processes. N-lactoyl-leucine and N-lactoyl-isoleucine sodium salts were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These modified amino acids proved to be bioavailable for various Chinese hamster ovary (CHO) cells and were suitable for replacement of canonical amino acids in cell culture feeds. The quality of the final recombinant protein was studied in bioprocesses using the derivatives, and the mechanism of cleavage was investigated in CHO cells. Altogether, both N-lactoyl amino acids represent an advantageous alternative to canonical amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.

3.
J Biotechnol ; 321: 1-12, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32580011

RESUMO

Highly concentrated cell culture media formulations are essential to develop next generation bioprocesses used to produce therapeutic monoclonal antibodies, fusion proteins, bispecific molecules and mAb fragments. Although cysteine/cystine and tyrosine are the first components preventing the development of highly concentrated complex cell culture media, leucine and isoleucine were identified as the next critical amino acids due to their limited solubility. This work sought to investigate highly soluble and readily bioavailable derivatives of both amino acids that may be used in batch, fed-batch or perfusion processes. The α-keto acids of Leu and Ile, keto leucine and keto isoleucine sodium salts, were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These keto acids were readily bioavailable for various CHO cells and can be used in both media and feeds. The quality of the final recombinant protein was studied in processes using the precursors and the mechanism of amination was investigated in CHO cells. Altogether, both keto acids represent an alternative to their respective amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.


Assuntos
Isoleucina , Cetoácidos , Leucina , Proteínas Recombinantes/metabolismo , Aminação , Animais , Disponibilidade Biológica , Reatores Biológicos , Células CHO , Cricetinae , Cricetulus , Meios de Cultura/química , Meios de Cultura/metabolismo , Isoleucina/química , Isoleucina/metabolismo , Cetoácidos/química , Cetoácidos/metabolismo , Leucina/química , Leucina/metabolismo , Solubilidade
4.
Clin Exp Allergy ; 49(3): 378-390, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30230051

RESUMO

BACKGROUND: Protein crystallographic studies suggest that the house dust mite (HDM) allergen Der p 5 potentially interacts with hydrophobic ligands. Der p 5, in association with its ligand(s), might therefore trigger innate immune signalling pathways in the airway epithelium and influence the initiation of the HDM-allergic response. OBJECTIVE: We investigated the lipid binding propensities of recombinant (r)Der p 5 and characterized the signalling pathways triggered by the allergen in airway epithelial cells. METHODS: rDer p 5 was produced in Pichia pastoris and characterized by mass spectrometry, multi-angle light scattering and circular dichroism. Its interactions with hydrophobic ligands were investigated in fluorescence-based lipid binding assays and in-silico docking simulations. Innate immune signalling pathways triggered by rDer p 5 were investigated in airway epithelial cell activation assays in vitro. RESULTS: Biophysical analysis showed that rDer p 5 was monomeric and adopted a similar α-helix-rich fold at both physiological and acidic pH. Spectrofluorimetry experiments showed that rDer p 5 is able to selectively bind lipid ligands, but only under mild acidic pH conditions. Computer-based docking simulations identified potential binding sites for these ligands. This allergen, with putatively associated lipid(s), triggered the production of IL-8 in respiratory epithelial cells through a TLR2-, NF-kB- and MAPK-dependent signalling pathway. CONCLUSIONS AND CLINICAL RELEVANCE: Despite the fact that Der p 5 represents a HDM allergen of intermediate prevalence, our findings regarding its lipid binding and activation of TLR2 indicate that it could participate in the initiation of the HDM-allergic state.


Assuntos
Antígenos de Dermatophagoides , Proteínas de Artrópodes , Brônquios , Células Epiteliais , Hipersensibilidade , Lipídeos , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Antígenos de Dermatophagoides/química , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/química , Proteínas de Artrópodes/imunologia , Brônquios/imunologia , Brônquios/patologia , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/patologia , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Ligantes , Lipídeos/química , Lipídeos/imunologia , Simulação de Acoplamento Molecular , Pyroglyphidae/química , Pyroglyphidae/imunologia
5.
PLoS One ; 12(10): e0185830, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28982170

RESUMO

BACKGROUND: House dust mites (HDMs) such as Dermatophagoides farinae and D. pteronyssinus represent major causes of perennial allergy. HDM proteomes are currently poorly characterized, with information mostly restricted to allergens. As of today, 33 distinct allergen groups have been identified for these 2 mite species, with groups 1 and 2 established as major allergens. Given the multiplicity of IgE-reactive mite proteins, potential additional allergens have likely been overlooked. OBJECTIVE: To perform a comprehensive characterization of the transcriptomes, proteomes and allergomes of D. farinae and D. pteronyssinus in order to identify novel allergens. METHODS: Transcriptomes were analyzed by RNA sequencing and de novo assembly. Comprehensive mass spectrometry-based analyses proteomes were combined with two-dimensional IgE reactivity profiling. RESULTS: Transcripts from D. farinae and D. pteronyssinus were assembled, translated into protein sequences and used to populate derived sequence databases in order to inform immunoproteomic analyses. A total of 527 and 157 proteins were identified by bottom-up MS analyses in aqueous extracts from purified HDM bodies and fecal pellets, respectively. Based on high sequence similarities (>71% identity), we also identified 2 partial and 11 complete putative sequences of currently undisclosed D. pteronyssinus counterparts of D. farinae registered allergens. Immunoprofiling on 2D-gels revealed the presence of unknown 23 kDa IgE reactive proteins in both species. Following expression of non-glycosylated recombinant forms of these molecules, we confirm that these new allergens react with serum IgEs from 42% (8/19) of HDM-allergic individuals. CONCLUSIONS: Using combined transcriptome and immunoproteome approaches, we provide a comprehensive characterization of D. farinae and D. pteronyssinus allergomes. We expanded the known allergen repertoire for D. pteronyssinus and identified two novel HDM allergens, now officially referred by the International Union of Immunological Societies (IUIS) Nomenclature Subcommittee as Der f 36 and Der p 36.


Assuntos
Alérgenos/metabolismo , Proteoma , Pyroglyphidae/metabolismo , Transcriptoma , Alérgenos/química , Alérgenos/genética , Sequência de Aminoácidos , Animais , Hipersensibilidade/sangue , Espectrometria de Massas , Pyroglyphidae/genética , Homologia de Sequência de Aminoácidos
6.
Curr Allergy Asthma Rep ; 16(9): 64, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27534655

RESUMO

Proteomics encompasses a variety of approaches unraveling both the structural features, post-translational modifications, and abundance of proteins. As of today, proteomic studies have shed light on the primary structure of about 850 allergens, enabling the design of microarrays for improved molecular diagnosis. Proteomic methods including mass spectrometry allow as well to investigate protein-protein interactions, thus yielding precise information on critical epitopes on the surface of allergens. Mass spectrometry is now being applied to the unambiguous identification, characterization, and comprehensive quantification of allergens in a variety of matrices, as diverse as food samples and allergen immunotherapy drug products. As such, it represents a method of choice for quality testing of allergen immunotherapy products.


Assuntos
Hipersensibilidade/diagnóstico , Imunoterapia/métodos , Proteômica/métodos , Humanos
7.
Bioconjug Chem ; 27(11): 2629-2645, 2016 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-27552359

RESUMO

ß-Lactam antibiotics allergy is recognized as a public health concern. By covalently binding to serum proteins, penicillins are known to form immunogenic complexes. The latter are recognized and digested by antigen-presenting cells into drug-hapten peptides leading to the immunization of treated persons and IgE-mediated hypersensitivity reactions encompassing anaphylaxis. If type I allergic reactions to drugs are often unpredictable, they are known to be dependent on CD4+ T-cells. This fundamental study revisits the chemical basis of the benzylpenicillin (BP) allergy with the aim of identifying immunologically relevant biomimetic benzylpenicilloylated peptides through the analysis of BP-conjugated human serum albumin (BP-HSA) profile and the evaluation of the naïve CD4+ T-cell responses to candidate BP-HSA-derived peptides. The chemical structures of BP-HSA bioconjugates synthesized in vitro at both physiological and basic pH were investigated by mass spectrometry. From the ten most representative lysine residues grafted by BP-hapten, HSA-bioinspired 15-mer peptide sequences were designed and the potential T-cell epitope profile of each peptide was predicted using two complementary in silico approaches, i.e., HLA class II binding prediction tools from the Immune Epitope Database and Analysis Resource (IEDB) and computational alanine scanning mutagenesis. Twelve structurally diversified benzylpenicilloylated peptides (BP-Ps) were selected and synthesized with the aid of a flexible synthesis pathway using an original benzylpenicilloylated lysine monomer as common precursor. In order to corroborate their predicted "epitope" profile, the naïve CD4+ T-cell response specific to BP was evaluated through a coculture approach. To our knowledge, this study showed for the first time the ability of bioinspired peptides structurally stemming from BP-HSA to be recognized by naïve CD4+ T-cells thus identifying a pre-existing T-cell repertoire for penicillin molecules bound to proteins. It also established a promising model approach expandable to other most frequently used penicillin classes of antibiotics to reveal biomimetic drug-modified antigenic peptides relevant for qualitative and quantitative drug allergy studies.


Assuntos
Biomimética , Desenho de Fármacos , Penicilina G/química , Peptídeos/química , Peptídeos/imunologia , Sequência de Aminoácidos , Técnicas de Química Sintética , Simulação por Computador , Epitopos/química , Epitopos/imunologia , Haptenos/química , Humanos , Imunização , Imunoglobulina E/imunologia , Lisina/química , Modelos Moleculares , Peptídeos/síntese química , Conformação Proteica , Albumina Sérica/química
8.
J Biol Chem ; 291(25): 13076-87, 2016 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-27129273

RESUMO

Allergy to the short ragweed (Ambrosia artemisiifolia) pollen is a major health problem. The ragweed allergen repertoire has been recently expanded with the identification of Amb a 11, a new major allergen belonging to the cysteine protease family. To better characterize Amb a 11, a recombinant proform of the molecule with a preserved active site was produced in Escherichia coli, refolded, and processed in vitro into a mature enzyme. The enzymatic activity is revealed by maturation following an autocatalytic processing resulting in the cleavage of both N- and C-terminal propeptides. The 2.05-Å resolution crystal structure of pro-Amb a 11 shows an overall typical C1A cysteine protease fold with a network of molecular interactions between the N-terminal propeptide and the catalytic triad of the enzyme. The allergenicity of Amb a 11 was confirmed in a murine sensitization model, resulting in airway inflammation, production of serum IgEs, and induction of Th2 immune responses. Of note, inflammatory responses were higher with the mature form, demonstrating that the cysteine protease activity critically contributes to the allergenicity of the molecule. Collectively, our results clearly demonstrate that Amb a 11 is a bona fide cysteine protease exhibiting a strong allergenicity. As such, it should be considered as an important molecule for diagnosis and immunotherapy of ragweed pollen allergy.


Assuntos
Antígenos de Plantas/imunologia , Cisteína Proteases/química , Precursores Enzimáticos/química , Extratos Vegetais/imunologia , Proteínas de Plantas/química , Rinite Alérgica Sazonal/imunologia , Alérgenos/química , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Cisteína Proteases/imunologia , Precursores Enzimáticos/imunologia , Feminino , Ligação de Hidrogênio , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/imunologia , Processamento de Proteína Pós-Traducional , Proteólise , Rinite Alérgica Sazonal/prevenção & controle
9.
Biochem J ; 473(12): 1805-19, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27099340

RESUMO

The control protein Factor H (FH) is a crucial regulator of the innate immune complement system, where it is active on host cell membranes and in the fluid phase. Mutations impairing the binding capacity of FH lead to severe autoimmune diseases. Here, we studied the solution structure of full-length FH, in its free state and bound to the C3b complement protein. To do so, we used two powerful techniques, hydroxyl radical protein footprinting (HRPF) and chemical cross-linking coupled with mass spectrometry (MS), to probe the structural rearrangements and to identify protein interfaces. The footprint of C3b on the FH surface matches existing crystal structures of C3b complexed with the N- and C-terminal fragments of FH. In addition, we revealed the position of the central portion of FH in the protein complex. Moreover, cross-linking studies confirmed the involvement of the C-terminus in the dimerization of FH.


Assuntos
Fator H do Complemento/química , Fator H do Complemento/metabolismo , Radical Hidroxila/química , Pegadas de Proteínas/métodos , Humanos , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína
10.
PLoS One ; 10(8): e0136258, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26317427

RESUMO

BACKGROUND: Allergy to short ragweed (Ambrosia artemisiifolia) pollen is a serious and expanding health problem in North America and Europe. Whereas only 10 short ragweed pollen allergens are officially recorded, patterns of IgE reactivity observed in ragweed allergic patients suggest that other allergens contribute to allergenicity. The objective of the present study was to identify novel allergens following extensive characterization of the transcriptome and proteome of short ragweed pollen. METHODS: Following a Proteomics-Informed-by-Transcriptomics approach, a comprehensive transcriptomic data set was built up from RNA-seq analysis of short ragweed pollen. Mass spectrometry-based proteomic analyses and IgE reactivity profiling after high resolution 2D-gel electrophoresis were then combined to identify novel allergens. RESULTS: Short ragweed pollen transcripts were assembled after deep RNA sequencing and used to inform proteomic analyses, thus leading to the identification of 573 proteins in the short ragweed pollen. Patterns of IgE reactivity of individual sera from 22 allergic patients were assessed using an aqueous short ragweed pollen extract resolved over 2D-gels. Combined with information derived from the annotated pollen proteome, those analyses revealed the presence of multiple unreported IgE reactive proteins, including new Amb a 1 and Amb a 3 isoallergens as well as 7 novel candidate allergens reacting with IgEs from 20-70% of patients. The latter encompass members of the carbonic anhydrase, enolase, galactose oxidase, GDP dissociation inhibitor, pathogenesis related-17, polygalacturonase and UDP-glucose pyrophosphorylase families. CONCLUSIONS: We extended the list of allergens identified in short ragweed pollen. These findings have implications for both diagnosis and allergen immunotherapy purposes.


Assuntos
Alérgenos/imunologia , Ambrosia/imunologia , Imunoglobulina E/imunologia , Proteínas de Plantas/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Alérgenos/química , Alérgenos/isolamento & purificação , Ambrosia/química , Feminino , Perfilação da Expressão Gênica , Humanos , Imunoglobulina E/química , Masculino , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Pólen/química , Proteômica
11.
J Allergy Clin Immunol ; 136(4): 1055-64, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25865353

RESUMO

BACKGROUND: Allergy to pollen from short ragweed (Ambrosia artemisiifolia) is a serious and expanding health problem in the United States and in Europe. OBJECTIVE: We sought to investigate the presence of undescribed allergens in ragweed pollen. METHODS: Ragweed pollen proteins were submitted to high-resolution gel electrophoresis and tested for IgE reactivity by using sera from 92 American or European donors with ragweed allergy. Pollen transcriptome sequencing, mass spectrometry (MS), and recombinant DNA technologies were applied to characterize new IgE-binding proteins. RESULTS: High-resolution IgE immunoblotting experiments revealed that 50 (54%) of 92 patients with ragweed allergy were sensitized to a 37-kDa allergen distinct from Amb a 1. The full-length cDNA sequence for this molecule was obtained by means of PCR cloning after MS sequencing of the protein combined with ragweed pollen RNA sequencing. The purified allergen, termed Amb a 11, was fully characterized by MS and confirmed to react with IgEs from 66% of patients. This molecule is a 262-amino-acid thiol protease of the papain family expressed as a combination of isoforms and glycoforms after proteolytic removal of N- and C-terminal propeptides from a proform. Three-dimensional modeling revealed a high structural homology with known cysteine proteases, including the mite Der p 1 allergen. The protease activity of Amb a 11, as well as its capacity to activate basophils from patients with ragweed allergy, were confirmed. The production of a nonglycosylated recombinant form of Amb a 11 in Escherichia coli established that glycosylation is not required for IgE binding. CONCLUSION: We identified the cysteine protease Amb a 11 as a new major allergen from ragweed pollen. Given the similar physicochemical properties shared by the 2 major allergens, we hypothesize that part of the allergenic activity previously ascribed to Amb a 1 is rather borne by Amb a 11.


Assuntos
Ambrosia , Cisteína Proteases , Proteínas de Plantas , Rinite Alérgica Sazonal/imunologia , Ambrosia/enzimologia , Ambrosia/genética , Ambrosia/imunologia , Sequência de Bases , Clonagem Molecular , Cisteína Proteases/genética , Cisteína Proteases/imunologia , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia
12.
Int Arch Allergy Immunol ; 168(3): 150-60, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26797104

RESUMO

BACKGROUND: The in-depth characterization of the recently identified house dust mite (HDM) major allergen Der p 23 requires the production of its recombinant counterpart because the natural allergen is poorly extractable from fecal pellets. This study aimed to provide a detailed physico-chemical characterization of recombinant Der p 23 (rDer p 23) as well as to investigate its IgE reactivity in a cohort of HDM-allergic patients from Thailand. METHODS: Purified rDer p 23, secreted from recombinant Pichia pastoris, was characterized by mass spectrometry and circular dichroism analyses as well as for its chitin-binding activity. The IgE-binding frequency and allergenicity of Der p 23 were determined by ELISA and RBL-SX38 degranulation assays, respectively. RESULTS: Purified intact rDer p 23 carried O-mannosylation and mainly adopted a random coil structure. Polyclonal antibodies to rDer p 23 can detect the corresponding natural allergen (nDer p 23) in aqueous fecal pellet extracts, suggesting that both forms of Der p 23 share common B-cell epitopes. Despite its homologies with chitin-binding proteins, both natural Der p 23 and rDer p 23 were unable to interact in vitro with chitin matrices. Of 222 Thai HDM-allergic patients tested, 54% displayed Der p 23-specific IgE responses. Finally, the allergenicity of rDer p 23 was confirmed by the degranulation of rat basophil leukemia cells. CONCLUSION: Our findings highlighted important levels of Der p 23 sensitizations in Thailand. Our study clearly suggested that rDer p 23 is likely more appropriate for HDM allergy component-resolved diagnosis than HDM extracts.


Assuntos
Antígenos de Dermatophagoides/imunologia , Quitina/metabolismo , Imunoglobulina E/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Dicroísmo Circular , Glicosilação , Humanos , Dados de Sequência Molecular , Ratos
13.
Protein Expr Purif ; 101: 8-13, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24874917

RESUMO

BACKGROUND: The development of recombinant house dust mite (HDM) allergens opened the way for the in-depth characterization of these molecules but also provided new opportunities to refine the diagnostic procedures of HDM allergy as well as the allergen-specific immunotherapy through tailor-made treatments. OBJECTIVE: In the present study, the HDM allergen Der p 21 was expressed in Pichia pastoris under a secreted form. The physico-chemical as well as the allergenic characterizations of recombinant Der p 21 (rDer p 21) were performed. METHODS: Purified rDer p 21, secreted from recombinant P. pastoris was characterized by CD and MS analysis and the frequency of IgE reactivity was determined by ELISA using 96 sera of HDM-allergic patients from Bangkok. The direct airway epithelial cell activation by rDer p 21 was also evaluated. RESULTS: rDer p 21 was highly expressed under a secreted form in P. pastoris. The physico-chemical characterization of purified rDer p 21 showed that the allergen displayed appropriate α-helix secondary structure content although a two amino acids truncation at the N-terminus of the protein was evidenced by MS. The prevalence of IgE reactivity to rDer p 21 reached 25% in the cohort of the HDM-allergic patients. rDer p 21 could trigger IL-8 production in airway epithelial cells through TLR2-dependent signaling. CONCLUSION: Properly folded rDer p 21 produced in P. pastoris is appropriate for HDM allergy diagnosis as well for future recombinant allergen-based specific immunotherapy.


Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Pichia/genética , Alérgenos/biossíntese , Alérgenos/genética , Animais , Antígenos de Dermatophagoides/biossíntese , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Clonagem Molecular , Humanos , Imunoglobulina E/imunologia , Interleucina-8/biossíntese , Pichia/metabolismo , Estrutura Secundária de Proteína , Pyroglyphidae/genética , Proteínas Recombinantes/genética , Mucosa Respiratória/imunologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologia
14.
J Allergy Clin Immunol ; 129(4): 1020-30, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22464673

RESUMO

BACKGROUND: Given their pivotal role in the polarization of T-cell responses, molecular changes at the level of dendritic cells (DCs) could represent an early signature indicative of the subsequent orientation of adaptive immune responses during immunotherapy. OBJECTIVE: We sought to investigate whether markers of effector and regulatory DCs are affected during allergen immunotherapy in relationship with clinical benefit. METHODS: Differential gel electrophoresis and label-free mass spectrometry approaches were used to compare whole proteomes from human monocyte-derived DCs differentiated toward either regulatory or effector functions. The expression of those markers was assessed by using quantitative PCR in PBMCs from 79 patients with grass pollen allergy enrolled in a double-blind, placebo-controlled clinical study evaluating the efficacy of sublingual tablets in an allergen exposure chamber over a 4-month period. RESULTS: We identified several markers associated with DC1 and/or DC17 effector DCs, including CD71, FSCN1, IRF4, NMES1, MX1, TRAF1. A substantial phenotypic heterogeneity was observed among various types of tolerogenic DCs, with ANXA1, Complement component 1 (C1Q), CATC, GILZ, F13A, FKBP5, Stabilin-1 (STAB1), and TPP1 molecules established as shared or restricted regulatory DC markers. The expression of 2 of those DCs markers, C1Q and STAB1, was increased in PBMCs from clinical responders in contrast to that seen in nonresponders or placebo-treated patients. CONCLUSION: C1Q and STAB1 represent candidate biomarkers of early efficacy of allergen immunotherapy as the hallmark of a regulatory innate immune response predictive of clinical tolerance.


Assuntos
Biomarcadores/metabolismo , Células Dendríticas/imunologia , Dessensibilização Imunológica/métodos , Administração Sublingual , Células Dendríticas/classificação , Células Dendríticas/metabolismo , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Hipersensibilidade/terapia , Tolerância Imunológica/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Proteoma/metabolismo , Resultado do Tratamento
15.
J Biol Chem ; 285(42): 32251-63, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20592021

RESUMO

C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca(2+)-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340-19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB(1)-EGF-CUB(2) interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model.


Assuntos
Complemento C1/química , Complemento C1r/química , Complemento C1s/química , Espectrometria de Massas/métodos , Estrutura Quaternária de Proteína , Sequência de Aminoácidos , Sítios de Ligação , Ativação do Complemento , Complemento C1/genética , Complemento C1/metabolismo , Complemento C1r/genética , Complemento C1r/metabolismo , Complemento C1s/genética , Complemento C1s/metabolismo , Humanos , Lisina/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Coloração e Rotulagem/métodos , Propriedades de Superfície
16.
Glycobiology ; 17(9): 932-44, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17591618

RESUMO

Human complement factor H (CFH) is a plasma glycoprotein involved in the regulation of the alternative pathway of the complement system. A deficiency in CFH is a cause of severe pathologies like atypical haemolytic uraemic syndrome (aHUS). CFH is a 155-kDa glycoprotein containing nine potential N-glycosylation sites. In the current study, we present a quantitative glycosylation analysis of CFH using capillary electrophoresis and a complete site-specific N-glycan characterization using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS). A 17.9-kDa mass decrease, observed after glycosidase treatment, indicated that N-glycosylation is the major post-translational modification of CFH. This mass difference is consistent with CFH glycosylation by diantennary disialylated glycans of 2204 Da on eight sites. CFH was not sensitive to endoglycosidase H (Endo H) deglycosylation, indicating the absence of hybrid and oligomannose structures. Quantitative analysis showed that CFH is mainly glycosylated by complex, diantennary disialylated, non-fucosylated glycans. Disialylated fucosylated and monosialylated non-fucosylated oligosaccharides were also identified. MS analysis allowed complete characterization of the protein backbone, verification of the glycosylation sites and site-specific N-glycan identification. The absence of glycosylation at Asn199 of the NGSP sequence of CFH is shown. Asn511, Asn700, Asn784, Asn804, Asn864, Asn893, Asn1011 and Asn1077 are glycosylated essentially by diantennary disialylated structures with a relative distribution varying between 45% for Asn804 and 75% for Asn864. Diantennary monosialylated glycans and triantennary trisialylated fucosylated and non-fucosylated structures have also been identified. Interestingly, the sialylation level along with the amount of triantennary structures decreases from the N- to the C-terminal side of the protein.


Assuntos
Fator H do Complemento/química , Polissacarídeos/química , Sequência de Aminoácidos , Cromatografia Líquida , Eletroforese Capilar , Glicosilação , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Oligossacarídeos/química , Mapeamento de Peptídeos , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo
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