| 1. |
- Samgina, Tatiana Y., et al.
(författare)
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Collision-Induced Dissociation Fragmentation Inside Disulfide C-Terminal Loops of Natural Non-Tryptic Peptides
- 2013
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Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305 .- 1879-1123. ; 24:7, s. 1037-1044
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Tidskriftsartikel (refereegranskat)abstract
- Collision-induced dissociation (CID) spectra of long non-tryptic peptides are usually quite complicated and rather difficult to interpret. Disulfide bond formed by two cysteine residues at C-terminus of frog skin peptides precludes one to determine sequence inside the forming loop. Thereby, chemical modification of S-S bonds is often used in "bottom up" sequencing approach. However, low-energy CID spectra of natural non-tryptic peptides with C-terminal disulfide cycle demonstrate an unusual fragmentation route, which may be used to elucidate the "hidden" C-terminal sequence. Low charge state protonated molecules experience peptide bond cleavage at the N-terminus of C-terminal cysteine. The forming isomeric acyclic ions serve as precursors for a series of b-type ions revealing sequence inside former disulfide cycle. The reaction is preferable for peptides with basic lysine residues inside the cycle. It may also be activated by acidic protons of Asp and Glu residues neighboring the loop. The observed cleavages may be quite competitive, revealing the sequence inside disulfide cycle, although S-S bond rupture does not occur in this case.
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| 2. |
- Albrecht, Inka, et al.
(författare)
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Development of autoantibodies against muscle-specific FHL1 in severe inflammatory myopathies
- 2015
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Ingår i: Journal of Clinical Investigation. - : AMER SOC CLINICAL INVESTIGATION INC. - 0021-9738 .- 1558-8238. ; 125:12, s. 4612-4624
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Tidskriftsartikel (refereegranskat)abstract
- Mutations of the gene encoding four-and-a-half LIM domain 1 (FHL1) are the causative factor of several X-linked hereditary myopathies that are collectively termed FHL1-related myopathies. These disorders are characterized by severe muscle dysfunction and damage. Here, we have shown that patients with idiopathic inflammatory myopathies (IIMs) develop autoimmunity to FHL1, which is a muscle-specific protein. Anti-FHL1 autoantibodies were detected in 25% of IIM patients, while patients with other autoimmune diseases or muscular dystrophies were largely anti-FHL1 negative. Anti-FHL1 reactivity was predictive for muscle atrophy, dysphagia, pronounced muscle fiber damage, and vasculitis. FHL1 showed an altered expression pattern, with focal accumulation in the muscle fibers of autoantibody-positive patients compared with a homogeneous expression in anti-FHL1-negative patients and healthy controls. We determined that FHL1 is a target of the cytotoxic protease granzyme B, indicating that the generation of FHL1 fragments may initiate FHL1 autoimmunity. Moreover, immunization of myositis-prone mice with FHL1 aggravated muscle weakness and increased mortality, suggesting a direct link between anti-FHL1 responses and muscle damage. Together, our findings provide evidence that FHL1 may be involved in the pathogenesis not only of genetic FHL1-related myopathies but also of autoimmune IIM. Importantly, these results indicate that anti-FHL1 autoantibodies in peripheral blood have promising potential as a biomarker to identify a subset of severe IIM.
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| 3. |
- Baumgarten, Thomas, et al.
(författare)
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Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress
- 2018
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 84:12
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Tidskriftsartikel (refereegranskat)abstract
- In Escherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coli.IMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.
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| 4. |
- Celorio-Mancera, Maria de la Paz, 1978-, et al.
(författare)
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Chemosensory proteins, major salivary factors in caterpillar mandibular glands
- 2012
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Ingår i: Insect Biochemistry and Molecular Biology. - : Elsevier BV. - 0965-1748 .- 1879-0240. ; 42:10, s. 796-805
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Tidskriftsartikel (refereegranskat)abstract
- Research in the field of insect-host plant interactions has indicated that constituents of insect saliva play an important role in digestion and affect host chemical defense responses. However, most efforts have focused on studying the composition and function of regurgitant or saliva produced in the labial glands. Acknowledging the need for understanding the role of the mandibular glands in herbivory, we sought to make a qualitative and semi-quantitative comparison of soluble luminal fractions between mandibular and labial glands of Vanessa gonerilla butterfly larvae. Amylase and lysozyme were inspected as possible major enzymatic activities in the mandibular glands aiding in pre-digestion and antimicrobial defense. Although detected, neither of these enzymatic activities was prominent in the luminal protein preparation of a particular type of gland. Proteins isolated from the glands were identified by mass spectrometry and by searching an EST-library database generated for four other nymphalid butterfly species, in addition to the public NCBI database. The identified proteins were also quantified from thedata using “Quanty”, an in-house program. The proteomic analysis detected chemosensory proteins as the most abundant luminal proteins in the mandibular glands. In comparison to these proteins, the relative amounts of amylase and lysozyme were much lower in both gland types. Therefore, we speculate that the primary role of the mandibular glands in Lepidopteran larvae is chemoreception which may include the detection of microorganisms on plant surfaces, host plant recognition and communication with conspecifics.
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| 5. |
- Celorio-Mancera, Maria de la Paz, 1978-, et al.
(författare)
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Effect of host plant and immune challenge on the levels of chemosensory and odorant-binding proteins in caterpillar salivary glands
- 2015
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Ingår i: Insect Biochemistry and Molecular Biology. - : Elsevier BV. - 0965-1748 .- 1879-0240. ; 61, s. 34-45
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Tidskriftsartikel (refereegranskat)abstract
- More than half of the proteome from mandibular glands in caterpillars is represented by chemosensory proteins. Based on sequence similarity, these proteins are putative transporters of ligands to gustatory receptors in sensory organs of insects. We sought to determine whether these proteins are inducible by comparing, both qualitatively and quantitatively, the salivary (mandibular and labial) proteomes from caterpillars (Vanessa cardui) reared on different plants and artificial diet containing either bacteria or bacterial cell-walls. We included a treatment where the caterpillars were switched from feeding on artificial diet to plant material at some point in their development. Additionally, we evaluated the degree of overlap between the proteomes in the hemolymph-filled coelom and salivary glands of caterpillars reared on plant material. We found that the quality and quantity of the identified proteins differed clearly between hemolymph-filled coelome, labial and mandibular glands. Our results indicated that even after molting and two-day feeding on a new diet, protein production is affected by the previous food source used by the caterpillar. Candidate proteins involved in chemosensory perception by insects were detected: three chemosensory (CSPs) and two odorant-binding proteins (OBPs). Using the relative amounts of these proteins across tissues and treatments as criteria for their classification, we detected hemolymph- and mandibular gland-specific CSPs and observed that their levels were affected by caterpillar diet. Moreover, we could compare the protein and transcript levels across tissues and treatment for at least one CSP and one OBP. Therefore, we have identified specific isoforms for testing the role of CSPs and OBPs in plant and pathogen recognition. We detected catalase, immune-related protein and serine proteases and their inhibitors in high relative levels in the mandibular glands in comparison to the labial glands. These findings suggest that the mandibular glands of caterpillars may play an important role protecting the caterpillar from oxidative stress, pathogens and aiding in digestion. Contamination with hemolymph proteins during dissection of salivary glands from caterpillars may occur but it is not substantial since the proteomes from hemolymph, mandibular and labial glands were easily discriminated from each other by principal component analysis of proteomic data.
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| 6. |
- Gerstner, Christina, et al.
(författare)
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Functional and Structural Characterization of a Novel HLA-DRB1*04:01-Restricted alpha-Enolase T Cell Epitope in Rheumatoid Arthritis
- 2016
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies to citrullinated proteins, common in rheumatoid arthritis (RA) patients, are strongly associated to a specific set of HLA-DR alleles including HLA-DRB1*04:01, *04:04, and *01:01. Here, we first demonstrate that autoantibody levels toward the dominant citrullinated B cell epitope from alpha-enolase are significantly elevated in HLA-DRB1*04:01-positive RA patients. Furthermore, we identified alpha-enolase-derived T cell epitopes and demonstrated that native and citrullinated versions of several peptides bind with different affinities to HLA-DRB1*04:01, *04:04, and *01:01. The citrulline residues in the eight identified peptides are distributed throughout the entire length of the presented epitopes and more specifically, localized at peptide positions p-2, p2, p4, p6, p7, p10, and p11. Importantly, in contrast to its native version peptide 26 (TSKGLFRAAVPSGAS), the HLA-DRB1*04:01-restricted citrullinated peptide Cit26 (TSKGLFCitAAVPSGAS) elicited significant functional T cell responses in primary cells from RA patients. Comparative analysis of the crystal structures of HLA-DRB1*04:01 in complex with peptide 26 or Cit26 demonstrated that the posttranslational modification did not alter the conformation of the peptide. And since citrullination is the only structural difference between the two complexes, this indicates that the neo-antigen Cit26 is recognized by T cells with high specificity to the citrulline residue.
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| 7. |
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| 8. |
- Ytterberg, A. Jimmy, et al.
(författare)
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Posttranslational Targeting of a Recombinant Protein Promotes Its Efficient Secretion into the Escherichia coli Periplasm
- 2019
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Ingår i: Applied and Environmental Microbiology. - : AMER SOC MICROBIOLOGY. - 0099-2240 .- 1098-5336. ; 85:13
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Tidskriftsartikel (refereegranskat)abstract
- Many recombinant proteins that are produced in Escherichia coli have to be targeted to the periplasm to be functional. N-terminal signal peptides can be used to direct recombinant proteins to the membrane-embedded Sec translocon, a multiprotein complex that translocates proteins across the membrane into the periplasm. We have recently shown that the cotranslational targeting of the single-chain variable antibody fragment BL1 saturates the capacity of the Sec translocon leading to impaired translocation of secretory proteins and protein misfolding/aggregation in the cytoplasm. In turn, protein production yields and biomass formation were low. Here, we study the consequences of targeting BL1 posttranslationally to the Sec translocon. Notably, the posttranslational targeting of BL1 does not saturate the Sec translocon capacity, and both biomass formation and protein production yields are increased. Analyzing the proteome of cells producing the posttranslationally targeted BL1 indicates that the decreased synthesis of endogenous secretory and membrane proteins prevents a saturation of the Sec translocon capacity. Furthermore, in these cells, highly abundant chaperones and proteases can clear misfolded/aggregated proteins from the cytoplasm, thereby improving the fitness of these cells. Thus, the posttranslational targeting of BL1 enables its efficient production in the periplasm due to a favorable adaptation of the E. coli proteome. We envisage that our observations can be used to engineer E. coli for the improved production of recombinant secretory proteins. IMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. To fold properly, many recombinant proteins have to be targeted to the E. coli periplasm, but so far the impact of the targeting pathway of a recombinant protein to the periplasm has not been extensively investigated. Here, we show that the targeting pathway of a recombinant antibody fragment has a tremendous impact on cell physiology, ultimately affecting protein production yields in the periplasm and biomass formation. This indicates that studying the targeting and secretion of proteins into the periplasm could be used to design strategies to improve recombinant protein production yields.
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| 9. |
- Ytterberg, A Jimmy, et al.
(författare)
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Shared immunological targets in the lungs and joints of patients with rheumatoid arthritis : identification and validation
- 2015
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ. - 0003-4967 .- 1468-2060. ; 74:9, s. 1772-1777
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: Immunological events in the lungs might trigger production of anti-citrullinated protein antibodies during early rheumatoid arthritis (RA). We investigated the presence of shared immunological citrullinated targets in joints and lungs of patients with RA.PATIENTS AND METHODS: Proteins extracted from bronchial (n=6) and synovial (n=7) biopsy specimens from patients with RA were investigated by mass spectrometry-based proteomics. One candidate peptide was synthesised and used to investigate by ELISA the presence of antibodies in patients with RA (n=393), healthy controls (n=152) and disease controls (n=236). HLA-DRB1 shared epitope (SE) alleles were detected in patients with RA.RESULTS: Ten citrullinated peptides belonging to seven proteins were identified, with two peptides shared between the synovial and bronchial biopsy samples. Further analysis, using accurate mass and retention time, enabled detection of eight citrullinated peptides in synovial and seven in bronchial biopsy specimens, with five peptides shared between the synovial and bronchial biopsy specimens. Two citrullinated vimentin (cit-vim) peptides were detected in the majority of synovial and lung tissues. Antibodies to a synthesised cit-vim peptide candidate (covering both cit-vim peptides identified in vivo) were present in 1.8% of healthy controls, 15% of patients with RA, and 3.4% of disease controls. Antibodies to cit-vim peptide were associated with the presence of the SE alleles in RA.CONCLUSIONS: Identical citrullinated peptides are present in bronchial and synovial tissues, which may be used as immunological targets for antibodies of patients with RA. The data provide further support for a link between lungs and joints in RA and identify potential targets for immunity that may mediate this link.
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| 10. |
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