| 1. |
- Guy, Jodie E, et al.
(författare)
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New insights into multiple coagulation factor deficiency from the solution structure of human MCFD2.
- 2008
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 381:4, s. 941-55
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Tidskriftsartikel (refereegranskat)abstract
- Human MCFD2 (multiple coagulation factor deficiency 2) is a 16-kDa protein known to participate in transport of the glycosylated human coagulation factors V and VIII along the secretory pathway. Mutations in MCFD2 or in its binding partner, the membrane-bound transporter ERGIC (endoplasmic reticulum-Golgi intermediate compartment)-53, cause a mild form of inherited hemophilia known as combined deficiency of factors V and VIII (F5F8D). While ERGIC-53 is known to be a lectin-type mannose binding protein, the role of MCFD2 in the secretory pathway is comparatively unclear. MCFD2 has been shown to bind both ERGIC-53 and the blood coagulation factors, but little is known about the binding sites or the true function of the protein. In order to facilitate understanding of the function of MCFD2 and the mechanism by which mutations in the protein cause F5F8D, we have determined the structure of human MCFD2 in solution by NMR. Our results show the folding of MCFD2 to be dependent on availability of calcium ions. The protein, which is disordered in the apo state, folds upon binding of Ca(2+) to the two EF-hand motifs of its C-terminus, while retaining some localized disorder in the N-terminus. NMR studies on two disease-causing mutant variants of MCFD2 show both to be predominantly disordered, even in the presence of calcium ions. These results provide an explanation for the previously observed calcium dependence of the MCFD2-ERGIC-53 interaction and, furthermore, clarify the means by which mutations in this protein result in inefficient secretion of blood coagulation factors V and VIII.
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| 2. |
- Idborg, Helena, et al.
(författare)
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Circulating Levels of Interferon Regulatory Factor-5 Associates With Subgroups of Systemic Lupus Erythematosus Patients
- 2019
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Ingår i: Frontiers in Immunology. - : FRONTIERS MEDIA SA. - 1664-3224. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann-Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (p(adjusted) = 3 x 10(-9), 3 x 10(-6), and 5 x 10(-6) respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identified three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by flow cytometry in a subset of patients to confirm the presence of IRF5 in plasma and detection of extracellular IRF5 was further confirmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials.
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| 3. |
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| 4. |
- Notarnicola, Antonella, et al.
(författare)
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Longitudinal assessment of reactivity and affinity profile of anti-Jo1 autoantibodies to distinct HisRS domains and a splice variant in a cohort of patients with myositis and anti-synthetase syndrome
- 2022
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Ingår i: Arthritis Research & Therapy. - : Springer Nature. - 1478-6354 .- 1478-6362. ; 24:1
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Tidskriftsartikel (refereegranskat)abstract
- Background To address the reactivity and affinity against histidyl-transfer RNA synthetase (HisRS) autoantigen of anti-Jo1 autoantibodies from serum and bronchoalveolar lavage fluid (BALF) in patients with idiopathic inflammatory myopathies/anti-synthetase syndrome (IIM/ASSD). To investigate the associations between the reactivity profile and clinical data over time. Methods Samples and clinical data were obtained from (i) 25 anti-Jo1(+) patients (19 sera with 16 longitudinal samples and 6 BALF/matching sera at diagnosis), (ii) 29 anti-Jo1(-) patients (25 sera and 4 BALF/matching sera at diagnosis), and (iii) 27 age/gender-matched healthy controls (24 sera and 3 BALF/matching sera). Reactivity towards HisRS full-length (HisRS-FL), three HisRS domains (WHEP, antigen binding domain (ABD), and catalytic domain (CD)), and the HisRS splice variant (SV) was tested. Anti-Jo1 IgG reactivity was evaluated by ELISA and western blot using IgG purified from serum by affinity chromatography. In paired serum-BALF, anti-Jo1 IgG and IgA reactivity was analyzed by ELISA. Autoantibody affinity was measured by surface plasmon resonance using IgG purified from sera. Correlations between autoantibody reactivity and clinical data were evaluated at diagnosis and longitudinally. Results Anti-Jo1 IgG from serum and BALF bound HisRS-FL, WHEP, and SV with high reactivity at the time of diagnosis and recognized both conformation-dependent and conformation-independent HisRS epitopes. Anti-HisRS-FL IgG displayed high affinity early in the disease. At the time of IIM/ASSD diagnosis, the highest autoantibody levels against HisRS-FL were found in patients ever developing interstitial lung disease (ILD) and arthritis, but with less skin involvement. Moreover, the reactivity of anti-WHEP IgG in BALF correlated with poor pulmonary function. Levels of autoantibodies against HisRS-FL, HisRS domains, and HisRS splice variant generally decreased over time. With some exceptions, longitudinal anti-HisRS-FL antibody levels changed in line with ILD activity. Conclusion High levels and high-affinity anti-Jo1 autoantibodies towards HisRS-FL were found early in disease in sera and BALF. In combination with the correlation of anti-HisRS-FL antibody levels with ILD and ILD activity in longitudinal samples as well as of anti-WHEP IgG in BALF with poor pulmonary function, this supports the previously raised hypothesis that the lung might have a role in the immune reaction in anti-Jo1-positive patients.
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| 5. |
- Preger, Charlotta, et al.
(författare)
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Autoantigenic properties of the aminoacyl tRNA synthetase family in idiopathic inflammatory myopathies
- 2023
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Ingår i: Journal of Autoimmunity. - : Elsevier BV. - 0896-8411 .- 1095-9157. ; 134
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: Autoantibodies are thought to play a key role in the pathogenesis of idiopathic inflammatory my-opathies (IIM). However, up to 40% of IIM patients, even those with clinical manifestations of anti-synthetase syndrome (ASSD), test seronegative to known myositis-specific autoantibodies. We hypothesized the existence of new potential autoantigens among human cytoplasmic aminoacyl tRNA synthetases (aaRS) in patients with IIM.Methods: Plasma samples from 217 patients with IIM according to 2017 EULAR/ACR criteria, including 50 pa-tients with ASSD, 165 without, and two with unknown ASSD status were identified retrospectively, as well as age and gender-matched sera from 156 population controls, and 219 disease controls. Patients with previously documented ASSD had to test positive for at least one of the five most common anti-aaRS autoantibodies (anti-Jo1,-PL7,-PL12,-EJ, and-OJ) and present with one or more of the following clinical manifestations: interstitial lung disease, myositis, arthritis, Raynaud's phenomenon, fever, or mechanic's hands. Demographics, laboratory, and clinical data of the IIM cohort (ASSD and non-ASSD) were compared. Samples were screened using a multiplex bead array assay for presence of autoantibodies against a panel of 117 recombinant protein variants, representing 33 myositis-related proteins, including all nineteen cytoplasmic aaRS. Prospectively collected clinical data for the IIM cohort were retrieved and compared between groups within the IIM cohort and correlated with the results of the autoantibody screening. Principal component analysis was used to analyze clinical manifestations between ASSD, non-ASSD groups, and individuals with novel anti-aaRS autoantibodies. Results: We identified reactivity towards 16 aaRS in 72 of the 217 IIM patients. Twelve patients displayed reactivity against nine novel aaRS. The novel autoantibody specificities were detected in four previously sero-negative patients for myositis-specific autoantibodies and eight with previously detected myositis-specific au-toantibodies. IIM individuals with novel anti-aaRS autoantibodies (n = 12) all had signs of myositis, and they had either muscle weakness and/or muscle enzyme elevation, 2/12 had mechanic's hands, 3/12 had interstitial lung disease, and 2/12 had arthritis. The individuals with novel anti-aaRS and a pathological muscle biopsy all presented widespread up-regulation of major histocompatibility complex class I. The reactivities against novel aaRS could be confirmed in ELISA and western blot. Using the multiplex bead array assay, we could confirm previously known reactivities to four of the most common aaRS (Jo1, PL12, PL7, and EJ (n = 45)) and identified patients positive for anti-Zo,-KS, and-HA (n = 10) that were not previously tested. A low frequency of anti-aaRS autoantibodies was also detected in controls.Conclusion: Our results suggest that most, if not all, cytoplasmic aaRS may become autoantigenic. Autoantibodies against new aaRS may be found in plasma of patients previously classified as seronegative with potential high clinical relevance.
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| 6. |
- Szykowska, Aleksandra, et al.
(författare)
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Selection and structural characterization of anti-TREM2 scFvs that reduce levels of shed ectodomain
- 2021
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 29:11, s. 1241-
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration, including Alzheimer's disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signaling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterized. Here, we present phage display generated antibody single-chain variable fragments (scFvs) to human TREM2 immunoglobulin-like domain. Co-crystal structures revealed the binding of two scFvs to an epitope on the TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species, which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterization of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.
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| 7. |
- Wigren, Edvard
(författare)
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Structural studies of the ERGIC-53/MCFD2 glycoprotein transport receptor complex
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The secretory pathway defines and maintains the intracellular architecture of the eukaryotic cell. Proteins targeted to either the plasma membrane, the extracellular medium or to specific organelles within the cell are dependent on the correct transfer along this pathway. The importance of its function has become increasingly clear and several proteins in this pathway have been linked to human diseases. One disease that is genetically associated with transport processes in the early secretory pathway is the combined blood coagulation factor V and VIII deficiency (F5F8D). It has been established that F5F8D is caused by mutations in the genes that code for the membrane bound glycoprotein receptor ERGIC-53 or its co-receptor protein MCFD2. Together these two proteins form a calcium dependent complex with 1:1 stoichiometry that specifically interacts and assist transport of the glycosylated blood coagulation proteins FV and FVIII from the endoplasmic reticulum. In this thesis, NMR-spectroscopy and X-ray crystallography have been applied in order to clarify the organisation of the ERGIC-53/MCFD2 glycoprotein transport receptor complex. The work resulted in the three-dimensional structure of MCFD2 in solution determined by NMR and the crystal structure of MCFD2 in complex with the carbohydrate recognition domain of ERGIC-53. These structures gave a first molecular view of the organisation of a cargo receptor complex in the early secretory pathway and revealed that MCFD2 undergoes significant conformational changes upon complex formation. NMR and CD-spectroscopy analysis showed MCFD2 to be disordered in the absence of Ca2+ ions, but to adopt a predominantly ordered structure upon binding Ca2+ ions. Hence, these data suggest that calcium binding and consequent folding of MCFD2 to be the underlying mechanism for the previously observed calcium dependence of the MCFD2/ERGIC-53 interaction. Moreover, the consequences of all known F5F8D causing missense mutations found in MCFD2 could be explained at a molecular level. The results highlight the importance of intact calcium binding EF-hand motifs for the structural stability of MCFD2 and point toward disruption of the ERGIC-53/MCFD2 interaction as the underlying mechanism for these mutations in causing F5F8D. Overall, this thesis work has provided new insights into the structural organisation of the ERGIC-53/MCFD2 transport receptor complex and highlighted the importance of calcium for the regulation of its function. By studying this complex the hope is that we have shed some light, not only on the mechanisms behind transport of FV and FVIII by ERGIC-53 and MCFD2, but also on general processes underlying the assisted glycoprotein transport in the early secretory pathway
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