| 1. |
- Baeckström, Dan, 1956, et al.
(författare)
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Expression of the leukocyte-associated sialoglycoprotein CD43 by a colon carcinoma cell line
- 1995
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258. ; 270, s. 13688-13692
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Tidskriftsartikel (refereegranskat)abstract
- The colon adenocarcinoma cell line COLO 205 secretes L-CanAg, a mucin- like glycoprotein carrying the carcinoma-associated sialyl-Lewis a carbohydrate epitope. In an attempt to identify its apoprotein, an NH2- terminal peptide sequence was obtained from purified L-CanAg. In all interpretable positions, this sequence showed 100% identity to the NH2- terminal of human CD43 (leukosialin, sialophorin), a plasma membrane-bound sialoglycoprotein hitherto only identified in leukocytes and other hematopoietic cells. An antiserum against deglycosylated L-CanAg and an anti- CD43 antiserum both immunoprecipitated a 61-kDa band, interpreted as the CD43 precursor, from COLO 205 cells as well as from the known CD43-expressing cell line HL-60. Results from immunoprecipitations following pulse-chase experiments and tunicamycin treatments were in agreement with earlier studies on the CD43 precursor. RNA blot analysis confirmed the expression of CD43 by the COLO 205 cell line, whereas three other colon carcinoma cell lines were negative. The glycosylation-dependent monoclonal antibody Leu-22, which recognizes leukocyte CD43, failed to bind L-CanAg, probably due to its much more extensive glycosylation. We conclude that L-CanAg is the secreted extracellular domain of a novel glycoform of CD43 and that CD43, if expressed in other carcinoma cells, may have escaped notice in studies relying on glycosylation-dependent monoclonal antibodies against leukocyte CD43.
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| 2. |
- Boreström, Cecilia, 1974, et al.
(författare)
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E2F1, ARID3A/Bright and Oct-2 factors bind to the Epstein-Barr virus C promoter, EBNA1 and oriP, participating in long-distance promoter-enhancer interactions.
- 2012
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Ingår i: The Journal of general virology. - : Microbiology Society. - 1465-2099 .- 0022-1317. ; 93, s. 1065-75
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Tidskriftsartikel (refereegranskat)abstract
- The Epstein-Barr virus (EBV) C promoter (Cp) regulates several genes required for B-cell proliferation in latent EBV infection. The family of repeats (FR) region of the latent origin of plasmid replication (oriP) functions as an Epstein-Barr nuclear antigen 1 (EBNA1)-dependent distant enhancer of Cp activity, and the enhancer-promoter interaction is mediated by a higher-order multi-protein complex containing several copies of EBNA1. Using DNA-affinity purification with a 170 bp region of the Cp in combination with mass spectrometry, we identified the cell cycle-regulatory protein E2F1, the E2F-binding protein ARID3A, and the B-cell-specific transcription factor Oct-2 as components of this multi-protein complex. Binding of the three factors to the FR region of oriP was determined by DNA-affinity and immunoblot analysis. Co-immunoprecipitation and proximity ligation analysis revealed that the three factors, E2F1, ARID3A and Oct-2, interact with each other as well as with EBNA1 in the nuclei of EBV-positive cells. Using the chromatin immunoprecipitation assay, we showed that E2F1 and Oct-2 interacted with the FR part of oriP and the Cp, but the ARID3A interaction was, however, only detected at the Cp. Our findings support the hypothesis that EBNA1 initiates transcription at the Cp via interactions between multiple EBNA1 homodimers and cellular transcription factors in a large molecular machinery that forms a dynamic interaction between Cp and FR.
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| 3. |
- Brinkmalm, Gunnar, et al.
(författare)
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An online nano-LC-ESI-FTICR-MS method for comprehensive characterization of endogenous fragments from amyloid β and amyloid precursor protein in human and cat cerebrospinal fluid.
- 2012
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Ingår i: Journal of mass spectrometry : JMS. - : Wiley. - 1096-9888 .- 1076-5174. ; 47:5, s. 591-603
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid precursor protein (APP) is the precursor protein to amyloid β (Aβ), the main constituent of senile plaques in Alzheimer's disease (AD). Endogenous Aβ peptides reflect the APP processing, and greater knowledge of different APP degradation pathways is important to understand the mechanism underlying AD pathology. When one analyzes longer Aβ peptides by low-energy collision-induced dissociation tandem mass spectrometry (MS/MS), mainly long b-fragments are observed, limiting the possibility to determine variations such as amino acid variants or post-translational modifications (PTMs) within the N-terminal half of the peptide. However, by using electron capture dissociation (ECD), we obtained a more comprehensive sequence coverage for several APP/Aβ peptide species, thus enabling a deeper characterization of possible variants and PTMs. Abnormal APP/Aβ processing has also been described in the lysosomal storage disease Niemann-Pick type C and the major large animal used for studying this disease is cat. By ECD MS/MS, a substitution of Asp7 → Glu in cat Aβ was identified. Further, sialylated core 1 like O-glycans at Tyr10, recently discovered in human Aβ (a previously unknown glycosylation type), were identified also in cat cerebrospinal fluid (CSF). It is therefore likely that this unusual type of glycosylation is common for (at least) species belonging to the magnorder Boreoeutheria. We here describe a detailed characterization of endogenous APP/Aβ peptide species in CSF by using an online top-down MS-based method.
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| 4. |
- Brinkmalm-Westman, Ann, 1966, et al.
(författare)
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Proteomics/peptidomics tools to find CSF biomarkers for neurodegenerative diseases.
- 2009
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Ingår i: Frontiers in bioscience : a journal and virtual library. - : IMR Press. - 1093-4715. ; 14, s. 1793-806
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Forskningsöversikt (refereegranskat)abstract
- Neurodegenerative diseases are characterized by premature neuronal loss in specific brain regions. During the past decades our knowledge on molecular mechanisms underlying neurodegeneration has increased immensely and resulted in promising drug candidates that might slow down or even stop the neuronal loss. These advances have put a strong focus on the development of diagnostic tools for early or pre-clinical detection of the disorders. In this review we discuss our experience in the field of neuroproteomics/peptidomics, with special focus on biomarker discovery studies that have been performed on CSF samples from well-defined patient and control populations.
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| 5. |
- Constantinescu, Radu, 1966, et al.
(författare)
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Proteomic profiling of cerebrospinal fluid in parkinsonian disorders.
- 2010
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Ingår i: Parkinsonism & related disorders. - : Elsevier BV. - 1873-5126 .- 1353-8020. ; :16, s. 545-49
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Tidskriftsartikel (refereegranskat)abstract
- Parkinson's disease (PD) and atypical parkinsonian disorders (APD), including multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD), are a group of neurodegenerative diseases sharing many similar signs and symptoms but distinguished by their particular clinical features, treatment response, prognosis and mortality. The differential diagnosis may be challenging, especially in early disease stages. Considering the importance of an accurate diagnosis both for clinical management and for research, new diagnostic tools are needed. In this study, we investigated 56 PD, 42 MSA, 39 PSP, 9 CBD patients, and 24 healthy controls. After screening the cerebrospinal fluid (CSF) proteome using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), we identified 4 proteins (ubiquitin [mass-to-charge ratio (m/z) 8590], beta2-microglobulin [m/z 11730], and 2 secretogranin 1 [chromogranin B] fragments [m/z 7260 and m/z 6250]) that differentiated healthy controls and PD patients from patients with APD. However, they could not differentiate PD patients from controls. As none of these changes were APD subgroup-specific, they most likely reflect the intensity and/or extent of the neurodegenerative process in general.
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| 6. |
- Dufva, Martin, 1967, et al.
(författare)
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Epstein-Barr virus nuclear antigen 5 inhibits pre-mRNA cleavage and polyadenylation.
- 2002
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Ingår i: Nucleic acids research. - 1362-4962. ; 30:10, s. 2131-43
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Tidskriftsartikel (refereegranskat)abstract
- The long-standing suspicion that Epstein-Barr virus nuclear antigen 5 (EBNA5) is involved in transcription regulation was recently confirmed by the observation by several groups that EBNA5 cooperates with EBNA2 in activation of the LMP1 promoter. In attempts to elucidate the molecular basis for the EBNA5-mediated enhancement of EBNA2 transactivation, we obtained evidence of an additional function of EBNA5: at high but still biologically relevant levels, EBNA5 acted as a repressor of gene expression by interfering with the processing of pre-mRNA. Transient transfections with reporter plasmids revealed that EBNA5 repressed reporter mRNA and protein expression in the cytoplasm, but did not lower the steady-state level of reporter RNA in the total cellular RNA fraction. We have excluded that repression occurred as a consequence of cell death induced by EBNA5. Using the RNase protection assay with a probe comprising the pre-mRNA cleavage and polyadenylation site, EBNA5 was found to inhibit 3'-end cleavage and polyadenylation of pre-mRNAs from the reporter plasmids investigated. The effect of inhibitory levels of EBNA5 on chromosomal genes was examined in transient transfections by expression profiling using a cDNA microarray panel containing 588 genes. The results showed that EBNA5 could also inhibit the expression of chromosomal genes and did it in a discriminatory manner. This is consistent with the notion that a regulatory mechanism exists in the cell that confers specificity to the selection by EBNA5 of target genes for repression.
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| 7. |
- Ekman, Stina, et al.
(författare)
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Effect of circadian rhythm, age, training and acute lameness on serum concentrations of cartilage oligomeric matrix protein (COMP) neo-epitope in horses
- 2019
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Ingår i: Equine Veterinary Journal. - : Wiley. - 0425-1644 .- 2042-3306. ; 51:5, s. 674-680
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Tidskriftsartikel (refereegranskat)abstract
- Background Molecular serum markers that can identify early reversible osteoarthritis (OA) in horses are lacking. Objectives We studied serum concentrations of a novel cartilage oligomeric matrix protein (COMP) neo-epitope in horses subjected to short-term exercise and with acute lameness. The effects of circadian rhythm and age were also evaluated. Study design Longitudinal studies in healthy horses and cross-sectional comparison of lame and non-lame horses. Methods Sera were collected from five horses before and after short-term interval exercise and during full-day box rest. Sera from 32 acutely lame horses were used to evaluate age-related effects. Independent samples from control horses (n = 41) and horses with acute lameness (n = 71) were included. COMP neo-epitope concentrations were analysed using custom-developed inhibition ELISAs validated for equine serum. The presence of COMP neo-epitope was delineated in healthy and osteoarthritic articular cartilage with immunohistochemistry. Results COMP neo-epitope concentrations decreased after speed training but returned to baseline levels post-exercise. No correlations between age and serum COMP neo-epitope concentrations were found (r = 0.0013). The mean (+/- s.d.) serum concentration of COMP neo-epitope in independent samples from non-lame horses was 0.84 +/- 0.38 mu g/mL, and for lame horses was 5.24 +/- 1.83 mu g/mL (P<0.001). Antibodies against COMP neo-epitope did not stain normal articular cartilage, but intracytoplasmic staining was found in superficial chondrocytes of mild OA cartilage and in the extracellular matrix of moderately osteoarthritic cartilage. Main limitations ELISA was based on polyclonal antisera rather than a monoclonal antibody. There is a sex and breed bias within the groups of horses, also it could have been of value to include horses with septic arthritis and tendonitis and investigated joint differences. Conclusions This COMP neo-epitope can be measured in sera, and results indicate that it could be a biomarker for pathologic fragmentation of cartilage in connection with acute joint lameness.
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| 8. |
- Forsman, Alma, 1979, et al.
(författare)
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Identification of intracellular proteins associated with the EBV-encoded nuclear antigen 5 using an efficient TAP procedure and FT-ICR mass spectrometry.
- 2008
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Ingår i: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:6, s. 2309-19
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Tidskriftsartikel (refereegranskat)abstract
- Epstein-Barr virus nuclear antigen 5 (EBNA5) is one of the first viral proteins detected after primary EBV infection and has been shown to be required for efficient transformation of B lymphocytes. EBNA5 is a protein that has many suggested functions but the underlying biology remains to be clarified. To gain further insight into the biological roles of the proposed multifunctional EBNA5, we isolated EBNA5 containing protein complexes using a modified tandem affinity purification (TAP) method and identified the protein components by LC-MS/MS analysis of tryptic digests on a LTQ-FT-ICR mass spectrometer. The modified TAP tag contained a Protein A domain and a StrepTagII sequence separated by two Tobacco Etch Virus protease cleavage sites and was fused to the C-terminus of EBNA5. Our results confirmed the wide applicability of this two-step affinity purification strategy for purification of protein complexes in mammalian cells. A total of 147 novel putative EBNA5 interaction partners were identified, 37 of which were validated with LC-MS/MS in split-tag experiments or in co-immuno precipitates from HEK293 cell extracts. This subgroup included the Bcl2-associated Athanogene 2 (BAG2) co-chaperone involved in protein folding and renaturation, the 26S proteasome subunit 2 involved in regulation of ubiquitin/proteasome protein degradation, and the heterogeneous ribonucleoprotein M (hnRNP M) involved in pre-mRNA processing. These EBNA5 interactors were further verified by co-immunoprecipitations from cell extracts of three EBV-positive lymphoblastoid lines. The combination of the Hsp70, Hsc70, BAG2 and 26S proteasome subunit 2 interactors suggests that EBNA5 might have a functional relationship with protein quality control systems that recognize proteins with abnormal structures and either refold them to normal conformation or target them for degradation. Our study also confirms previously identified interactors including HA95, Hsp70, Hsc70, Hsp27, HAX-1, Prolyl 4-hydroxylase, S3a, and alpha- and beta-tubulin.
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| 9. |
- Granéli, Cecilia, 1981, et al.
(författare)
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Novel markers of osteogenic and adipogenic differentiation of human bone marrow stromal cells identified using a quantitative proteomics approach.
- 2014
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Ingår i: Stem cell research. - : Elsevier BV. - 1876-7753 .- 1873-5061. ; 12:1, s. 153-165
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Tidskriftsartikel (refereegranskat)abstract
- Today, the tool that is most commonly used to evaluate the osteogenic differentiation of bone marrow stromal cells (BMSCs) in vitro is the demonstration of the expression of multiple relevant markers, such as ALP, RUNX2 and OCN. However, as yet, there is no single surface marker or panel of markers which clearly defines human BMSCs (hBMSCs) differentiating towards the osteogenic lineage. The aim of this study was therefore to examine this issue. Stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was utilized to investigate differently expressed surface markers in osteogenically differentiated and undifferentiated hBMSCs. Labeled membrane proteins were analyzed by mass spectrometry (MS) and 52 proteins with an expression ratio above 2, between osteogenically differentiated and undifferentiated cells, were identified. Subsequent validation, by flow cytometry and ELISA, of the SILAC expression ratios for a number of these proteins and investigations of the lineage specificity of three candidate markers were performed. The surface markers, CD10 and CD92, demonstrated significantly increased expression in hBMSCs differentiated towards the osteogenic and adipogenic lineages. In addition, there was a slight increase in CD10 expression during chondrogenic differentiation. Furthermore, the expression of the intracellular protein, crystalline-αB (CRYaB), was only significantly increased in osteogenically differentiated hBMSCs and not affected during differentiation towards the chondrogenic or adipogenic lineages. It has been concluded from the present results that CD10 and CD92 are potential markers of osteogenic and adipogenic differentiation and that CRYaB is a potential novel osteogenic marker specifically expressed during the osteogenic differentiation of hBMSCs in vitro.
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| 10. |
- Halim, Adnan, et al.
(författare)
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Assignment of Saccharide Identities through Analysis of Oxonium Ion Fragmentation Profiles in LC-MS/MS of Glycopeptides.
- 2014
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Ingår i: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3907 .- 1535-3893. ; 13:12, s. 6024-32
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Tidskriftsartikel (refereegranskat)abstract
- Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure-function relationships is important to better understand these events. However, characterization of glycan and glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβ-O- structures. The difference in the oxonium ion fragmentation profiles of such glycopeptides may thus be used to distinguish among these glycan structures and could be of importance in LC-MS/MS-based glycoproteomic studies.
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