| 1. |
- Ganfornina, Maria D., et al.
(författare)
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Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress
- 2008
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Ingår i: Aging Cell. - : Wiley. - 1474-9726 .- 1474-9718. ; 7:4, s. 506-515
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Tidskriftsartikel (refereegranskat)abstract
- Many nervous system pathologies are associated with increased levels of apolipoprotein D (ApoD), a lipocalin also expressed during normal development and aging. An ApoD homologous gene in Drosophila, Glial Lazarillo, regulates resistance to stress, and neurodegeneration in the aging brain. Here we study for the first time the protective potential of ApoD in a vertebrate model organism. Loss of mouse ApoD function increases the sensitivity to oxidative stress and the levels of brain lipid peroxidation, and impairs locomotor and learning abilities. Human ApoD overexpression in the mouse brain produces opposite effects, increasing survival and preventing the raise of brain lipid peroxides after oxidant treatment. These observations, together with its transcriptional up-regulation in the brain upon oxidative insult, identify ApoD as an acute response protein with a protective and therefore beneficial function mediated by the control of peroxidated lipids.
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| 2. |
- Sjögren, Jonathan, et al.
(författare)
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EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans.
- 2015
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 25:10, s. 1053-1063
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Tidskriftsartikel (refereegranskat)abstract
- Enzymes that affect glycoproteins of the human immune system, and thereby modulate defense responses, are abundant among bacterial pathogens. Two endoglycosidases from the human pathogen Streptococcus pyogenes, EndoS and EndoS2, have recently been shown to hydrolyze N-linked glycans of human IgG. However, detailed characterization and comparison of the hydrolyzing activities have not been performed. In the present study we set out to characterize the enzymes by comparing the activities of EndoS and EndoS2 on a selection of therapeutic monoclonal antibodies (mAbs), cetuximab, adalimumab, panitumumab, and denosumab. By analyzing the glycans hydrolyzed by EndoS and EndoS2 from the antibodies using MALDI-TOF, we found that both enzymes cleaved complex glycans, and that EndoS2 hydrolyzed hybrid and oligomannose structures to a greater extent compared to EndoS. A comparison of UHPLC profiles of the glycan pool of cetuximab hydrolyzed with EndoS and EndoS2 showed that EndoS2 hydrolyzed hybrid and oligomannose glycans whereas these peaks were missing in the EndoS chromatogram. We utilized this difference in glycoform selectivity and developed a liquid chromatography separation method to quantify high-mannose content in the Fc fragments of the selected mAbs. We conclude that EndoS and EndoS2 hydrolyze different glycoforms from the Fc-glycosylation site on therapeutic mAbs, and that this can be used for rapid quantification of high-mannose content.
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| 3. |
- Åkerström, Bo, et al.
(författare)
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rA1M-035, a Physicochemically Improved Human Recombinant α-Microglobulin, Has Therapeutic Effects in Rhabdomyolysis-Induced Acute Kidney Injury
- 2019
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Ingår i: Antioxidants and Redox Signaling. - : Mary Ann Liebert Inc. - 1557-7716 .- 1523-0864. ; 30:4, s. 489-504
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Tidskriftsartikel (refereegranskat)abstract
- AIMS: Human α1-microglobulin (A1M) is an endogenous reductase and radical- and heme-binding protein with physiological antioxidant protective functions. Recombinant human A1M (rA1M) has been shown to have therapeutic properties in animal models of preeclampsia, a pregnancy disease associated with oxidative stress. Recombinant A1M, however, lacks glycosylation, and shows lower solubility and stability than A1M purified from human plasma. The aims of this work were to (i) use site-directed mutagenesis to improve the physicochemical properties of rA1M, (ii) demonstrate that the physicochemically improved rA1M displays full in vitro cell protective effects as recombinant wild-type A1M (rA1M-wt), and (iii) show its therapeutic potential in vivo against acute kidney injury (AKI), another disease associated with oxidative stress.RESULTS: A novel recombinant A1M-variant (rA1M-035) with three amino acid substitutions was constructed, successfully expressed, and purified. rA1M-035 had improved solubility and stability compared with rA1M-wt, and showed intact in vitro heme-binding, reductase, antioxidation, and cell protective activities. Both rA1M-035 and rA1M-wt showed, for the first time, potential in vivo protective effects on kidneys using a mouse rhabdomyolysis glycerol injection model of AKI.INNOVATION: A novel recombinant A1M-variant, rA1M-035, was engineered. This protein showed improved solubility and stability compared with rA1M-wt, full in vitro functional activity, and potential protection against AKI in an in vivo rhabdomyolysis mouse model.CONCLUSION: The new rA1M-035 is a better drug candidate than rA1M-wt for treatment of AKI and preeclampsia in human patients. Antioxid. Redox Signal. 00, 000-000.
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| 4. |
- Allhorn, Maria, et al.
(författare)
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A novel enzyme with antioxidant capacity produced by the ubiquitous skin colonizer Propionibacterium acnes
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- The role of the skin microbiota in human health is poorly understood. Here, we identified and characterized a novel antioxidant enzyme produced by the skin microbiota, designated RoxP for radical oxygenase of Propionibacterium acnes. RoxP is uniquely produced by the predominant skin bacterium P. acnes, with no homologs in other bacteria; it is highly expressed and strongly secreted into culture supernatants. We show that RoxP binds heme, reduces free radicals, and can protect molecules from oxidation. Strikingly, RoxP is crucial for the survival of P. acnes in oxic conditions and for skin colonization of P. acnes ex vivo. Taken together, our study strongly suggests that RoxP facilitates P. acnes' survival on human skin, and is an important beneficial factor for the host-commensal interaction. Thus, RoxP is the first described skin microbiota-derived mutualistic factor that potentially can be exploited for human skin protection.
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| 5. |
- Allhorn, Maria
(författare)
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Alpha-1-microglobulin, heme-binding protein, reductase and antioxidant
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes structural and functional studies of alpha-1-microglobulin (a1m), a 26 kDa plasma and tissue protein that is evolutionarily well-conserved and belongs to the lipocalin superfamily. The lipocalins are polypeptides of 160-190-amino acids that are folded into an 8-stranded beta-barrel forming a pocket with a hydrophobic interior. a1m carries heterogeneous yellow-brown chromophores covalently bound to one unpaired cysteinyl (C34) and three lysyl residues (K92, 118, 130) located at the entrance to the pocket. a1m is found both in free form and as a complex with IgA in blood and interstitial tissues. We show here that a1m is involved in heme metabolism. The protein binds to heme and, when exposed to hemoglobin or erythrocyte membranes, a processed form of a1m, t-a1m (t=truncated), with a free thiol group in position 34 and lacking the C-terminal tetrapeptide, is released from free a1m and the IgA-complex. The processed t-a1m has an intense yellow-brown colour and has properties suggestive of heme-degradation enzyme activity. The processed t-a1m-form is found in urine as well as in chronic ulcer fluids, where it is co-localized with heme. Chronic ulcers are characterized by long-standing inflammation and hemolysis with a continuous release of heme and iron, which are considered to be pathogenetic factors. These findings suggest that a1m is involved in the defence against heme-mediated oxidation. We also found that a1m has enzymatic reductase and dehydrogenase properties. Thus, a1m reduces cytochrome c, methemoglobin, and free iron to their non-oxidized forms, using the biological electron donors NADH and ascorbic acid as electron-supplying co-factors. Recombinant a1m-variants lacking C34 and/or the chromophore-carrying K92, 118 and 130 have less reducing activity, suggesting that the thiol group in position 34 as well as the nearby located lysyl groups are involved in the reaction. The protein has antioxidation properties, i.e. it inhibits the oxidation of biological targets as collagen and LDL, by the pro-oxidants heme, oxidized hemoglobin, hydrogen peroxide and oxidized iron. a1m can even remove some of the oxidation products on collagen and LDL after they have been formed. a1m has been found in LDL from human plasma, suggesting that a1m is a naturally occurring component of LDL. In vitro, a1m binds specifically to LDL-particles. a1m inhibits the hydroxyl radical-induced peroxidation of purified erythrocyte membranes. a1m can also inhibit the generation of intracellular reactive oxygen species in K562 cells after exposure of the cells to heme and hydrogen peroxide as well as in resting cells. In conclusion, a1m may be regarded as a heme-metabolising protein, a new regulator of oxidation and an extracellular antioxidant.
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| 6. |
- Allhorn, Maria, et al.
(författare)
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EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity
- 2008
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Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- ABSTRACT: BACKGROUND: The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS. RESULTS: We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity. CONCLUSIONS: We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself.
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| 7. |
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| 8. |
- Allhorn, Maria, et al.
(författare)
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Heme-Scavenging Role of alpha1-Microglobulin in Chronic Ulcers.
- 2003
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Ingår i: Journal of Investigative Dermatology. - : Elsevier BV. - 1523-1747 .- 0022-202X. ; 121:3, s. 640-646
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Tidskriftsartikel (refereegranskat)abstract
- Chronic venous ulcers are characterized by chronic inflammation. Heme and iron, originating from blood cell hemolysis as well as extravascular necrosis, have been implicated as important pathogenic factors due to their promotion of oxidative stress. It was recently reported that the plasma and tissue protein alpha1-microglobulin is involved in heme metabolism. The protein binds heme, and a carboxy-terminally processed form, truncated alpha1-microglobulin, also degrades heme. Here, we show the presence of micromolar levels of heme and free iron in chronic leg ulcer fluids. Micromolar amounts of alpha1-microglobulin was also present in the ulcer fluids and bound to added radiolabeled heme. Truncated alpha1-microglobulin was found in the ulcer fluids and exogenously added alpha1-microglobulin was processed into the truncated alpha1-microglobulin form. Histochemical analysis of chronic wound tissue showed the presence of iron deposits, heme/porphyrins in infiltrating cells basement membranes and fibrin cuffs around vessels, and alpha1-microglobulin ubiquitously distributed but especially abundant in basement membranes around vessels and at fibrin cuffs. Our results suggest that alpha1-microglobulin constitutes a previously unknown defense mechanism against high heme and iron levels during skin wound healing. Excessive heme and iron, which are not buffered by alpha1-microglobulin, may underlie the chronic inflammation in chronic ulcers.
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| 9. |
- Allhorn, Maria, et al.
(författare)
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Human IgG/FcgammaR Interactions Are Modulated by Streptococcal IgG Glycan Hydrolysis.
- 2008
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The human pathogen Streptococcus pyogenes produces an endoglycosidase, EndoS that hydrolyzes the chitobiose core of the asparagine-linked glycan on the heavy chain of human IgG. IgG-binding to Fc gamma receptors (FcgammaR) on leukocytes triggers effector functions including phagocytosis, oxidative burst and the release of inflammatory mediators. The interactions between FcgammaR and the Fc domain of IgG depend on the IgG glycosylation state. METHODOLOGY/PRINCIPAL FINDINGS: Here we show for the first time that EndoS hydrolyzes the heavy chain glycan of all four human IgG subclasses (IgG1-4), in purified form and in a plasma environment. An inactive form of EndoS, obtained by site-directed mutagenesis, binds IgG with high affinity, in contrast to wild type EndoS that only transiently interacts with IgG, as shown by Slot-blotting and surface plasmon resonance technology. Furthermore, EndoS hydrolysis of the IgG glycan influences the binding of IgG to immobilized soluble FcgammaR and to an erythroleukemic cell line, K562, expressing FcgammaRIIa. Incubation of whole blood with EndoS results in a dramatic decrease of IgG binding to activated monocytes as analyzed by flow cytometry. Moreover, the IgG bound to K562 cells dissociates when cells are treated with EndoS. Likewise, IgG bound to immobilized FcgammaRIIa and subsequently treated with EndoS, dissociates from the receptor as analyzed by surface plasmon resonance and Western blot. CONCLUSIONS/SIGNIFICANCE: We provide novel information about bacterial enzymatic modulation of the IgG/FcgammaR interaction that emphasizes the importance of glycosylation for antibody effector functions. Moreover, EndoS could be used as a biochemical tool for specific IgG N-glycan hydrolysis and IgG purification/detection, or as a potential immunosuppressing agent for treatment of antibody-mediated pathological processes.
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| 10. |
- Allhorn, Maria, et al.
(författare)
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Processing of the lipocalin alpha(1)-microglobulin by hemoglobin induces heme-binding and heme-degradation properties.
- 2002
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Ingår i: Blood. - 1528-0020. ; 99:6, s. 1894-1901
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Tidskriftsartikel (refereegranskat)abstract
- alpha(1)-Microglobulin is a 26-kd protein, widespread in plasma and tissues and well-conserved among vertebrates. alpha(1)-Microglobulin belongs to the lipocalins, a protein superfamily with highly conserved 3-dimensional structures, forming an internal ligand binding pocket. The protein, isolated from urine, has a heterogeneous yellow-brown chromophore bound covalently to amino acid side groups around the entrance of the lipocalin pocket. alpha(1)-Microglobulin is found in blood both in free form and complex-bound to immunoglobulin A (IgA) via a half-cystine residue at position 34. It is shown here that an alpha(1)-microglobulin species, which we name t-alpha(1)-microglobulin (t = truncated), with a free Cys34 thiol group, lacking its C-terminal tetrapeptide, LIPR, and with a more polar environment around the entrance of the lipocalin pocket, is released from IgA-alpha(1)-microglobulin as well as from free alpha(1)-microglobulin when exposed to the cytosolic side of erythrocyte membranes or to purified oxyhemoglobin. The processed t-alpha(1)-microglobulin binds heme and the alpha(1)-microglobulin-heme complex shows a time-dependent spectral rearrangement, suggestive of degradation of heme concomitantly with formation of a heterogeneous chromophore associated with the protein. The processed t-alpha(1)-microglobulin is found in normal and pathologic human urine, indicating that the cleavage process occurs in vivo. The results suggest that alpha(1)-microglobulin is involved in extracellular heme catabolism. (Blood. 2002;99:1894-1901)
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