Publications by authors named "Friedrich Buck"

61 Publications

Model prediction of a Kunitz-type trypsin inhibitor protein from seeds of Acacia nilotica L. with strong antimicrobial and insecticidal activity.

Turk J Biol 2020 19;44(4):188-200. Epub 2020 Aug 19.

Botany Division, Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan Pakistan.

A Kunitz-type trypsin inhibitor protein has been purified and characterized from seeds of L. LC-MS/MS analysis of trypsin inhibitor (TI) provided the N-terminal fragment of 11 amino acids which yielded 100% identity with already reported Kunitz-type trypsin inhibitor protein of (TI) in UniProtKB database search. SDS-PAGE showed a single band of ~21 kDa under nonreduced condition and appearance of a daughter band (17 kDa) in the presence of -mercaptoethanol indicating the presence of interchain disulfide linkage typical for Kunitz-type trypsin inhibitors. TI was purified from seed extract by using a combination of anion exchange and gel filtration chromatography. Since TI showed maximum homology with TI, a molecular structure of TI was predicted which showed highly -sheeted molecular conformation similar to crystallographic structure of trypsin inhibitor (TI). TI (20 µg) produces significant population inhibition against different human pathogenic bacteria along strong antifungal activity (50 µg). Entomotoxin potential of TI was evaluated against two stored grain insect pests (Herbst) (Tenebrionidae: Coleoptera) and (Linnaeus) (Curculionidae: Coleoptera). Statistically significant mortality of adults was observed at 1.5 mg after 15 days in comparison to control. Additionally, number of total eggs, larvae, pupae, adults, and their male/female ratio were also severely reduced in comparison to control. Similarly, two generation progeny of was studied after mixing TI with rice kernels. Mean percent mortality of adult population was significantly higher after 9 days of exposure in comparison to control group. TI significantly reduced the F1 generation while little mortality was observed for F2 generation. Exploration of such potent molecules is the prerequisite of our time regarding the anticipation of postantibiotic era and the development of insect resistance against chemical pesticides.
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http://dx.doi.org/10.3906/biy-2002-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478134PMC
August 2020

The cell adhesion molecule L1 interacts with nuclear proteins via its intracellular domain.

FASEB J 2020 08 13;34(8):9869-9883. Epub 2020 Jun 13.

Zentrum für Molekulare Neurobiologie, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

Proteolytic cleavage of the cell adhesion molecule L1 (L1) in brain tissue and in cultured cerebellar neurons results in the generation and nuclear import of a 30 kDa fragment comprising most of L1's C-terminal, intracellular domain. In search of molecules that interact with this domain, we performed affinity chromatography with the recombinant intracellular L1 domain and a nuclear extract from mouse brains, and identified potential nuclear L1 binding partners involved in transcriptional regulation, RNA processing and transport, DNA repair, chromatin remodeling, and nucleocytoplasmic transport. By co-immunoprecipitation and enzyme-linked immunosorbent assay using recombinant proteins, we verified the direct interaction between L1 and the nuclear binding partners non-POU domain containing octamer-binding protein and splicing factor proline/glutamine-rich. The proximity ligation assay confirmed this close interaction in cultures of cerebellar granule cells. Our findings suggest that L1 fragments regulate multiple nuclear functions in the nervous system. We discuss possible physiological and pathological roles of these interactions in regulation of chromatin structure, gene expression, RNA processing, and DNA repair.
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http://dx.doi.org/10.1096/fj.201902242RDOI Listing
August 2020

Irisin: Still chasing shadows.

Mol Metab 2020 04 31;34:124-135. Epub 2020 Jan 31.

Institute of Muscle Biology and Growth, Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany. Electronic address:

Objective: Considerable uncertainty remains regarding the veracity of measuring myokine irisin more than seven years after its original description. Unresolved issues include the nature of transcription of the irisin precursor fibronectin type III domain containing 5 (FNDC5) gene across species, the reliability of irisin levels measured with commercial enzyme-linked immunosorbent assays (ELISAs), and the overall validity of the recently published reference values for human serum measured with quantitative mass spectrometry. We utilized multiple species and measures to evaluate the robustness of commonly used reagents and methods for reporting irisin.

Methods: Amplification of cDNA was used to assess the FNDC5 transcript patterns in humans and mice. The specificity and sensitivity of different irisin antibodies were examined via western blotting. Quantification of circulating native irisin was conducted with mass spectrometry using an absolute quantification peptide for irisin.

Results: We show that there is a greater transcript diversity of human FNDC5 than currently annotated, but no indication of the expression of transcripts leading to a truncated form of irisin. Available irisin antibodies still bind to patterns of unspecific serum proteins, which compromise reliable measurements of irisin with ELISAs. Absolute quantification of irisin with labeled peptides by mass spectrometry is an advanced method but requires a multi-step sample preparation introducing uncontrollable variations in the measurement.

Conclusion: Our data represent an explicit warning against measuring circulating irisin using available methods. Measuring irisin is akin to chasing shadows.
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http://dx.doi.org/10.1016/j.molmet.2020.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033458PMC
April 2020

Graphene quantum dots as cysteine protease nanocarriers against stored grain insect pests.

Sci Rep 2020 02 26;10(1):3444. Epub 2020 Feb 26.

Department of Entomology, Faculty of Agricultural Sciences & Technology, Bahauddin Zakariya University, 60800, Multan, Pakistan.

Storing grains remain vulnerable to insect pest attack. The present study developed a biopesticide using biomolecules and their encapsulation in nanoparticles. A 25 kDa cysteine protease extracted from seeds of Albizia procera (ApCP) was encapsulated in graphene quantum dots (GQDs). The insecticidal activity of ApCP, with or without GQDs, against two stored grain insect pests, Tribolium castaneum (Herbst) and Rhyzopertha dominica (Fabricius) was explored. Insects were exposed to three concentrations 7.0, 3.5 and 1.7 mg of ApCP per a gram of wheat flour and grains. The insecticidal activity of ApCP encapsulated with GQDs was improved compared to that of ApCP without GQDs for both insect pests. The number of eggs and larvae of T. castaneum was reduced by 49% and 86%, respectively. Larval mortality was increased to 72%, and adult eclosion of T. castaneum was reduced by 98% at a 7.0 mg/g concentration of ApCP with GQDs compared to that of ApCP without GQDs. Exposure to 7.0 mg/g ApCP with GQDs, the number of R. dominica eggs and larvae was reduced by 72% and 92% respectively, larval mortality was increased by 90%, and eclosion was reduced by 97%. The extraction, purification, characterization, quantification and encapsulation of ApCP with GQDs were also studied. Cysteine protease nanocarriers have the potential to control stored grain insect pests.
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http://dx.doi.org/10.1038/s41598-020-60432-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044290PMC
February 2020

Computational modeling and functional characterization of a GgChi: A class III chitinase from corms of Gladiolus grandiflorus.

Kaohsiung J Med Sci 2018 Dec 12;34(12):673-683. Epub 2018 Oct 12.

Botany Division, Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan. Electronic address:

The present study describes the predicted model and functional characterization of an endochitinase (30 kDa) from corms of Gladiolus grandiflorus. ESI-QTOF-MS generated peptide showed 96% sequence homology with family 18, Class III acidic endochitinase of Gladiolus gandavensis. Purified G. grandiflorus chitinase (GgChi) hydrolyzed 4-methylumbelliferyl β-d-N,N',N''-triacetylchitotriose substrate showing specific endochitinase activity. Since no structural details of GgChi were available in the Protein Data Bank (PDB), a homology model was predicted using the coordinate information of Crocus vernus chitinase (PDB ID: 3SIM). Ramachandran plot indicated 84.5% in most favored region, 14.8% in additional and 0.6% in generously allowed region while no residue in disallowed region. The predicted structure indicated a highly conserved (β/α) (TIM barrel) structure similar to the family 18, class III chitinases. The GgChi also showed sequence and structural homologies with other active chitinases. The GgChi (50 μg/disc) showed no antibacterial activity, but did provide mild growth inhibition of phytopathogenic fungus Fusarium oxysporum at a concentration of 500 μg/well Similarly, insect toxicity bioassays of GgChi (50 μg) against nymphs of Bemisia tabaci showed 14% reduction in adult emergence and 14% increase in mortality rate in comparison to control values. The GgChi (1.5 mg) protein showed significant reduction in a population of flour beetle (Tribolium castaneum) after 35 days, but lower reactivity against rice weevil (Sitophilus oryzae). The results of this study provide detai.led insight on functional characterization of a family 18 class III acidic plant endochitinase.
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http://dx.doi.org/10.1016/j.kjms.2018.08.003DOI Listing
December 2018

Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells.

PLoS Pathog 2016 06 14;12(6):e1005660. Epub 2016 Jun 14.

Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany.

Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines.
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http://dx.doi.org/10.1371/journal.ppat.1005660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907486PMC
June 2016

Improvement of neuronal cell survival by astrocyte-derived exosomes under hypoxic and ischemic conditions depends on prion protein.

Glia 2016 Jun 16;64(6):896-910. Epub 2016 Mar 16.

Zentrum Für Molekulare Neurobiologie, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

Prion protein (PrP) protects neural cells against oxidative stress, hypoxia, ischemia, and hypoglycemia. In the present study we confirm that cultured PrP-deficient neurons are more sensitive to oxidative stress than wild-type neurons and present the novel findings that wild-type, but not PrP-deficient astrocytes protect wild-type cerebellar neurons against oxidative stress and that exosomes released from stressed wild-type, but not from stressed PrP-deficient astrocytes reduce neuronal cell death induced by oxidative stress. We show that neuroprotection by exosomes of stressed astrocytes depends on exosomal PrP but not on neuronal PrP and that astrocyte-derived exosomal PrP enters into neurons, suggesting neuronal uptake of astrocyte-derived exosomes. Upon exposure of wild-type astrocytes to hypoxic or ischemic conditions PrP levels in exosomes were increased. By mass spectrometry and Western blot analysis, we detected increased levels of 37/67 kDa laminin receptor, apolipoprotein E and the ribosomal proteins S3 and P0, and decreased levels of clusterin/apolipoprotein J in exosomes from wild-type astrocytes exposed to oxygen/glucose deprivation relative to exosomes from astrocytes maintained under normoxic conditions. The levels of these proteins were not altered in exosomes from stressed PrP-deficient astrocytes relative to unstressed PrP-deficient astrocytes. These results indicate that PrP in astrocytes is a sensor for oxidative stress and mediates beneficial cellular responses, e.g. release of exosomes carrying PrP and other molecules, resulting in improved survival of neurons under hypoxic and ischemic conditions.
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http://dx.doi.org/10.1002/glia.22963DOI Listing
June 2016

S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure.

FASEB J 2016 05 2;30(5):1849-64. Epub 2016 Feb 2.

Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, Frankfurt, Germany;

Cardiac myosin-binding protein C (cMyBP-C) regulates actin-myosin interaction and thereby cardiac myocyte contraction and relaxation. This physiologic function is regulated by cMyBP-C phosphorylation. In our study, reduced site-specific cMyBP-C phosphorylation coincided with increased S-glutathiolation in ventricular tissue from patients with dilated or ischemic cardiomyopathy compared to nonfailing donors. We used redox proteomics, to identify constitutive and disease-specific S-glutathiolation sites in cMyBP-C in donor and patient samples, respectively. Among those, a cysteine cluster in the vicinity of the regulatory phosphorylation sites within the myosin S2 interaction domain C1-M-C2 was identified and showed enhanced S-glutathiolation in patients. In vitro S-glutathiolation of recombinant cMyBP-C C1-M-C2 occurred predominantly at Cys(249), which attenuated phosphorylation by protein kinases. Exposure to glutathione disulfide induced cMyBP-C S-glutathiolation, which functionally decelerated the kinetics of Ca(2+)-activated force development in ventricular myocytes from wild-type, but not those from Mybpc3-targeted knockout mice. These oxidation events abrogate protein kinase-mediated phosphorylation of cMyBP-C and therefore potentially contribute to the reduction of its phosphorylation and the contractile dysfunction observed in human heart failure.-Stathopoulou, K., Wittig, I., Heidler, J., Piasecki, A., Richter, F., Diering, S., van der Velden, J., Buck, F., Donzelli, S., Schröder, E., Wijnker, P. J. M., Voigt, N., Dobrev, D., Sadayappan, S., Eschenhagen, T., Carrier, L., Eaton, P., Cuello, F. S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure.
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http://dx.doi.org/10.1096/fj.201500048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836368PMC
May 2016

Disseminated Tumor Cells Persist in the Bone Marrow of Breast Cancer Patients through Sustained Activation of the Unfolded Protein Response.

Cancer Res 2015 Dec 16;75(24):5367-77. Epub 2015 Nov 16.

Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Disseminated tumor cells (DTC), which share mesenchymal and epithelial properties, are considered to be metastasis-initiating cells in breast cancer. However, the mechanisms supporting DTC survival are poorly understood. DTC extravasation into the bone marrow may be encouraged by low oxygen concentrations that trigger metabolic and molecular alterations contributing to DTC survival. Here, we investigated how the unfolded protein response (UPR), an important cytoprotective program induced by hypoxia, affects the behavior of stressed cancer cells. DTC cell lines established from the bone marrow of patients with breast cancer (BC-M1), lung cancer, (LC-M1), and prostate cancer (PC-E1) were subjected to hypoxic and hypoglycemic conditions. BC-M1 and LC-M1 exhibiting mesenchymal and epithelial properties adapted readily to hypoxia and glucose starvation. Upregulation of UPR proteins, such as the glucose-regulated protein Grp78, induced the formation of filamentous networks, resulting in proliferative advantages and sustained survival under total glucose deprivation. High Grp78 expression correlated with mesenchymal attributes of breast and lung cancer cells and with poor differentiation in clinical samples of primary breast and lung carcinomas. In DTCs isolated from bone marrow specimens from breast cancer patients, Grp78-positive stress granules were observed, consistent with the likelihood these cells were exposed to acute cell stress. Overall, our findings provide the first evidence that the UPR is activated in DTC in the bone marrow from cancer patients, warranting further study of this cell stress pathway as a predictive biomarker for recurrent metastatic disease.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-3728DOI Listing
December 2015

Activation of Cell Surface Bound 20S Proteasome Inhibits Vascular Cell Growth and Arteriogenesis.

Biomed Res Int 2015 4;2015:719316. Epub 2015 Jun 4.

Department of Cardiology, University Hospital Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg, Germany.

Arteriogenesis is an inflammatory process associated with rapid cellular changes involving vascular resident endothelial progenitor cells (VR-EPCs). Extracellular cell surface bound 20S proteasome has been implicated to play an important role in inflammatory processes. In our search for antigens initially regulated during collateral growth mAb CTA 157-2 was generated against membrane fractions of growing collateral vessels. CTA 157-2 stained endothelium of growing collateral vessels and the cell surface of VR-EPCs. CTA 157-2 bound a protein complex (760 kDa) that was identified as 26 kDa α7 and 21 kDa β3 subunit of 20S proteasome in mass spectrometry. Furthermore we demonstrated specific staining of 20S proteasome after immunoprecipitation of VR-EPC membrane extract with CTA 157-2 sepharose beads. Functionally, CTA 157-2 enhanced concentration dependently AMC (7-amino-4-methylcoumarin) cleavage from LLVY (N-Succinyl-Leu-Leu-Val-Tyr) by recombinant 20S proteasome as well as proteasomal activity in VR-EPC extracts. Proliferation of VR-EPCs (BrdU incorporation) was reduced by CTA 157-2. Infusion of the antibody into the collateral circulation reduced number of collateral arteries, collateral proliferation, and collateral conductance in vivo. In conclusion our results indicate that extracellular cell surface bound 20S proteasome influences VR-EPC function in vitro and collateral growth in vivo.
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http://dx.doi.org/10.1155/2015/719316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4471257PMC
March 2016

Tuning IL-2 signaling by ADP-ribosylation of CD25.

Sci Rep 2015 Mar 10;5:8959. Epub 2015 Mar 10.

1] Institute of Immunology, University Medical Center, 20246 Hamburg, Germany [2] Normandy University, Institute for Research and Innovation in Biomedicine, 76183 Rouen, France.

Control of immunologic tolerance and homeostasis rely on Foxp3(+)CD4(+)CD25(+) regulatory T cells (Tregs) that constitutively express the high affinity receptor for Interleukin-2, CD25. Tregs proliferate in response to injections of IL-2/anti-IL-2 antibody complexes or low doses of IL-2. However, little is known about endogenous mechanisms that regulate the sensitivity of CD25 to signaling by IL-2. Here we demonstrate that CD25 is ADP-ribosylated at Arg35 in the IL-2 binding site by ecto-ADP-ribosyltransferase ARTC2.2, a toxin-related GPI-anchored ecto-enzyme. ADP-ribosylation inhibits binding of IL-2 by CD25, IL-2- induced phosphorylation of STAT5, and IL-2-dependent cell proliferation. Our study elucidates an as-yet-unrecognized mechanism to tune IL-2 signaling. This newly found mechanism might thwart Tregs at sites of inflammation and thereby permit a more potent response of activated effector T cells.
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http://dx.doi.org/10.1038/srep08959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354014PMC
March 2015

Interaction of the cell adhesion molecule CHL1 with vitronectin, integrins, and the plasminogen activator inhibitor-2 promotes CHL1-induced neurite outgrowth and neuronal migration.

J Neurosci 2014 Oct;34(44):14606-23

Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854, Center for Neuroscience, Shantou University Medical College, Shantou 515041, People's Republic of China, and

The cell adhesion molecule close homolog of L1 (CHL1) plays important functional roles in the developing and adult nervous system. In search of the binding partners that mediate the diverse and sometimes opposing functions of CHL1, the extracellular matrix-associated proteins vitronectin and plasminogen activator inhibitor-2 (PAI-2) were identified as novel CHL1 interaction partners and tested for involvement in CHL1-dependent functions during mouse cerebellar development. CHL1-induced cerebellar neurite outgrowth and cell migration at postnatal days 6-8 were inhibited by a CHL1-derived peptide comprising the integrin binding RGD motif, and by antibodies against vitronectin or several integrins, indicating a vitronectin-dependent integrin-mediated pathway. A PAI-2-derived peptide, or antibodies against PAI-2, urokinase type plasminogen activator (uPA), uPA receptor, and several integrins reduced cell migration. CHL1 colocalized with vitronectin, PAI-2, and several integrins in cerebellar granule cells, suggesting an association among these proteins. Interestingly, at the slightly earlier age of 4-5 d, cerebellar neurons did not depend on CHL1 for neuritogenesis and cell migration. However, differentiation of progenitor cells into neurons at this stage was dependent on homophilic CHL1-CHL1 interactions. These observations indicate that homophilic CHL1 trans-interactions regulate differentiation of neuronal progenitor cells at early postnatal stages, while heterophilic trans-interactions of CHL1 with vitronectin, integrins, and the plasminogen activator system regulate neuritogenesis and neuronal cell migration at a later postnatal stage of cerebellar morphogenesis. Thus, within very narrow time windows in postnatal cerebellar development, distinct types of molecular interactions mediated by CHL1 underlie the diverse functions of this protein.
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http://dx.doi.org/10.1523/JNEUROSCI.3280-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608427PMC
October 2014

Identification and analysis of ADP-ribosylated proteins.

Curr Top Microbiol Immunol 2015 ;384:33-50

Institute of Immunology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246, Hamburg, Germany.

The analysis of ADP-ribosylated proteins is a challenging task, on the one hand because of the diversity of the target proteins and the modification sites, on the other hand because of the particular problems posed by the analysis of ADP-ribosylated peptides. ADP-ribosylated proteins can be detected in in vitro experiments after the incorporation of radioactively labeled or chemically modified ADP-ribose. Endogenously ADP-ribosylated proteins may be detected and enriched by antibodies directed against the ADP-ribosyl moiety or by ADP-ribosyl binding macro domains. The determination of the exact attachment site of the modification, which is a prerequisite for the understanding of the specificity of the various ADP-ribosyl transferases and the structural consequences of ADP-ribosylation, necessitates the proteolytic cleavage of the proteins. The resulting peptides can afterwards be enriched either by IMAC (using the affinity of the pyrophosphate group for heavy metal ions) or by immobilized boronic acid beads (using the affinity of the vicinal ribose hydroxy groups for boronic acid). The identification of the modified peptides usually requires tandem mass spectrometric measurements. Problems that hamper the mass spectrometric analysis by collision-induced decay (CID) can be circumvented either by the application of different fragmentation techniques (electron transfer or electron capture dissociation; ETD or ECD) or by enzymatic cleavage of the ADP-ribosyl group to ribosyl-phosphate.
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http://dx.doi.org/10.1007/82_2014_424DOI Listing
March 2015

Alterations in Soluble Class III Peroxidases of Maize Shoots by Flooding Stress.

Proteomes 2014 Jun 26;2(3):303-322. Epub 2014 Jun 26.

University of Hamburg, Biocenter Klein Flottbek and Botanical Garden, Oxidative Stress and Plant Proteomics Group, Ohnhorststraße 18, D-22609 Hamburg, Germany.

Due to changing climate, flooding (waterlogged soils and submergence) becomes a major problem in agriculture and crop production. In the present study, the effect of waterlogging was investigated on peroxidases of maize ( L.) leaves. The plants showed typical adaptations to flooding stress, , alterations in chlorophyll ratios and increased basal shoot diameter. Seven peroxidase bands could be detected by first dimension modified SDS-PAGE and 10 bands by first dimension high resolution Clear Native Electrophoresis that altered in dependence on plant development and time of waterlogging. Native isoelectric focusing revealed three acidic to neutral and four alkaline guaiacol peroxidases that could be further separated by high resolution Clear Native Electrophorese in the second dimension. One neutral peroxidase (pI 7.0) appeared to be down-regulated within four hours after flooding, whereas alkaline peroxidases (pI 9.2, 8.0 and 7.8) were up-regulated after 28 or 52 h. Second dimensions revealed molecular masses of 133 kDa and 85 kDa for peroxidases at pI 8.0 and 7.8, respectively. Size exclusion chromatography revealed native molecular masses of 30-58 kDa for peroxidases identified as class III peroxidases and ascorbate peroxidases by mass spectrometry. Possible functions of these peroxidases in flooding stress will be discussed.
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http://dx.doi.org/10.3390/proteomes2030303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302756PMC
June 2014

Preliminary crystallographic analysis of a cruciferin protein from seeds of Moringa oleifera.

Protein J 2014 Jun;33(3):253-7

Laboratory for Structural Biology of Infection and Inflammation, Department of Chemistry, c/o DESY, University of Hamburg, Notkestrasse 85, 22603, Hamburg, Germany,

A 55 kDa cruciferin protein has been purified and characterized from seeds of Moringa oleifera plant. Protein blast of N-terminal amino-acid sequence showed 60 % sequence similarity with cruciferin from Brassica napus. The M. oleifera protein has been crystallized applying the sitting drop method using 5 % polyethylene glycol 8,000, 38.5 % 3-methyl-1,5-pentanediol and 0.1 M sodium cacodylate pH 6.5. The crystals belonged to the P6322 hexagonal space group with cell dimensions, a = b = 98.4, c = 274.3 Å. Initial diffraction data have been collected to a resolution of 6 Å.
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http://dx.doi.org/10.1007/s10930-014-9558-xDOI Listing
June 2014

A non-canonical initiation site is required for efficient translation of the dendritically localized Shank1 mRNA.

PLoS One 2014 12;9(2):e88518. Epub 2014 Feb 12.

Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

Local protein synthesis in dendrites enables neurons to selectively change the protein complement of individual postsynaptic sites. Though it is generally assumed that this mechanism requires tight translational control of dendritically transported mRNAs, it is unclear how translation of dendritic mRNAs is regulated. We have analyzed here translational control elements of the dendritically localized mRNA coding for the postsynaptic scaffold protein Shank1. In its 5' region, the human Shank1 mRNA exhibits two alternative translation initiation sites (AUG⁺¹ and AUG⁺²¹⁴), three canonical upstream open reading frames (uORFs1-3) and a high GC content. In reporter assays, fragments of the 5'UTR with high GC content inhibit translation, suggesting a contribution of secondary structures. uORF3 is most relevant to translation control as it overlaps with the first in frame start codon (AUG⁺¹), directing translation initiation to the second in frame start codon (AUG⁺²¹⁴). Surprisingly, our analysis points to an additional uORF initiated at a non-canonical ACG start codon. Mutation of this start site leads to an almost complete loss of translation initiation at AUG⁺¹, demonstrating that this unconventional uORF is required for Shank1 synthesis. Our data identify a novel mechanism whereby initiation at a non-canonical site allows for translation of the main Shank1 ORF despite a highly structured 5'UTR.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0088518PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3922875PMC
January 2015

Subcellular sorting of the G-protein coupled mouse somatostatin receptor 5 by a network of PDZ-domain containing proteins.

PLoS One 2014 11;9(2):e88529. Epub 2014 Feb 11.

Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

PSD-95/discs large/ZO-1 (PDZ) domain proteins integrate many G-protein coupled receptors (GPCRs) into membrane associated signalling complexes. Additional PDZ proteins are involved in intracellular receptor trafficking. We show that three PDZ proteins (SNX27, PIST and NHERF1/3) regulate the mouse somatostatin receptor subtype 5 (SSTR5). Whereas the PDZ ligand motif of SSTR5 is not necessary for plasma membrane targeting or internalization, it protects the SSTR5 from postendocytic degradation. Under conditions of lysosomal inhibition, recycling of the SSTR5 to the plasma membrane does not depend on the PDZ ligand. However, recycling of the wild type receptor carrying the PDZ binding motif depends on SNX27 which interacts and colocalizes with the receptor in endosomal compartments. PIST, implicated in lysosomal targeting of some membrane proteins, does not lead to degradation of the SSTR5. Instead, overexpressed PIST retains the SSTR5 at the Golgi. NHERF family members release SSTR5 from retention by PIST, allowing for plasma membrane insertion. Our data suggest that PDZ proteins act sequentially on the GPCR at different stages of its subcellular trafficking.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0088529PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3921201PMC
October 2014

Cell death triggered by Yersinia enterocolitica identifies processing of the proinflammatory signal adapter MyD88 as a general event in the execution of apoptosis.

J Immunol 2014 Feb 20;192(3):1209-19. Epub 2013 Dec 20.

Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Eppendorf, 20246 Hamburg, Germany;

Many pathogenic microorganisms have evolved tactics to modulate host cell death or survival pathways for establishing infection. The enteropathogenic bacterium Yersinia enterocolitica deactivates TLR-induced signaling pathways, which triggers apoptosis in macrophages. In this article, we show that Yersinia-induced apoptosis of human macrophages involves caspase-dependent cleavage of the TLR adapter protein MyD88. MyD88 was also cleaved when apoptosis was mediated by overexpression of the Toll-IL-1R domain-containing adapter inducing IFN-β in epithelial cells. The caspase-processing site was mapped to aspartate-135 in the central region of MyD88. MyD88 is consequently split by caspases in two fragments, one harboring the death domain and the other the Toll-IL-1R domain. Caspase-3 was identified as the protease that conferred the cleavage of MyD88 in in vitro caspase assays. In line with a broad role of caspase-3 in the execution of apoptosis, the processing of MyD88 was not restricted to Yersinia infection and to proapoptotic Toll-IL-1R domain-containing adapter inducing IFN-β signaling, but was also triggered by staurosporine treatment. The cleavage of MyD88 therefore seems to be a common event in the advanced stages of apoptosis, when caspase-3 is active. We propose that the processing of MyD88 disrupts its scaffolding function and uncouples the activation of TLR and IL-1Rs from the initiation of proinflammatory signaling events. The disruption of MyD88 may consequently render dying cells less sensitive to proinflammatory stimuli in the execution phase of apoptosis. The cleavage of MyD88 could therefore be a means of conferring immunogenic tolerance to apoptotic cells to ensure silent, noninflammatory cell demise.
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http://dx.doi.org/10.4049/jimmunol.1203464DOI Listing
February 2014

Proteome analysis of the effects of all-trans retinoic acid on human germ cell tumor cell lines.

J Proteomics 2014 Jan 22;96:300-13. Epub 2013 Nov 22.

Department of Oncology, Hematology and Bone Marrow Transplantation with section Pneumology, Hubertus Wald-Tumor Zentrum (UCCH), University Hospital Eppendorf (UKE), Hamburg, Germany; Division of Hematology, University Hospital Zürich, Zürich, Switzerland. Electronic address:

Unlabelled: We analysed the effects of all-trans retinoic acid (ATRA) on proliferation and changes in the global proteome of the nullipotent human embryonal carcinoma cell line 2102Ep and the pluripotent cell line NTERA2 cl.D1 (NT2). Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of proteins of the retinoid pathway. We established a proteome map of the germ cell tumor (GCT) cell line NT2 showing neuronal differentiation under ATRA treatment for 7days. Using bioinformatic analyses, we identified functional groups of altered proteins and potentially involved pathways, of which changes to the organization of the cytoskeleton and anti-apoptotic effects were the most prominent. Changes observed in the expression of factors involved in the retinoid pathway under ATRA, namely an upregulation of CRBP and CRABP2, were also reflected in GCT tissues of different histologies, providing further insight into factors involved in the differentiation of these pluripotent tumors.

Biological Significance: Treatment of NT2 germ cell tumor cells with all-trans retinoic acid (ATRA) is a model to investigate differentiation. We analysed differentially expressed proteins by 2D-PAGE and mass spectrometry and provide a proteome map of NT2 cells under 7days of ATRA. By bioinformatic analyses, functional groups of proteins and involved pathways like changes to the cytoskeleton and anti-apoptotic effects were identified. Factors involved in the retinoid pathway, in particular upregulation of CRBP, CRABP1 and CRABP2, also showed differential expression in tumors with different histological subtypes, which provides insight into gene regulation under induced and spontaneous differentiation in germ cell tumors.
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http://dx.doi.org/10.1016/j.jprot.2013.11.010DOI Listing
January 2014

Generation of amyloid-β is reduced by the interaction of calreticulin with amyloid precursor protein, presenilin and nicastrin.

PLoS One 2013 9;8(4):e61299. Epub 2013 Apr 9.

Zentrum für Molekulare Neurobiologie, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer's disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca(2+)- and N-glycan-independent interaction is mediated by amino acids 330-344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β42 levels in culture supernatants, while transfection with the P-domain increases amyloid-β40 levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330-344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β40 and amyloid-β42 levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer's disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061299PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3621835PMC
October 2013

Tumor-associated Neu5Ac-Tn and Neu5Gc-Tn antigens bind to C-type lectin CLEC10A (CD301, MGL).

Glycobiology 2013 Jul 18;23(7):844-52. Epub 2013 Mar 18.

Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.

In human tumors, glycoproteins often exhibit abnormal glycosylation patterns, e.g. certain Lewis structures, TF antigen, Tn antigen and/or their sialylated forms, creating additional binding sites for glycoreceptors. In the present study, we have analyzed the carbohydrate specificity of the C-type lectin CLEC10A using glycan profiling by enzyme-linked immunosorbent assay (ELISA). In addition to the known ligands, we show binding to two tumor-associated antigens, namely Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn, with an affinity of CLEC10A in the micromolar range. Detailed analyses of the glycan-lectin interactions were carried out by surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR. CLEC10A binds Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn with dissociation constants of 297 and 80 µM, respectively, as determined by SPR. Comparison of the STD nuclear magnetic resonance (NMR) binding epitopes of Tn and Neu5Acα2,6-Tn revealed a constant binding mode of the N-acetylgalactosamine moiety. This finding is in good agreement with binding studies of CLEC10A transfectomas, which show a well-defined interaction of transmembrane CLEC10A with 6-sialylated-Tn structures. Since both Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn together with the previously known Tn antigen are expressed in human tumors such as mammary carcinoma, the interaction with CLEC10A expressed by macrophages and dendritic cells could be of major functional significance in tumor progression.
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http://dx.doi.org/10.1093/glycob/cwt021DOI Listing
July 2013

Plasma membrane-associated malate dehydrogenase of maize (Zea mays L.) roots: native versus recombinant protein.

J Proteomics 2013 Mar 10;80:66-77. Epub 2013 Jan 10.

University of Hamburg, Biocentre Klein Flottbek and Botanical Garden, Plant Physiology, Ohnhorststraße 18, D-22609 Hamburg, Germany. Electronic address:

Malate dehydrogenase (MDH, EC 1.1.1.37) is involved in several cellular processes including plant development, nutrient uptake and oxidative stress. Evidence for a plasma membrane-associated MDH has been presented for maize (Zea mays L.) roots. In the present study isoenzymes of MDH were purified from highly enriched plasma membrane preparations of maize and compared with soluble isoenzymes (Km, pH optima, pI and molecular masses). Modified SDS-PAGE analyses revealed monomers of 41 kDa for membrane-associated MDH, whereas monomers (35 kDa) and dimers (70 kDa) were detected for soluble isoenzymes. Membrane-associated MDH of cauliflower (Brassica oleracea L.) inflorescences and spinach (Spinacia oleracea L.) leaves showed molecular masses similar to the membrane-associated MDH of maize. The specific maize MDH involved was identified by mass spectrometry (ESI-QTOF-MS/MS, MALDI-TOF-MS). The corresponding gene was cloned and the protein was characterised after heterologous expression in Escherichia coli. Enzyme kinetics and properties of the recombinant and native proteins were compared. The function of thiol groups and the presence of disulphide bonds were analysed by the effect of N-ethylmaleimide, diagonal electrophoresis and labelling. Semiquantitative reverse transcription polymerase chain reaction of maize root transcripts demonstrated a constitutive expression of the gene encoding plasma membrane-associated MDH.
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http://dx.doi.org/10.1016/j.jprot.2012.12.015DOI Listing
March 2013

The RNA-binding protein MARTA2 regulates dendritic targeting of MAP2 mRNAs in rat neurons.

J Neurochem 2013 Mar 20;124(5):670-84. Epub 2013 Jan 20.

Institute for Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Dendritic targeting of mRNAs encoding the microtubule-associated protein 2 (MAP2) in neurons involves a cis-acting dendritic targeting element. Two rat brain proteins, MAP2-RNA trans-acting protein (MARTA)1 and MARTA2, bind to the cis-element with both high affinity and specificity. In this study, affinity-purified MARTA2 was identified as orthologue of human far-upstream element binding protein 3. In neurons, it resides in somatodendritic granules and dendritic spines and associates with MAP2 mRNAs. Expression of a dominant-negative variant of MARTA2 disrupts dendritic targeting of endogenous MAP2 mRNAs, while not noticeably altering the level and subcellular distribution of polyadenylated mRNAs as a whole. Finally, MAP2 transcripts associate with the microtubule-based motor KIF5 and inhibition of KIF5, but not cytoplasmic dynein function disrupts extrasomatic trafficking of MAP2 mRNA granules. Thus, in neurons MARTA2 appears to represent a key trans-acting factor involved in KIF5-mediated dendritic targeting of MAP2 mRNAs.
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http://dx.doi.org/10.1111/jnc.12079DOI Listing
March 2013

sarA negatively regulates Staphylococcus epidermidis biofilm formation by modulating expression of 1 MDa extracellular matrix binding protein and autolysis-dependent release of eDNA.

Mol Microbiol 2012 Oct 10;86(2):394-410. Epub 2012 Sep 10.

Institute for Medical Microbiology, Virology and Hygiene, University Medical Centre Hamburg-Eppendorf, Martinistraße 52, 20246, Hamburg, Germany.

Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant-associated infections. Nonetheless, large proportions of invasive Staphylococcus epidermidis isolates fail to form a biofilm in vitro. We here tested the hypothesis that this apparent paradox is related to the existence of superimposed regulatory systems suppressing a multicellular biofilm life style in vitro. Transposon mutagenesis of clinical significant but biofilm-negative S. epidermidis 1585 was used to isolate a biofilm positive mutant carrying a Tn917 insertion in sarA, chief regulator of staphylococcal virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via overexpression of the giant 1 MDa extracellular matrix binding protein (Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA (eDNA) release significantly contributed to biofilm formation in mutant 1585ΔsarA. Increased eDNA amounts indirectly resulted from upregulation of metalloprotease SepA, leading to boosted processing of autolysin AtlE, in turn inducing augmented autolysis and release of eDNA. Hence, this study identifies sarA as a negative regulator of Embp- and eDNA-dependent biofilm formation. Given the importance of SarA as a positive regulator of polysaccharide mediated cell aggregation, the regulator enables S. epidermidis to switch between mechanisms of biofilm formation, ensuring S. epidermidis adaptation to hostile environments.
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http://dx.doi.org/10.1111/j.1365-2958.2012.08203.xDOI Listing
October 2012

Purification, crystallization and preliminary X-ray diffraction analysis of crotamine, a myotoxic polypeptide from the Brazilian snake Crotalus durissus terrificus.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2012 Sep 30;68(Pt 9):1052-4. Epub 2012 Aug 30.

Centro Multiusuário de Inovação Biomolecular, Departamento de Física, Universidade Estadual Paulista (UNESP), São José do Rio Preto-SP 15054-000, Brazil.

Crotamine, a highly basic myotoxic polypeptide (molecular mass 4881 Da) isolated from the venom of the Brazilian rattlesnake Crotalus durissus terrificus, causes skeletal muscle contraction and spasms, affects the functioning of voltage-sensitive sodium channels by inducing sodium influx and possesses antitumour activity, suggesting potential pharmaceutical applications. Crotamine was purified from C. durissus terrificus venom; the crystals diffracted to 1.9 Å resolution and belonged to the orthorhombic space group I2(1)2(1)2(1) or I222, with unit-cell parameters a = 67.75, b = 74.4, c = 81.01 Å. The self-rotation function indicated that the asymmetric unit contained three molecules. However, structure determination by molecular replacement using NMR-determined coordinates was unsuccessful and a search for potential derivatives has been initiated.
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http://dx.doi.org/10.1107/S1744309112032721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433195PMC
September 2012

High phosphate directly affects endothelial function by downregulating annexin II.

Kidney Int 2013 Feb 22;83(2):213-22. Epub 2012 Aug 22.

Department of Internal Medicine D, University of Münster, Münster, Germany.

Hyperphosphatemia is associated with increased cardiovascular risk in patients with renal disease and in healthy individuals. Here we tested whether high phosphate has a role in the pathophysiology of cardiovascular events by interfering with endothelial function, thereby impairing microvascular function and angiogenesis. Protein expression analysis found downregulation of annexin II in human coronary artery endothelial cells, an effect associated with exacerbated shedding of annexin II-positive microparticles by the cells exposed to high phosphate media. EAhy926 endothelial cells exposed to sera from hyperphosphatemic patients also display decreased annexin II, suggesting a negative correlation between serum phosphate and annexin II expression. By using endothelial cell-based assays in vitro and the chicken chorioallantoic membrane assay in vivo, we found that angiogenesis, vessel wall morphology, endothelial cell migration, capillary tube formation, and endothelial survival were impaired in a hyperphosphatemic milieu. Blockade of membrane-bound extracellular annexin II with a specific antibody mimicked the effects of high phosphate. In addition, high phosphate stiffened endothelial cells in vitro and in rats in vivo. Thus, our results link phosphate and adverse clinical outcomes involving the endothelium in both healthy individuals and patients with renal disease.
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http://dx.doi.org/10.1038/ki.2012.300DOI Listing
February 2013

Comparison of displacement versus gradient mode for separation of a complex protein mixture by anion-exchange chromatography.

J Chromatogr B Analyt Technol Biomed Life Sci 2012 Jul 6;901:34-40. Epub 2012 Jun 6.

University Medical Center Hamburg-Eppendorf, Department of Clinical Chemistry, Mass Spectrometric Proteomics, Campus Forschung, N27 Room 00.08, Martinistr. 52, 20246 Hamburg, Germany.

Liquid chromatography is often the method of choice for the analysis of proteins in their native state. Nevertheless compared to two-dimensional electrophoresis, the resolution of common chromatographic techniques is low. Liquid chromatography in the displacement mode has previously been shown to offer higher resolution and to elute proteins in the high concentrations. In this study we compared to what extend displacement mode was a suitable alternative to gradient mode for the separation of a complex protein mixture using anion-exchange displacement chromatography and if it is therefore helpful for proteomic investigations. Hence we analyzed the qualitative protein composition of each fraction by tryptic digestion of the proteins, analysis of the tryptic peptides by liquid chromatography coupled to mass spectrometry followed by data base analysis and by measuring the elution profiles of 22 selected proteins with selected reaction monitoring mass spectrometry. In the fractions of displacement mode a significantly higher number of identified proteins (51 versus 16) was yielded in comparison to gradient mode. The resolution of displacement chromatography was slightly lower than of gradient chromatography for many but not for all proteins. The selectivities of displacement mode and gradient mode are very different. In conclusion displacement chromatography is a well suited alternative for top-down proteomic approaches which start with separating intact proteins first prior to mass spectrometric analysis of intact or digested proteins. The significant orthogonality of both modes may be used in the future for combining them in multidimensional fractionation procedures.
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http://dx.doi.org/10.1016/j.jchromb.2012.05.037DOI Listing
July 2012

Crystal structure of a dimeric Ser49- PLA₂-like myotoxic component of the Vipera ammodytes meridionalis venomics reveals determinants of myotoxicity and membrane damaging activity.

Mol Biosyst 2012 Apr 23;8(5):1405-11. Epub 2012 Feb 23.

Institute of Biochemistry and Molecular Biology, University of Hamburg, Laboratory of Structural Biology of Infection and Inflammation, c/o DESY, Build. 22a, 22603 Hamburg, Germany.

Myotoxicity and membrane damage play a central role in the life-threatening effects of the viper envenomation. Myotoxins are an important part of the viper venomics. A Ser49 PLA₂-like myotoxin from the venom of Vipera ammodytes meridionalis, the most venomous snake in Europe, was crystallized and its three-dimensional structure determined. The toxin is devoid of phospholipolytic activity. The structure demonstrates a formation of dimers. In the dimers functionally important peptide segments, located on the protein surface, point in the same direction which can strengthen the pharmacological effect. This supports the hypothesis about the physiological importance of the toxin oligomerization for the myotoxicity and membrane damage. The crystallographic model revealed that the structural determinants of myotoxicity (a positively charged C-terminal region and a hydrophobic knuckle) are fully exposed on the protein surface and accessible for interactions with target membranes. Distortion of the catalytic site region explains the absence of enzymatic activity. The structure reveals anion-binding sites which can be considered as possible sites of interactions of the toxin with a negatively charged membrane surface. The high structural similarity of the Ser49 myotoxin and Asp49 PLA₂ from the same venom suggests an evolutionary relationship: probably, the Ser49 myotoxin is a product of evolution of the catalytically active phospholipase A₂. The first toxin lost the enzymatic activity which is not necessary for the myotoxicity but preserved the cytotoxicity and membrane damaging activity as important components of the venom toxicity.
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http://dx.doi.org/10.1039/c2mb05490fDOI Listing
April 2012

Combining lectin affinity chromatography and immunodepletion - A novel method for the enrichment of disease-specific glycoproteins in human plasma.

Methods 2012 Feb 22;56(2):254-9. Epub 2011 Dec 22.

Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany.

Drastic enrichment of potential disease-specific glycoprotein markers in human plasma can be achieved by the combination of affinity- and immuno-depletion. In the affinity-fractionation step all glycoproteins carrying a certain glycostructure are isolated by lectin affinity chromatography, thus depleting other components. Against the respective glycoprotein fraction isolated from the plasma of healthy individuals antibodies are raised in llamas. The llama heavy chain antibodies (which are particularly stable) directed at the isolated plasma glycoprotein fraction are immobilized and the immunoaffinity column thus obtained is used to deplete the respective glycoprotein fraction of patient plasma samples. Depletion of proteins normally found in human plasma by 99.8-99.9% can be achieved, resulting in a 800-1000-fold enrichment of potential disease-specific proteins in the flow-through of the immunoaffinity column.
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http://dx.doi.org/10.1016/j.ymeth.2011.12.004DOI Listing
February 2012

Monoclonal antibodies for the identification and purification of vNAR domains and IgNAR immunoglobulins from the horn shark Heterodontus francisci.

Hybridoma (Larchmt) 2011 Aug;30(4):323-9

Department of Marine Biotechnology, Centro de Investigacion Cientifica y de Educacion Superior de Ensenada, Ensenada, Baja California, Mexico.

In addition to conventional antibodies, cartilaginous fish have evolved a distinctive type of immunoglobulin, designated as IgNAR, which lacks the light polypeptide chains and is composed entirely by heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce the variable domain of a single heavy chain polypeptide (vNARs). These, together with the VHH camel domains, constitute the smallest naturally occurring domains able to recognize an antigen. Their special features, such as small size, long extended finger-like CDR3, and thermal and chemical stability, make them suitable candidates for biotechnological purposes. Here we describe the generation of two mouse monoclonal antibodies (MAbs), MAb 370-12 and MAb 533-10, that both specifically react with vNAR domains of the horn shark Heterodontus francisci. While the former recognizes a broad spectrum of recombinant vNAR proteins, the latter is more restricted. MAb 370-12 precipitated a single band from whole shark serum, which was identified as IgNAR by mass spectrometry. Additionally, we used MAb 370-12 to follow the IgNAR-mediated immune response of sharks during immunization protocols with two different antigens (complete cells and a synthethic peptide), thus corroborating that MAb 370-12 recognizes both isolated vNAR domains and whole IgNAR molecules. Both MAbs represent an affordable molecular, biochemical, and biotechnological tool in the field of shark single-domain antibodies.
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http://dx.doi.org/10.1089/hyb.2011.0010DOI Listing
August 2011
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