Carsten Schmelter

University Medical Center of the Johannes Gutenberg University Mainz

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Norbert Pfeiffer

Johannes Gutenberg University Mainz

Natarajan Perumal

University Medical Center of the Johannes Gutenberg University Mainz

Franz H. Grus

Johannes Gutenberg University Mainz

Sebastian Funke

University Medical Center of the Johannes Gutenberg University Mainz

Franz H. Grus

University Medical Center of the Johannes Gutenberg University Mainz

Katharina Bell

National Health and Medical Research Council

Caroline Manicam

University Medical Center of the Johannes Gutenberg University Mainz

Vanessa M. Beutgen

Philipps University of Marburg

Kristian Nzogang Fomo

University Medical Center of the Johannes Gutenberg University Mainz

Sascha D. Markowitsch

Johannes Gutenberg University Mainz

Cooperative Institutions

Johannes Gutenberg University Mainz

University Medical Center of the Johannes Gutenberg University Mainz

University of Würzburg

University Hospital Münster

University of Münster

Charité - Universitätsmedizin Berlin

Humboldt-Universität zu Berlin

Filatov Institute of Eye Diseases and Tissue Therapy of the National Academy of Medical Sciences of Ukraine

University of Tübingen

Universitätsklinikum Würzburg

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Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels

Biochimica et Biophysica Acta (BBA) - Biomembranes (2011)

Nicole Bobak Stefan Bittner Joseph Andronic Susanne Hartmann Friederike Mühlpfordt

BK channel

Osmoregulation

Homeostasis

10.1016/j.bbamem.2011.04.013

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Citations (45)

Cell Death Discovery (2020)

Katharina Bell Ines Rosignol Elena Sierra‐Filardi Natalia Rodríguez‐Muela Carsten Schmelter

Glaucoma is a common age-related disease leading to progressive retinal ganglion cell (RGC) death, visual field defects and vision loss and is the second leading cause of blindness in the elderly worldwide. Mitochondrial dysfunction and impaired autophagy have been linked to glaucoma and induction of autophagy shows neuroprotective effects in glaucoma animal models. We have shown that autophagy decreases with aging in the retina and that autophagy can be neuroprotective for RGCs, but it is currently unknown how aging and autophagy deficiency impact RGCs susceptibility and survival. Using the optic nerve crush model in young and olWelcome@1234d Ambra1+/gt (autophagy/beclin-1 regulator 1+/gt) mice we analysed the contribution of autophagy deficiency on retinal ganglion cell survival in an age dependent context. Interestingly, old Ambra1+/gt mice showed decreased RGC survival after optic nerve crush in comparison to old Ambra1+/+, an effect that was not observed in the young animals. Proteomics and mRNA expression data point towards altered oxidative stress response and mitochondrial alterations in old Ambra1+/gt animals. This effect is intensified after RGC axonal damage, resulting in reduced oxidative stress response showing decreased levels of Nqo1, as well as failure of Nrf2 induction in the old Ambra1+/gt. Old Ambra1+/gt also failed to show increase in Bnip3l and Bnip3 expression after optic nerve crush, a response that is found in the Ambra1+/+ controls. Primary RGCs derived from Ambra1+/gt mice show decreased neurite projection and increased levels of apoptosis in comparison to Ambra1+/+ animals. Our results lead to the conclusion that oxidative stress response pathways are altered in old Ambra1+/gt mice leading to impaired damage responses upon additional external stress factors.

10.1038/s41420-020-0257-4

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Contribution of the Commensal Microflora to the Immunological Homeostasis and the Importance of Immune-Related Drug Development for Clinical Applications

International Journal of Molecular Sciences (2021)

Vanessa M. Beutgen Carsten Schmelter Norbert Pfeiffer Franz H. Grus

Not long ago, self-reactive immune activity was considered as pathological trait. A paradigm shift has now led to the recognition of autoimmune processes as part of natural maintenance of molecular homeostasis. The immune system is assigned further roles beneath the defense against pathogenic organisms. Regarding the humoral immune system, the investigation of natural autoantibodies that are frequently found in healthy individuals has led to further hypotheses involving natural autoimmunity in other processes as the clearing of cellular debris or decrease in inflammatory processes. However, their role and origin have not been entirely clarified, but accumulating evidence links their formation to immune reactions against the gut microbiome. Antibodies targeting highly conserved proteins of the commensal microflora are suggested to show self-reactive properties, following the paradigm of the molecular mimicry. Here, we discuss recent findings, which demonstrate potential links of the commensal microflora to the immunological homeostasis and highlight the possible implications for various diseases. Furthermore, specific components of the immune system, especially antibodies, have become a focus of attention for the medical management of various diseases and provide attractive treatment options in the future. Nevertheless, the development and optimization of such macromolecules still represents a very time-consuming task, shifting the need to more medical agents with simple structural properties and low manufacturing costs. Synthesizing only the biologically active sites of antibodies has become of great interest for the pharmaceutical industry and offers a wide range of therapeutic application areas as it will be discussed in the present review article.

Molecular mimicry

10.3390/ijms22168896

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Citations (1)

Synthetic antibody-derived immunopeptide provides neuroprotection in glaucoma through molecular interaction with retinal protein histone H3.1

Frontiers in Medicine (2022)

Kristian Nzogang Fomo Carsten Schmelter Joshua Atta Vanessa M. Beutgen R. Schwarz

Glaucoma is a group of optic neuropathies characterized by the progressive degeneration of retinal ganglion cells (RGCs) as well as their axons leading to irreversible loss of sight. Medical management of the intraocular pressure (IOP) still represents the gold standard in glaucoma therapy, which only manages a single risk factor and does not directly address the neurodegenerative component of this eye disease. Recently, our group showed that antibody-derived immunopeptides (encoding complementarity-determining regions, CDRs) provide attractive glaucoma medication candidates and directly interfere its pathogenic mechanisms by different modes of action. In accordance with these findings, the present study showed the synthetic complementary-determining region 2 (CDR2) peptide (INSDGSSTSYADSVK) significantly increased RGC viability in vitro in a concentration-dependent manner (p < 0.05 using a CDR2 concentration of 50 μg/mL). Employing state-of the-art immunoprecipitation experiments, we confirmed that synthetic CDR2 exhibited a high affinity toward the retinal target protein histone H3.1 (HIST1H3A) (p < 0.001 and log2-fold change > 3). Furthermore, molecular dynamics (MD) simulations along with virtual docking analyses predicted potential CDR2-specific binding regions of HIST1H3A, which might represent essential post-translational modification (PTM) sites for epigenetic regulations. Quantitative mass spectrometry (MS) analysis of retinas demonstrated 39 proteins significantly affected by CDR2 treatment (p < 0.05). An up-regulation of proteins involved in the energy production (e.g., ATP5F1B and MT-CO2) as well as the regulatory ubiquitin proteasome system (e.g., PSMC5) was induced by the synthetic CDR2 peptide. On the other hand, CDR2 reduced metabolic key enzymes (e.g., DDAH1 and MAOB) as well as ER stress-related proteins (e.g., SEC22B and VCP) and these data were partially confirmed by microarray technology. Our outcome measurements indicate that specific protein-peptide interactions influence the regulatory epigenetic function of HIST1H3A promoting the neuroprotective mechanism on RGCs in vitro. In addition to IOP management, such synthetic peptides as CDR2 might serve as a synergistic immunotherapy for glaucoma in the future.

10.3389/fmed.2022.993351

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Lectin-Based Affinity Enrichment and Characterization of N-Glycoproteins from Human Tear Film by Mass Spectrometry

Molecules (2023)

Carsten Schmelter Alina Brueck Natarajan Perumal Sichang Qu Norbert Pfeiffer

The glycosylation of proteins is one of the most common post-translational modifications (PTMs) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome, maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of tear film might promote the development of chronic eye diseases, indicating glycoproteins as a valuable source for biomarker discovery or drug target identification. Therefore, the present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and to characterize their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we first accumulated native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA, and UEA I, termed 4L) was applied to glycopeptide enrichment from human tear sample digests, followed by MS-based detection and localization of their specific N-glycosylation sites. As the main result, our study identified a total of 26 N glycosylation sites of 11 N-glycoproteins in the tear sample pools of healthy individuals (n = 3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP), and extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, already associated with the pathogenesis of various chronic eye diseases such as dry eye syndrome (DES). In conclusion, the results of the present study will serve as an important tear film N-glycoprotein catalog for future studies focusing on human tear film and ocular surface-related inflammatory diseases.

PNGase F

Proteome

10.3390/molecules28020648

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Citations (5)

Tränenfilmspezifische Biomarker bei Glaukompatienten

Klinische Monatsblätter für Augenheilkunde (2022)

Kristian Nzogang Fomo Carsten Schmelter Norbert Pfeiffer Franz H. Grus

Glaucoma is a group of chronic eye diseases that lead to degeneration of retinal ganglion cells (RGCs) and their axons followed by irreversible loss of vision in the patient. Glaucoma is a disease that initially evolves asymptomatically with the first symptoms appearing only at an advanced stage of this eye disease. For this reason, it is always necessary to develop state-of-the-art technologies and methods for the identification and characterization of new, specific biomarkers for the early diagnosis of glaucoma. Therefore, the analysis of biological fluids, as in this case the tear fluid of patients, represents an attractive source to identify new specific as well as sensitive biomarkers in glaucoma. These biomarkers could be involved in the pathophysiological processes of glaucoma or possibly serve for diagnostic differentiation of various types of glaucoma.Das Glaukom bezeichnet eine Gruppe chronischer Augenkrankheiten, die zu einer Degeneration der retinalen Ganglienzellen (RGZ) und ihrer Axone führen, gefolgt von einem irreversiblen Verlust des Sehvermögens des Patienten. Das Glaukom ist eine Krankheit, die sich zunächst asymptomatisch entwickelt, wobei die ersten Symptome erst in einem fortgeschrittenen Stadium dieser Augenkrankheit auftreten. Aus diesem Grund ist es stets notwendig, modernste Technologien und Methoden zur Identifizierung und Charakterisierung neuer, spezifischer Biomarker für die Frühdiagnose des Glaukoms zu entwickeln. Daher stellt die Analyse biologischer Flüssigkeiten, wie in diesem Fall der Tränenflüssigkeit von Patienten, eine attraktive Quelle für die Identifizierung neuer spezifischer sowie sensitiver Biomarker bei Glaukom dar. Diese Biomarker könnten an den pathophysiologischen Prozessen des Glaukoms beteiligt sein oder möglicherweise zur diagnostischen Differenzierung verschiedener Glaukomtypen dienen.

10.1055/a-1749-6972

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Citations (2)

Follicular lymphoma grade 3B is a distinct neoplasm according to cytogenetic and immunohistochemical profiles

Haematologica (2011)

Heike Horn Carsten Schmelter Ellen Leich Itziar Salaverría Tiemo Katzenberger

Background According to the current World Health Organization Classification of Lymphoid Tumours, follicular lymphoma is subclassified into three grades according to the number of centroblasts. Follicular lymphoma grade 3 can be further divided into types A and B. Almost all available genetic data on grade 3B follicular lymphomas have been generated from tumors with an additional diffuse large B-cell lymphoma component. The purely follicular type of follicular lymphoma grade 3B is a rare neoplasm.Design and Methods We performed a detailed immunohistochemical (CD10, IRF4/MUM1, BCL2, Ig light chains) and genetic (translocations of BCL2, BCL6, MYC, IRF4) characterization of the largest series of purely follicular cases of grade 3B follicular lymphoma available to date, comprising 23 tumor samples. We also included 25 typical grade 1 or 2 follicular lymphomas, 9 follicular lymphomas with large centrocytes and/or high proliferation indices (FL/LCC), 12 cases of follicular lymphoma grade 3A, 16 cases of diffuse large B-cell lymphoma/follicular lymphoma grade 3B and 15 follicular lymphomas in which a straightforward distinction between grades 3A and 3B was not possible.Results Translocations affecting BCL2 and BCL6 genes are rare in grade 3B follicular lymphomas (2/23, 9% and 4/23, 17%) when compared with grade 1 or 2 follicular lymphomas (22/25, 88%, P

Follicular lymphoma

Neoplasm

10.3324/haematol.2011.042531

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Citations (158)

Peptides of the variable IgG domain as potential biomarker candidates in primary open-angle glaucoma (POAG)

Human Molecular Genetics (2017)

Carsten Schmelter Natarajan Perumal Sebastian Funke Katharina Bell Norbert Pfeiffer

Autoantibody profiling has gained increasing interest in the research field of glaucoma promising the detection of highly specific and sensitive marker candidates for future diagnostic purposes. Recent studies demonstrated that immune responses are characterized by the expression of congruent or similar complementarity determining regions (CDR) in different individuals and could be used as molecular targets in biomarker discovery. Main objective of this study was to characterize glaucoma-specific peptides from the variable region of sera-derived immunoglobulins using liquid chromatography--mass spectrometry (LC-MS)-based quantitative proteomics. IgG was purified from sera of 13 primary open-angle glaucoma patients (POAG) and 15 controls (CTRL) and subsequently digested into Fab and Fc by papain. Fab was further purified, tryptic digested and measured by LC-MS/MS. Discovery proteomics revealed in total 75 peptides of the variable IgG domain showing significant glaucoma-related level changes (P < 0.05; log2 fold change ≥ 0.5): 6 peptides were high abundant in POAG sera, whereas 69 peptides were low abundant in comparison to CTRL group. Via accurate inclusion mass screening strategy 28 IgG V domain peptides were further validated showing significantly decreased expression levels in POAG sera. Amongst others 5 CDR1, 2 CDR2 and 1 CDR3 sequences. In addition, we observed significant shifts in the variable heavy chain family distribution and disturbed κ/λ ratios in POAG patients in contrast to CTRL. These findings strongly indicate that glaucoma is accompanied by systemic effects on antibody production and B cell maturation possibly offering new prospects for future diagnostic or therapy purposes.

Biomarker Discovery

Fold change

10.1093/hmg/ddx332

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Citations (28)

Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma

International Journal of Molecular Sciences (2022)

Henrik Tonner Selina Hunn Nadine Auler Carsten Schmelter Norbert Pfeiffer

Slow and progressive loss of retinal ganglion cells (RGCs) is the main characteristic of glaucoma, the second leading cause of blindness worldwide. Previous studies have shown that impaired mitochondrial dynamics could facilitate retinal neurodegeneration. Mitochondrial dynamics are regulated directly (fission) or more indirectly (fusion) by dynamin-like protein 1 (DNML1). Therefore, DNM1L might be a promising target for an antibody-based approach to treat glaucoma. The consequences of targeting endogenous DNM1L by antibodies in a glaucoma animal model have not been investigated yet. Here, we show that the intravitreal application of an anti-DNM1L antibody showed protective effects regarding the survival of RGCs and their axons in the retinal nerve fiber layer (RNFL). Antibody treatment also improved retinal functionality, as observed by electroretinography (Ganzfeld ERG). Western blot analysis revealed altered DNM1L phosphorylation and altered expression of proteins related to apoptosis suggesting a decreased apoptosis rate. Mass spectrometry analysis revealed 28 up-regulated and 21 down-regulated proteins (p < 0.05) in both experimental groups. Protein pathway analysis showed that many proteins interacted directly with the target protein DNM1L and could be classified into three main protein clusters: Vesicle traffic-associated (NSF, SNCA, ARF1), mitochondrion-associated (HSP9A, SLC25A5/ANT2, GLUD1) and cytoskeleton-associated (MAP1A) signaling pathway. Our results demonstrate that DNM1L is a promising target for an antibody-based approach to glaucoma therapy.

DNM1L

10.3390/ijms232113618

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Citations (5)

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

Journal of Visualized Experiments (2019)

Natarajan Perumal Lars Straßburger Carsten Schmelter Adrian Gericke Norbert Pfeiffer

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome

Sample Preparation

Orbitrap

10.3791/59140-v

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Citations (8)