Biology subjects

Gramlich, M.

Publications and source records attributed to Gramlich, M..

7 recordsLinked to original sources

bioRxiv2026

Presynaptic Terminals Dynamically Modulate Spontaneous Release Frequency During Early Synaptic Plasticity Through and Entropic Force Framework

Spontaneous synaptic transmission has been established as essential for the maintenance of synaptic weights during action potential-induced transmission. However, spontaneous transmission also changes during synaptic plasticity and has been shown to, in part, mediate changes in synaptic weights. Despite decades of research, a coherent framework for understanding the complex molecular processes that support presynaptic spontaneous transmission during maintenance and plasticity has remained elusive. We show here that presynapses modulate spontaneous transmission frequency during the early time-course of plasticity following entropic force theory. We use live primary hippocampal cultures as a model system and induce plasticity using an established Long-Term Potentiation (LTP) protocol. We then use a combination of electron microscopy, fluorescence microscopy, and computational modeling to show how spontaneous release frequency dynamically changes during early plasticity. We use our entropic force theory to show how the dynamically changing synaptic vesicle pool structure mediates spontaneous release changes. Lastly, we show how these changes are altered in the presence of P301L tau leading to degeneration. The results from this study provide new insights that not only help understand normal synaptic function but also aid in understanding neurodegeneration.

neuroscience↗

bioRxiv2023

Development of a PNGase Rc column for online deglycosylation of complex glycoproteins during HDX-MS

Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins.

biochemistry↗

bioRxiv2023

Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells

Signal-regulatory protein (SIRP) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRP and the "dont eat me" ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRP (hSIRP)-specific nanobodies (Nbs). We identified three high-affinity Nbs targeting the hSIRP/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis (ADCP). For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography (PET) in novel hSIRP/hCD47 knock-in (KI) mice, we demonstrated the applicability of 64Cu-hSIRP-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRP-Nbs presented in this study have potential as versatile probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies.

immunology↗

bioRxiv2022

A novel PNGase Rc for improved protein N-deglycosylation in bioanalytics and HDX-MS epitope mapping under challenging conditions

N-linked glycosylation is a ubiquitous posttranslational modification of proteins. While it plays an important role in the biological function of proteins, it often poses a major challenge for their analytical characterization. Currently available peptide N-glycanases (PNGases) are often inefficient at deglycosylating proteins due to sterically inaccessible N-glycosylation sites. This usually leads to poor sequence coverage in bottom-up analysis using liquid chromatography with tandem mass spectrometry (LC-MS) and makes it impossible to obtain an intact mass signal in top-down MS analysis. In addition, most PNGases operate optimally only in the neutral to slightly acidic pH range and are severely compromised in the presence of reducing and denaturing substances, which limits their use for advanced bioanalysis based on hydrogen-deuterium exchange in combination with mass spectrometry (HDX-MS). Here, we present a novel peptide N-glycanase from Rudaea cellulosilytica (PNGase Rc) for which we demonstrate broad substrate specificity for N-glycan hydrolysis from multiply occupied and natively folded proteins. Our results show that PNGase Rc is functional even under challenging, HDX quench conditions (pH 2.5, 0 {degrees}C) and in the presence of 0.4 M Tris(2-carboxyethyl)phosphine (TCEP), 4 M urea and 1 M guanidinium chloride (GdmCl). Most importantly, we successfully applied the PNGase Rc in an HDX-MS workflow to determine the epitope of a nanobody targeting the extracellular domain of human signal-regulating protein alpha (SIRP).

biochemistry↗

bioRxiv2021

Single-domain antibodies for targeting, detection and in vivo imaging of human CD4+ cells

The advancement of new immunotherapies necessitates appropriate probes to monitor the presence and distribution of distinct immune cell populations. Considering the key role of CD4+ T cells in regulating immunological processes, we generated novel single-domain antibodies (nanobodies, Nbs) that specifically recognize human CD4. After in depth analysis of their binding properties, recognized epitopes, and effects on T cell proliferation, activation and cytokine release, we selected CD4 Nbs that did not interfere with crucial T cell processes in vitro and converted them into immune tracers for non-invasive molecular imaging. By optical imaging, we demonstrate the ability of a high-affinity CD4-Nb to specifically visualize CD4+ cells in vivo using a xenograft model. Furthermore, time-resolved immune positron emission tomography (immunoPET) of a human CD4 knock-in mouse model showed rapid accumulation of 64Cu-radiolabeled CD4-Nb in CD4+ T cell-rich tissues. We propose that the CD4 Nbs presented here could serve as versatile probes for stratifying patients and monitoring individual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.

immunology↗

bioRxiv2020

Myosin V regulates spatial localization of different forms of neurotransmitter release in central synapses

Synaptic active zone (AZ) contains multiple specialized release sites for vesicle fusion. The utilization of release sites is regulated to determine spatiotemporal organization of the two main forms of synchronous release, uni-vesicluar (UVR) and multi-vesicular (MVR). We previously found that the vesicle-associated molecular motor myosin V regulates temporal utilization of release sites by controlling vesicle anchoring at release sites (Maschi et al, 2018). Here we show that acute inhibition of myosin V shifts preferential location of vesicle docking away from AZ center towards periphery, and results in a corresponding spatial shift in utilization of release sites during UVR. Similarly, inhibition of myosin V also reduces preferential utilization of central release sites during MVR, leading to more spatially distributed and temporally uniform MVR that occurs farther away from the AZ center. Thus myosin V regulates both temporal and spatial utilization of release sites during two main forms of synchronous release.

neuroscience↗

bioRxiv2020

NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response

As the COVID-19 pandemic escalates, the need for effective vaccination programs, therapeutic intervention, and diagnostic tools increases. Here, we identified 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD) of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following a detailed epitope determination and characterization of the binding mode by structural analysis, we constructed a hetero-bivalent Nb targeting two different epitopes within the RBD:ACE2 interface. This resulted in a high-affinity binder with a viral neutralization efficacy in the picomolar range. Using the bivalent Nb as a surrogate, we established a competitive multiplex binding assay ("NeutrobodyPlex") for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in the serum of convalescent or vaccinated patients. As demonstrated, the NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, helping to monitor immune status or guide vaccine design. This approach is easily transferrable to diagnostic laboratories worldwide.

immunology↗