Biology subjects

Marino, S. F.

Publications and source records attributed to Marino, S. F..

6 recordsLinked to original sources

bioRxiv2026

Identification of targetable epitope surfaces from the high resolution structure of the superantigen Staphylococcal Enterotoxin L

Emetic exotoxins secreted by Staphylococcus species (Staphylococcal enterotoxins, SEs) are a major cause of food poisoning cases worldwide and many are additionally classified as superantigens - able to potently activate T cells in an antigen independent manner. Fewer than half of the gene products of the known SE genes have been extensively characterized. The gene for Staphylococcal enterotoxin L (SEL) occurs in both foodborne and clinical isolates but no detailed structural characterization has yet been available. We report here the crystal structure of SEL and confirm its function as a superantigen via direct T cell activation assays. By comparison of the SEL sequence with that of its four closest homologues (SEI, SEK, SEM and SEQ), we have identified binding epitopes unique for SEL and mapped these regions onto the structure. These data provide the first high resolution view of SEL and the basis for the development of diagnostic procedures for its specific detection.

molecular biology↗

bioRxiv2023

Structure-based humanization of a therapeutic antibody for Multiple Myeloma

The optimal efficacy of xenogeneically generated proteins intended for application in humans requires that their own antigenicity be minimized. This necessary adaptation of antibodies to a humanized version poses challenges since modifications even distant from the binding sites can greatly influence antigen recognition and this is the primary feature that must be maintained during all modifications. Current strategies often rely on grafting and/or randomization/selection to arrive at a humanized variant retaining the binding properties of the original molecule. However, in terms of speed and efficiency, rationally directed approaches can be superior, provided the requisite structural information is available. We present here a humanization procedure based on the high-resolution X-ray structure of a chimeric IgG against a marker for Multiple Myeloma. Based on in silico modelling of humanizing amino acid substitutions identified from sequence alignments, we devised a straightforward cloning procedure to rapidly evaluate the proposed sequence changes. Careful inspection of the structure allowed identification of a potentially problematic amino acid change that indeed disrupted antigen binding. Subsequent optimization of the antigen binding loop sequences resulted in substantial recovery of binding affinity lost in the completely humanized antibody. X-ray structures of the humanized and optimized variants demonstrate that the antigen binding mode is preserved, with surprisingly few direct contacts to antibody atoms. These results underline the importance of structural information for the efficient optimization of protein therapeutics.

biophysics↗

bioRxiv2022

SARS-CoV-2 has not emerged in roe, red or fallow deer in Germany or Austria during the COVID 19 pandemic

Spillover of SARS-CoV-2 to North American white tailed deer (Odocoileus virginianus) has been documented. We evaluated pre and pandemic exposure of German and Austrian deer species using a SARS-CoV-2 pseudoneutralization assay. In stark contrast to North American white tailed deer, we found no evidence of SARS-CoV-2 exposure. Article Summary LineUsing a sensitive serological assay, 433 pre and pandemic deer samples from Germany and Austria tested negative for SARS-CoV-2-specific antibodies highlighting a major difference between central European and North American deer exposure and in their epidemiologic roles.

microbiology↗

bioRxiv2021

Disrupting the CD177:proteinase 3 membrane complex reduces anti-PR3 antibody-induced neutrophil activation

CD177 is a neutrophil-specific receptor presenting proteinase 3 (PR3) autoantigen on the neutrophil surface. CD177 expression is restricted to a neutrophil subset resulting in CD177pos/mPR3high and CD177neg/mPR3low populations. The size of the CD177pos/mPR3high subset has implications for anti-neutrophil cytoplasmic autoantibody (ANCA)-associated autoimmune vasculitis (AAV) where patients harbor PR3-specific ANCA that activate neutrophils. We generated high affinity anti-CD177 monoclonal antibodies, some of which interfered with PR3 binding to CD177 (PR3 "blockers") as determined by surface plasmon resonance spectroscopy, and used them to test the effect of competing PR3 from the surface of CD177pos neutrophils. Because intact anti-CD177 antibodies also caused neutrophil activation, we prepared non-activating Fab fragments of a PR3 blocker and non-blocker that bound specifically to CD177pos neutrophils by flow cytometry. We observed that Fab blocker clone 40, but not non-blocker clone 80, dose-dependently reduced anti-PR3 antibody binding to CD177pos neutrophils. Importantly, preincubation with clone 40 significantly reduced respiratory burst in primed neutrophils challenged either with monoclonal antibodies to PR3 or PR3-ANCA IgG from AAV patients. After separating the two CD177/mPR3 neutrophil subsets from individual donors by magnetic sorting, we found that PR3-ANCA provoked significantly more superoxide production in CD177pos/mPR3high than in CD177neg/mPR3low neutrophils, and that anti-CD177 Fab clone 40 reduced the superoxide production of CD177pos cells to the level of the CD177neg cells. Our data demonstrate the importance of the CD177:PR3 membrane complex in maintaining a high ANCA epitope density and thereby underscore the contribution of CD177 to the severity of PR3-ANCA diseases.

immunology↗

bioRxiv2018

Structure of a cytochrome-based bacterial nanowire

Electrically conductive pili from Geobacter species, termed bacterial \"nanowires\", are intensely studied for their biological significance and potential in the development of new materials. We have characterized a unique nanowire from conductive G. sulfurreducens pili preparations by cryo-electron microscopy composed solely of the c-type cytochrome OmcS. We present here, at 3.4 [A] resolution, a novel structure of a cytochrome-based filament and discuss its possible role in long-range biological electron transport.\n\nSummary sentenceCryo-electron microscopy reveals the remarkable assembly of a c-type cytochrome into filaments comprising a heme-based bacterial nanowire.

biophysics↗

bioRxiv2018

Rapid and efficient C-terminal labeling of nanobodies for DNA-PAINT

Single molecule localization-based approaches to superresolution microscopy (SMLM) create images that resolve features smaller than the diffraction limit of light by rendering them from the sequentially measured positions of thousands of individual molecules. New SMLM approaches based on the transient binding of very bright dyes via DNA-DNA interaction (DNA-PAINT) allow the resolution of dyes only a few nanometers apart in vitro. This imaging of cellular structures requires the specific association of dyes to their targets, which results in an additional \"linkage error\". This error can be minimized by using extremely small, single-domain antibody-based binders such as nanobodies, but the DNA-oligomers used in DNA-PAINT are of significant size in comparison to nanobodies and may interfere with binding. We have here developed an optimized procedure based on enzymatic labeling and click-chemistry for the coupling of DNA oligomers to the nanobody C-terminus, which is located on the opposite side of the epitope-binding domain. Our approach allows for straightforward labeling, purification and DNA-PAINT imaging. We performed high efficiency labeling of two different nanobodies and show dual color multiplexed SMLM to demonstrate the general applicability of our labeling scheme.

biophysics↗