Biology subjects

Varon Silva, D.

Publications and source records attributed to Varon Silva, D..

4 recordsLinked to original sources

bioRxiv2024

Detection of Antibodies Against the African Parasite Trypanosoma brucei Using Synthetic glycosylphosphatidylinositol oligosaccharide fragments

Trypanosoma brucei (T. brucei) parasites cause two major infectious diseases in Africa: African trypanosomiasis in humans (HAT) and Nagana in animals. Despite the enormous economic and social impact, vaccines and reliable diagnostic measures are still lacking for these diseases. The main obstacle to developing accurate diagnostic methods and an active vaccine is the parasites ability for antigenic variation and impairment of B cell maturation, which prevents the development of a long-lasting, effective immune response. The antigenic variation is sustained by random gene switching, segmental gene conversion, and altered glycosylation states of solvent-exposed regions of the corresponding variant surface glycoproteins (VSG). These glycoproteins use a glycosylphosphatidylinositol (GPI) anchor for attachment to the membrane. GPIs of T. brucei have specific branched structures that are further heterogeneously galactosylated. We synthesized a glycan fragment library containing T. brucei GPIs most prominent structural features and performed an epitope mapping using mice and human sera of infected specimens using glycan microarrays. The studies indicate that in contrast to VSG, T. brucei GPIs are recognized by both short-lived IgM and long-lasting IgG, indicating a specific immune response against GPI structures. These findings enable the development of diagnostic tests based on synthetic antigens for reliable diagnosis of human African trypanosomiasis and Nagana.

biochemistry↗

bioRxiv2019

A novel, ultrasensitive approach for quantitative carbohydrate composition and linkage analysis using LC-ESI ion trap tandem mass spectrometry

Glycan identification and characterisation is essential to correlate glycoconjugate structure to biological function. The structural assignment of carbohydrates is often based on MS composition analyses and knowledge on well-studied glycosylation pathways. Nevertheless, many monosaccharide building blocks are indistinguishable by mass alone and detailed linkage information is also not easily obtained by MS/MS analyses, in particular when organisms are studied where the glycosylation pathways are less well defined. Here, we present a novel, simple and sensitive method using Reversed Phase (RP) - Liquid Chromatography Electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) for unambiguous identification and linkage determination of monosaccharides including N-acetylneuraminic acids. Sequential permethylation and reductive amination steps are employed prior and after acid hydrolysis to enable separation and differentiation of the various monosaccharides and their respective linkage positions. The well-established, monosaccharide specific methylation patterns allowed for the identification of the various derivatised monosaccharide alditols based on their retention time and tandem mass spectrometry fingerprint. Absolute quantitation can also be accomplished by including a set of internal standards, thus simultaneously providing qualitative and quantitative information on the monosaccharide residues present.

biochemistry↗

bioRxiv2019

Glycan size and attachment site location affect electron transfer dissociation (ETD) fragmentation and automated glycopeptide identification

We used a small synthetic glycopeptide library to systematically evaluate the effect of glycosylation site location and glycan size on the efficiency of ETD MS/MS fragmentation and subsequent automated identification. Understanding how the physico-chemical properties of glycopeptides influence glycopeptide fragmentation allows for optimizing fragmentation conditions and software assisted data analyses, which rely on informative fragmentation spectra for subsequent data processing to identify glycopeptides. Often, mis-assignment of glycopeptides occurs due to uncertainties such as failure to produce sufficient peptide backbone fragment ions. Our synthetic glycopeptide library contained glycopeptides differing in glycosylation site position within the peptide as well as glycan size (from the pentasaccharide N-glycan core to fully sialylated, biantennary N-glycans). Different software solutions such as SEQUEST and Amanda were compared for ETD glycopeptide identification. We found that all, glycan size, glycosylation site position within a glycopeptide and individual precursor m/z significantly impacted the number and quality of assignable glycopeptide backbone fragments, and thus the likelihood to be correctly identified in software assisted data analyses.

biochemistry↗

bioRxiv2019

Structural Determinants of Coiled Coil Mechanics

The natural abundance of coiled coil (CC) motifs in the cytoskeleton and the extracellular matrix suggests that CCs play a crucial role in the bidirectional mechanobiochemical signaling between cells and the matrix. Their functional importance and structural simplicity has allowed the development of numerous applications, such as protein-origami structures, drug delivery systems and biomaterials. With the goal of establishing CCs as nanomechanical building blocks, we investigated the importance of helix propensity and hydrophobic core packing on the mechanical stability of 4-heptad CC heterodimers. Using single-molecule force spectroscopy, we show that both parameters determine the force-induced dissociation in shear loading geometry; however, with different effects on the energy landscape. Decreasing the helix propensity lowers the transition barrier height, leading to a concomitant decrease in the distance to the transition state. In contrast, a less tightly packed hydrophobic core increases the distance to the transition state. We propose that this sequence-structure-mechanics relationship is evolutionarily optimized in natural CCs and can be used for tuning their mechanical properties in applications.

biophysics↗