Biology subjects

Taron, C. H.

Publications and source records attributed to Taron, C. H..

6 recordsLinked to original sources

bioRxiv2025

Application of post glycosylation modifying enzymes for mass spectrometry imaging of modified N-glycans in situ.

Glycans are essential components of cells and are involved in innumerable biological processes. Their structural diversity and complexity present unique analytical challenges. Glycans are comprised of various types of monosaccharides that are linked together at different positions and with varied stereochemistry. In addition, glycans are frequently decorated with a diverse set of chemical modifications, termed post-glycosylation modifications (PGMs). Characterization of PGMs is essential for a thorough understanding of glycans, however, the technical challenges and low throughput of current methodologies have limited our understanding of these modifications. Here we demonstrate a novel approach for rapid visualization of specific PGMs present in tissue N-glycans by applying PGM-targeting enzymes to mass spectrometry imaging (MSI). The method enables in situ investigation of glycans with PGMs en masse, identifying the sugar residue and position modified, as well as visualizing the spatial distribution of each modified N-glycan in tissues. As the repertoire of PGM-targeting enzymes expands, we anticipate this approach will enable a better understanding of PGM distribution within a dynamic N-glycome. This may yield both new biological insights and the potential for identification of novel disease biomarkers.

molecular biology↗

bioRxiv2024

High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies

Fosmids and cosmids are vectors frequently used in functional metagenomic studies. With a large insert capacity (around 30 kb) they can encode dozens of cloned genes or in some cases, entire biochemical pathways. Fosmids with cloned inserts can be transferred to heterologous hosts and propagated to enable screening for new enzymes and metabolites. After screening, fosmids from clones with an activity of interest must be de novo sequenced, a critical step towards identification of the gene(s) of interest. In this work, we present a new approach for rapid and high-throughput fosmid sequencing directly from Escherichia coli colonies without liquid culturing or fosmid purification. Our sample preparation involves fosmid amplification with phi29 polymerase and then direct nanopore sequencing using the Oxford Nanopore Technologies system. We also present a bioinformatics pipeline termed "phiXXer" that facilitates both de novo read assembly and vector trimming to generate a linear sequence of the fosmid insert. Finally, we demonstrate accurate sequencing of 96 fosmids in a single run and validate the method using two fosmid libraries that contain cloned large insert ([~]30-40 kb) genomic or metagenomic DNA. ImportanceLarge-insert clone (fosmids or cosmids) sequencing is challenging and arguably the most limiting step of functional metagenomic screening workflows. Our study establishes a new method for high-throughput nanopore sequencing of fosmid clones directly from lysed E. coli cells. It also describes a companion bioinformatic pipeline that enables de novo assembly of fosmid DNA insert sequences. The devised method widens the potential of functional metagenomic screening by providing a simple, high-throughput approach to fosmid clone sequencing that dramatically speeds the pace of discovery.

molecular biology↗

bioRxiv2023

A novel family of sugar-specific phosphodiesterases that remove zwitterionic modifications of N-acetylglucosamine

The zwitterions phosphorylcholine (PC) and phosphoethanolamine (PE) are often found esterified to certain sugars in polysaccharides and glycoconjugates in a wide range of biological species. One such modification involves PC attachment to the 6-carbon of N-acetylglucosamine (GlcNAc-6-PC) in N-glycans and glycosphingolipids (GSLs) of parasitic nematodes, a modification that helps the parasite evade host immunity. Knowledge of enzymes involved in the synthesis and degradation of PC and PE modifications is limited. More detailed studies on such enzymes would contribute to a better understanding of the function of PC modifications and have potential application in the structural analysis of zwitterion-modified glycans. In this study, we used functional metagenomic screening to identify phosphodiesterases encoded in a human fecal DNA fosmid library that remove PC from GlcNAc-6-PC. A novel bacterial phosphodiesterase was identified and biochemically characterized. This enzyme (termed GlcNAc-PDase) shows remarkable substrate preference for GlcNAc-6-PC and GlcNAc-6-PE, with little or no activity on other zwitterion-modified hexoses. The identified GlcNAc-PDase protein sequence is a member of the large endonuclease/exonuclease/phosphatase (EEP) superfamily where it defines a distinct subfamily of related sequences of previously unknown function, mostly from Clostridium bacteria species. Finally, we demonstrate use of GlcNAc-PDase to confirm the presence of GlcNAc-6-PC in N-glycans and GSLs of the parasitic nematode Brugia malayi in a glycoanalytical workflow.

biochemistry↗

bioRxiv2023

The AMP deaminase of the mollusk Helix pomatia is an unexpected member of the adenosine deaminase-related growth factor (ADGF) family

We report here the first occurrence of an adenosine deaminase-related growth factor (ADGF) that deaminates adenosine 5 monophosphate (AMP) in preference to adenosine. The ADGFs are a group of secreted deaminases found throughout the animal kingdom that affect the extracellular concentration of adenosine by converting it to inosine. The AMP deaminase studied here was first isolated and biochemically characterized from the roman snail Helix pomatia in 1983. Determination of the amino acid sequence of the AMP deaminase enabled sequence comparisons to protein databases and revealed it as a member of the ADGF family. Cloning and expression of its cDNA in Pichia pastoris allowed the comparison of the biochemical characteristics of the native and recombinant forms of the enzyme and confirmed they correspond to the previously reported activity. Uncharacteristically, the H. pomatia AMP deaminase was determined to be dissimilar to the AMP deaminase family by sequence comparison while demonstrating similarity to the ADGFs despite having AMP as its preferred substrate rather than adenosine.

biochemistry↗

bioRxiv2022

Comparative analyses of the N-glycoproteomes in HCT116 cancer cells and their non-tumorigenic DNMT1/3b double knockout (DKO1) cells and insight into the Mannose-6-phosphate pathway

N-glycoproteomic analyses provide valuable resources for investigation of cancer mechanisms, biomarkers, and therapeutic targets. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 display hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells.

biochemistry↗

bioRxiv2021

A broad-specificity O-glycoprotease that enables improved analysis of glycoproteins and glycopeptides containing intact complex O-glycans

Analysis of mucin type O-glycans linked to serine/threonine of glycoproteins is technically challenging, in part, due to a lack of effective enzymatic tools that enable their analysis. Recently, several O-glycan-specific endoproteases that can cleave the protein adjacent to the appended glycan have been described. Despite significant progress in understanding the biochemistry of these enzymes, known O-glycoproteases have specificity constrains, such as inefficient cleavage of glycoproteins bearing sialylated O-glycans, high selectivity for certain type of glycoproteins or protein sequence bias, that limit their analytical application. In this study, we examined the capabilities of an immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa. The peptide substrate sequence selectivity and its impact on IMPa activity was interrogated using an array of synthetic peptides and their glycoforms. We show that IMPa has no specific P1 residue preference and can tolerate most amino acids at the P1 position, except aspartic acid. The enzyme does not cleave between two adjacent O-glycosites, indicating that O-glycosylated serine/threonine is not allowed at position P1. Glycopeptides with as few as two amino acids on either side of an O-glycosite were specifically cleaved by IMPa. Finally, IMPa efficiently cleaved peptides and proteins carrying sialylated and asialylated O-glycans of varying complexity. We present the use of IMPa in a one-step O-glycoproteomics workflow for glycoprofiling of individual purified glycoproteins granulocyte colony-stimulating factor (G-CSF) and receptor-type tyrosine-protein phosphatase C (CD45) without the need for glycopeptide enrichment. In these examples, IMPa enabled identification of O-glycosites and the range of complex O-glycan structures at each site.

biochemistry↗