Biology subjects

Capicciotti, C. J.

Publications and source records attributed to Capicciotti, C. J..

6 recordsLinked to original sources

bioRxiv2025

Characterization of bacterial fucokinase/GDP-fucose pyrophosphorylase (FKP) enzymes supports the evolution of interdomain communication and modularity in the FKP family

L-fucokinase (FUK) and GDP-fucose pyrophosphorylase (GFPP) salvage free L-fucose and synthesize the valuable nucleotide-sugar GDP-L-fucose (GDP-Fuc). Some organisms express these enzymes as one bifunctional polypeptide called L-fucokinase/GDP-fucose pyrophosphorylase (FKP), which has attracted attention for use in the chemoenzymatic synthesis of GDP-Fuc. Despite the documented use of the FKP from Bacteroides fragilis (BfFKP), the evolutionary origins of these enzymes and their relationships to monofunctional FUKs and GFPPs are poorly understood. We hypothesized that biochemical characterization of these proteins coupled with an evolutionary analysis would uncover the natural diversity of FKPs, facilitating the discovery of new biocatalysts. Phylogenetic and sequence similarity network (SSN) analyses distinguished FKPs from FUKs and GFPPs, suggesting that FKPs originate from one ancestral fusion event between these domains. To evaluate how environmental factors might select for functional diversity within the FKP family, we recombinantly expressed and purified a putative FKP from the thermophilic bacterium Thermophagus xiamenensis (TxFKP). This enzyme exhibited in vitro kinase and pyrophosphorylase activities and demonstrated subtle kinetic differences compared to BfFKP. While alanine scanning mutational analysis of the TxFKP FUK and GFPP domains supported the role of conserved residues that TxFKP uses to coordinate substrate binding and catalysis, other mutations in the TxFKP GFPP domain influenced kinase activity differentially for the substrates L-fucose and D-arabinose, showing an unprecedented role for the GFPP domain in FUK substrate specificity. Finally, thermal shift profiles of TxFKP and BfFKP were biphasic and provided new insights into how these enzymes have evolved to respond to different sugar substrates.

biochemistry↗

bioRxiv2024

Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2

Cell entry of severe acute respiratory coronavirus-2 (SARS-CoV-2) and other CoVs can occur via two distinct routes. Following receptor binding by the spike glycoprotein, membrane fusion can be triggered by spike cleavage either at the cell surface in a transmembrane serine protease 2 (TMPRSS2)-dependent manner or within endosomes in a cathepsin-dependent manner. Cellular sialoglycans have been proposed to aid in CoV attachment and entry, although their functional contributions to each entry pathway are unknown. In this study, we used genetic and enzymatic approaches to deplete sialic acid from cell surfaces and compared the requirement for sialoglycans during endosomal and cell-surface CoV entry, primarily using lentiviral particles pseudotyped with the spike proteins of different sarbecoviruses. We show that entry of SARS-CoV-1, WIV1-CoV and WIV16-CoV, like the SARS-CoV-2 omicron variant, depends on endosomal cathepsins and requires cellular sialoglycans for entry. Ancestral SARS-CoV-2 and the delta variant can use either pathway for entry, but only require sialic acid for endosomal entry in cells lacking TMPRSS2. Binding of SARS-CoV-2 spike protein to cells did not require sialic acid, nor was sialic acid required for SARS-CoV-2 entry in TMRPSS2-expressing cells. These findings suggest that cellular sialoglycans are not strictly required for SARS-CoV-2 attachment, receptor binding or fusion, but rather promote endocytic entry of SARS-CoV-2 and related sarbecoviruses. In contrast, the requirement for sialic acid during entry of MERS-CoV pseudoparticles and authentic HCoV-OC43 was not affected by TMPRSS2 expression, consistent with a described role for sialic acid in merbecovirus and embecovirus cell attachment. Overall, these findings clarify the role of sialoglycans in SARS-CoV-2 entry and suggest that cellular sialoglycans mediate endosomal, but not cell-surface, SARS-CoV-2 entry. Thus, it may be important to consider both cell entry pathways when developing sarbecovirus entry inhibitors targeting virus-sialoglycan interactions. Author summaryThe COVID-19 pandemic, caused by SARS-CoV-2, has resulted in over 676 million infections and 6.8 million deaths so far, demonstrating the threat posed by emerging CoVs. In humans, SARS-CoV-2 and related coronaviruses cause respiratory tract infections, such as the common cold, as well as more severe disease in some individuals. To prepare for future outbreaks, conserved steps in the CoV replication could be considered for antiviral prophylactic or therapeutic approaches. One such process is CoV cell entry, which occurs via two main routes: At the cell surface or within endosomes. Cellular receptors, proteases and complex sugars, known as glycans, mediate CoV entry steps. In this study, we compared the role of a specific glycan subset, sialoglycans, in endosomal and cell surface CoV entry. We show that sialoglycans are required for entry of various CoVs that are mainly dependent on the endosomal route, but in the case of SARS-CoV-2, sialoglycans were not required when the cell-surface entry route was available. Our findings contribute to understanding the mechanisms of CoV entry, which could inform development of pan-CoV antivirals that target CoV entry steps.

microbiology↗

bioRxiv2023

Glyco-Engineering Cell Surfaces by Exo-Enzymatic Installation of GlcNAz and LacNAz Motifs

Exo-enzymatic glyco-engineering of cell-surface glycoconjugates enables the selective display of well-defined glyco-motifs bearing bioorthogonal functional groups which can be used to study glycans and their interactions with glycan-binding proteins. While the installation of monosaccharides and their derivatives using glycosyltransferase enzymes has rapidly evolved, similar strategies to introduce chemical-reporter functionalized Type 2 LacNAc motifs have not been reported. Herein, we report the chemo-enzymatic synthesis of unnatural UDP-GlcNAc and UDP-GalNAc nucleotide-sugars, and the donor and acceptor substrate tolerance of the human glycosyltransferases B3GNT2 and B4GalT1, respectively, to form derivatized LacNAc moieties. We also demonstrate that B3GNT2 can be used to exo-enzymatically install GlcNAc and GlcNAz onto cell-surface glycans. GlcNAc- or GlcNAz-engineered cells can be further extended by B4GalT1, producing LacNAc or LacNAz-engineered cells. Our glyco-engineering labeling strategy is amenable to different cell types and our work expands the exo-enzymatic glycan editing toolbox to selectively introduce unnatural Type 2 LacNAc motifs.

biochemistry↗

bioRxiv2023

One-Step Selective Labeling of Native Cell-Surface Sialoglycans by Exogenous α2,8-Sialylation

Exo-enzymatic glycan labeling strategies have emerged as versatile tools for efficient and selective installation of glycan terminal motifs onto live cell-surfaces. Through employing specific enzymes and nucleotide sugar probes, cells can be equipped with defined glyco-epitopes for modulating cell function or selective visualization and enrichment of glycoconjugates. Here, we identify Campylobacter jejuni sialyltransferase Cst-II I53S as a tool for cell-surface glycan modification, expanding the exo-enzymatic labeling toolkit to include installation of 2,8-disialyl epitopes. Labeling with Cst-II was achieved with biotin- and azide-tagged CMP-Neu5Ac derivatives on a model glycoprotein and on native sialylated cell-surface glycans across a panel of cell lines. The introduction of modified Neu5Ac derivatives onto cells by Cst-II was also retained on the surface for 6 h. By examining the specificity of Cst-II on cell surfaces, it was revealed that the 2,8-sialyltransferase primarily labeled N-glycans, with O-glycans labeled to a lesser extent, and there was an apparent preference for 2,3-linked sialosides. This approach thus broadens the scope of tools for selective exo-enzymatic labeling of native sialylated glycans and is highly amenable for construction of cell-based arrays.

biochemistry↗

bioRxiv2022

Exo-Enzymatic Cell-Surface Glycan Labeling for Capturing Glycan-Protein Interactions through Photo-Crosslinking

Tools to interrogate glycoconjugate-protein interactions in the context living cells are highly attractive for the identification of critically important functional binding partners of glycan-binding proteins. These interactions are challenging to interrogate due to low affinity and rapid dissociation rates of glycan-protein binding events. The use of photo-crosslinkers to capture glycan-protein interaction complexes has shown great promise for identifying binding partners involved in these interactions. Current methodologies use metabolic oligosaccharide engineering (MOE) to incorporate photo-crosslinking sugars. However, these MOE strategies are not amenable to all cell types and can result in low incorporation and cell-surface display of the photo-crosslinking probe, limiting their utility for studying many types of interactions. We describe here an exo-enzymatic strategy for selectively introducing photo-crosslinking probes into cell-surface glycoconjugates using the recombinant human sialyltransferase ST6GAL1 and a diazirine-linked CMP-Neu5Ac derivative. Probe introduction is highly efficient, amenable to different cell types and resulted in improved crosslinking when compared to MOE. This exo-enzymatic labeling approach can selectively introduce the photo-crosslinking sugar on to specific glycan epitopes and subclasses by harnessing the specificity of the sialyltransferase employed, underscoring its potential as a tool to interrogate and identify glycoconjugate ligands for diverse glycan-binding proteins.

biochemistry↗

bioRxiv2021

Cell Surface Glycan Engineering Reveals that Matriglycan Alone can Recapitulate Dystroglycan Binding and Function

-Dystroglycan (-DG) is uniquely modified on O-mannose sites by a repeating disaccharide (-Xyl1,3-GlcA{beta}1,3-)n termed matriglycan, which is a receptor for laminin-G domain-containing proteins and employed by old-world arenaviruses for infection. Using chemoenzymatically synthesized matriglycans printed as a microarray, we demonstrated length-dependent binding to Laminin, Lassa virus GP1, and the clinically-important antibody IIH6. Utilizing an enzymatic engineering approach, an N-linked glycoprotein was converted into a IIH6-positive Laminin-binding glycoprotein. Engineering of the surface of cells deficient for either -DG or O-mannosylation with matriglycans of sufficient length recovered infection with a Lassa-pseudovirus. Finally free matriglycan in a dose and length dependent manner inhibited viral infection of wildtype cells. These results indicate that matriglycan alone is necessary and sufficient for IIH6 staining, Laminin and LASV GP1 binding, and Lassa-pseudovirus infection and support a model in which it is a tunable receptor for which increasing chain length enhances ligand-binding capacity. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Graphical Abstract C_FIG_DISPLAY

biochemistry↗