Biology subjects

Samara, N. L.

Publications and source records attributed to Samara, N. L..

5 recordsLinked to original sources

bioRxiv2026

An in vivo chemical genetic approach for targeted glycoproteome analysis in Drosophila melanogaster

Glycans impact every aspect of physiology. Despite the rapid increase in relevance and translational potential, there remains a lack of tools for studying glycosylation in vivo. Here, we present FlyMOE, a chemical-genetic platform for targeted glycoprotein profiling in Drosophila melanogaster. Transgenic flies are equipped with biosynthetic capabilities to install bioorthogonally-tagged monosaccharides into the cellular glycoproteome. The genetic tractability of Drosophila allows for versatile application based on developmental stages, genetic drivers and glycosyltransferase enzymes. We employ FlyMOE to create an overview of the glycoproteome of Drosophila tissues and embryos based on mass spectrometry glycoproteomics, and investigate the substrates of individual members of the PGANT glycosyltransferase family in vivo. FlyMOE will allow the detailed investigation of the glycoproteome in Drosophila and similarly genetically modifiable in vivo model systems.

bioengineering↗

bioRxiv2025

A single catalytic domain residue regulates the substrate specificity of a mucin-type O-glycosyltransferase in salivary gland function

Human GalNAc-T1 is a ubiquitously expressed mucin-type O-glycosyltransferase that modulates various biological pathways, including immunity, extracellular matrix formation, and salivary gland development and function. However, substrates of GalNAc-T1 are mostly unknown and the mechanistic details of regulation are not well characterized. Here, we investigate the role of the Drosophila melanogaster GalNAc-T1 ortholog PGANT5 in salivary glands and provide structural and biochemical details into its mechanism of O-glycosylation. We find that loss of pgant5 causes irregular secretory granule morphology, altered mucin packaging, and disruption of secretion. Alternative splicing of pgant5 yields two isoforms, pgant5A and pgant5B, with higher expression of pgant5A in salivary glands. Rescue with pgant5A, but not pgant5B, partially restores secretory granule morphology and salivary gland function. A single residue difference within the substrate binding pockets of PGANT5A and PGANT5B increases the in vitro activity and specificity of PGANT5A towards salivary gland mucins relative to PGANT5B, corroborating the in vivo expression and role of pgant5A in salivary glands. Our data additionally provide new insights into a catalytic domain motif that regulates GalNAc-T activity. Overall, our studies illustrate how alternative splicing of an O-glycosyltransferase that results in modest amino acid changes can influence substrate specificity to regulate tissue-specific biological functions.

biochemistry↗

bioRxiv2023

A Toxoplasma gondii O-glycosyltransferase that modulates bradyzoite cyst wall rigidity is structurally and functionally distinct from host homologues

Infection with the Apicomplexan protozoan Toxoplasma gondii can be life-threatening in immunocompromised hosts. Transmission frequently occurs through the oral ingestion of T. gondii bradyzoite cysts, which transition to tachyzoites, disseminate, and then form cysts containing bradyzoites in the central nervous system, resulting in latent infection. There are currently no effective treatments to cure latent infection. Bradyzoites are encapsulated by a cyst wall that is critical for immune evasion, survival, and transmission. Cyst wall rigidity is influenced by the O-glycosylation of the mucin domain of the cyst wall protein CST1 by mucin-type O-glycosyltransferases (GalNAc-Ts). Here, we report the first structures of a protozoan GalNAc-T, T.gondii-GalNAc-T3 in the apo state and in complex with glycopeptide substrates. The structures reveal features that are strictly conserved in Apicomplexan homologues of T.gondii-GalNAc-T3, including a unique 2nd metal binding site that is coupled to substrate binding and enzymatic activity in vitro and cyst wall O-glycosylation in T. gondii. Additional structural features illustrate the divergence of GalNAc-Ts from parasite to host and highlight multiple druggable sites in T.gondii-GalNAc-T3 and its homologues in Apicomplexa that are responsible for a wide range of parasitic diseases.

biochemistry↗

bioRxiv2023

Shared and Distinct Mechanisms of UBA1 Inactivation Across Different Diseases

Most cellular ubiquitin signaling is initiated by UBA1, which activates and transfers ubiquitin to tens of E2 enzymes. Clonally acquired UBA1 missense mutations cause an inflammatory-hematologic overlap disease called VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic) syndrome. Despite extensive clinical investigation into this lethal disease, little is known about the underlying molecular mechanisms. Here, by dissecting VEXAS-causing UBA1 mutations, we discovered that p.Met41 mutations alter cytoplasmic isoform expression, whereas other mutations reduce catalytic activity of nuclear and cytoplasmic isoforms by diverse mechanisms, including aberrant oxyester formation. Strikingly, non-p.Met41 mutations most prominently affect transthioesterification, revealing ubiquitin transfer to cytoplasmic E2 enzymes as a shared property of pathogenesis amongst different VEXAS syndrome genotypes. A similar E2 charging bottleneck exists in some lung cancer-associated UBA1 mutations, but not in spinal muscular atrophy-causing UBA1 mutations, which instead, render UBA1 thermolabile. Collectively, our results highlight the precision of conformational changes required for faithful ubiquitin transfer, define distinct and shared mechanisms of UBA1 inactivation in diverse diseases, and suggest that specific E1-E2 modules control different aspects of tissue differentiation and maintenance.

biochemistry↗

bioRxiv2021

Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation

The SARS-CoV-2 coronavirus responsible for the global pandemic contains a unique furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation. Here, we show that O-glycosylation near the furin cleavage site is mediated by specific members of the GALNT enzyme family and is dependent on the novel proline at position 681 (P681). We further demonstrate that O-glycosylation of S decreases furin cleavage. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the potential role of P681 mutations in the recently identified, highly transmissible B.1.1.7 variant.

biochemistry↗