Biology subjects

Pepi, L. E.

Publications and source records attributed to Pepi, L. E..

6 recordsLinked to original sources

bioRxiv2026

An atlas of the aging glycoproteome reveals how glycosylation modulates autoantibody binding in the brain

N-glycosylation generates substantial molecular diversity, yet how glycan composition varies throughout aging remains poorly understood. We mapped the N-glycoproteome of brain, liver, spleen, kidney, and plasma across nine ages spanning the mouse lifespan, identifying 80,618 unique N-glycopeptides at 9,211 glycosites on 4,391 glycoproteins, together with matched proteomic trajectories (glycoproteinatlas.org). Organ identity was the dominant determinant of glycan composition, but numerous glycosites nevertheless underwent remodeling with age. The brain showed a prominent age-associated increase in short paucimannose glycans, driven in part by the age-associated increase in the glycan-processing enzyme HEXB. In white matter, a hotspot for this change, aging increased the paucimannosylation of Myelin oligodendrocyte glycoprotein (MOG), a major target of central nervous system autoimmunity. We identified MOG-reactive autoantibodies in aged individuals and patients with MOG antibody-associated disease (MOGAD) and showed that the glycan at MOG N60 modulates autoantibody binding. Experimental autoimmune encephalomyelitis (EAE) recapitulated a subset of the age-associated glycosite changes, revealing shared glycosylation signatures between aging and autoimmune targeting of MOG. Together, these findings reveal site-specific N-glycosylation as both a marker of age and a regulator of molecular interactions.

neuroscience↗

bioRxiv2024

Vibrio cholerae RbmB is an α-1,4-polysaccharide lyase with biofilm-dispersal activity against Vibrio Polysaccharide (VPS)

Many pathogenic bacteria form biofilms as a protective measure against environmental and host hazards. The underlying structure of the biofilm matrix consists of secreted macromolecules, usually including exopolysaccharides. To escape the biofilm, bacteria may produce a number of matrix-degrading enzymes, including glycosidic enzymes that digest exopolysaccharide scaffolds. The human pathogen Vibrio cholerae assembles and secretes an exopolysaccharide called VPS (Vibrio polysaccharide) which is essential in most cases for the formation of biofilms and consists of a repeating tetrasaccharide unit. Previous studies have indicated that a secreted glycosidase called RbmB is involved in Vibrio cholerae biofilm dispersal, although the mechanism by which this occurs is not understood. To approach the question of RbmB function, we recombinantly expressed and purified RbmB and tested its activity against purified VPS. Using a fluorescence-based biochemical assay, we show that RbmB specifically cleaves VPS in vitro under physiological conditions. Analysis of the cleavage products using mass spectrometry, solid-state NMR, and solution NMR indicates that RbmB cleaves VPS at a specific site (at the -1,4 linkage between D-galactose and a modified L-guluronic acid) into a mixture of tetramers and octamers. We demonstrate that the product of the cleavage contains a double bond in the modified-guluronic acid ring, strongly suggesting that RbmB is cleaving using a glycoside lyase mechanism. Finally, we show that recombinant RbmB from Vibrio cholerae and the related aquatic species Vibrio coralliilyticus are both able to disperse living Vibrio cholerae biofilms. Our results support the role of RbmB as a polysaccharide lyase involved in biofilm dispersal as well as an additional glycolytic enzyme to add to the toolbox of potential therapeutic antibacterial enzymes. Author summaryBiofilms are protective sheaths produced by many bacteria that play important roles in survival in the environment and in hosts during infection. Understanding how biofilms form and disperse is essential in the fight against harmful bacterial pathogens. Vibrio cholerae is a historically significant human pathogen that forms biofilms made of a polysaccharide called VPS and secreted accessory proteins. Within the V. cholerae biofilm gene cluster is a gene encoding a protein called RbmB, which is known to play a role in biofilm dispersal. Using a combination of biochemical and analytical tools, we show that RbmB indeed cleaves VPS into 4- and 8-monosaccharide fragments at a unique site and demonstrate that it falls into the family of polysaccharide lyase enzymes (as opposed to the more common hydrolases). Furthermore, recombinant RbmB disperses living V. cholerae biofilms further indicating that it may play a role in VPS processing or degradation.

biochemistry↗

bioRxiv2024

Comparative Analysis of Glycoproteomic Software Using a Tailored Glycan Database

Glycoproteomics is a rapidly developing field, and data analysis has been stimulated by several technological innovations. As a result, there are many software tools from which to choose; and each comes with unique features that can be difficult to compare. This work presents a head-to-head comparison of five modern analytical software: Byonic, Protein Prospector, MSFraggerGlyco, pGlyco3, and GlycoDecipher. To enable a meaningful comparison, parameter variables were minimized. One potential confounding variable is the glycan database that informs glycoproteomic searches. We performed glycomic profiling of the samples and used the output to construct matched glycan databases for each software. Up to 19,000 glycopeptide spectra were identified across three replicates of wild-type SH-SY5Y cells. There was substantial overlap among most software for glycoproteins identified, locations of glycosites, and glycans, although Byonic reported a suspiciously large number of glycoproteins and glycosites of questionable reliability. We show that Protein Prospector identified the most glycopeptide spectrum matches with high agreement to known glycosites in UniProt. Overall, our results indicate that glycoproteomic searches should involve more than one software to generate confidence. It may be useful to consider software with peptide-first approaches and with glycan-first approaches.

bioinformatics↗

bioRxiv2023

Siglecs in the Porcine Oviduct and Sialylated Ligands on Sperm: Roles in the Formation of the Sperm Reservoir

AbstractDuring mammalian insemination, most of the deposited sperm are lost by retrograde flow or the female reproductive tracts immune response. Once semen enters the uterus, seminal fluid and sperm elicit leukocyte infiltration that contributes to the elimination of sperm in the uterus. However, unlike the uterus, invading sperm do not trigger a phagocytic response in the oviduct in the absence of dysfunction or disease states. Thus, the oviduct possesses a distinct immunological microenvironment that tolerates sperm while maintaining the capacity to respond to pathogens. It has been suggested that sperm glycocalyx contributes to innate oviductal tolerance, but the cell and molecular mechanisms are not understood. The current investigation focused on the role of sialic acid-containing glycoconjugates on sperm and their potential to elicit innate tolerance via cognate sialic acid-binding immunoglobulin-type lectins (Siglecs) expressed in the oviduct. In this manuscript, we report our discovery of eight Siglecs (Siglecs-1, -2, -3, -5, -10, -11, -14, -15) expressed in the lower pig oviduct, five of which are known for immune inhibitory functions (Siglecs-2, -3, -5, -10, and -11) and how these may play a role in achieving sperm-induced immune suppression in the oviduct microenvironment. Mass spectrometry profiling of porcine sperm revealed the presence of a mixture of 2,3 and 2,6 linked sialic acids with 2,3-linked sialic acids as the dominant linkage. Of the detected glycans, several sialic acid-containing glycoconjugates were identified as potential ligands for Siglecs (among O-linked glycans: NeuAc1GalNAc1, NeuGc1GalNAc1, NeuAc2Gal1GalNAc1; attached to glycolipids: NeuAc2Gal1GalNAc1Gal1Glc1, Fuc1Gal1GalNAc1NeuAc1Gal1Glc1). This is the first report of Siglec expression in the mammalian oviduct and total glycan analysis of porcine sperm. The results of this study reveal the potential for a sperm-sialoglycan and oviductal-Siglec axis that may contribute to the distinct immunophysiology of the oviduct fundamentally required for undisrupted reproduction in mammals.

biochemistry↗

bioRxiv2022

Comparative Mucomic Analysis of Three Functionally Distinct Cornu aspersum Secretions

Every animal secretes mucus, placing them among the most diverse biological materials. Mucus hydrogels are complex mixtures of water, ions, carbohydrates, and proteins. Uncertainty surrounding their composition and how interactions between components contribute to mucus function complicates efforts to exploit their properties. There is substantial interest in commercializing mucus from the garden snail, Cornu aspersum, for skincare, drug delivery, tissue engineering, and composite materials. C. asperum secretes three mucus -- one shielding the animal from environmental threats, one adhesive mucus from the pedal surface of the foot, and another pedal mucus that is lubricating. It remains a mystery how compositional differences account for their substantially different properties. Here, we characterize mucus proteins, glycosylation, ion content, and mechanical properties to understand structure-function relationships through an integrative "mucomics" approach. We identify new macromolecular components of these hydrogels, including a novel protein class termed Conserved Anterior Mollusk Proteins (CAMPs). Revealing differences between C. aspersum mucus shows how considering structure at all levels can inform the design of mucus-inspired materials.

biochemistry↗

bioRxiv2021

Metabolic heritage mapping: heterogenous pools of cytoplasmic nucleotide sugars are selectively utilized by various glycosyltransferases

Biosynthesis of macromolecules requires precursors such as sugars or amino acids, originating from exogenous/dietary sources, reutilization/salvage of degraded molecules or de novo synthesis. Since these sources are assumed to contribute to one homogenous pool, their individual contributions are often overlooked. Protein glycosylation uses monosaccharides from all the above sources to produce nucleotide sugars required to assemble hundreds of distinct glycans. Here we demonstrate that cells identify the origin/heritage of the monosaccharide, fucose, for glycosylation. We measured the contribution of GDP-fucose from each of these sources for glycan synthesis and found that different fucosyltransferases, individual glycoproteins, and linkage-specific fucose residues identify and select different GDP-fucose pools dependent on their heritage. This supports the hypothesis that GDP-fucose exists in multiple, distinct pools, not as a single homogenous pool. The selection is tightly regulated since the overall pool size remains constant. We present novel perspectives on monosaccharide metabolism, which may have general applicability.

biochemistry↗