Biology subjects

Shajahan, A.

Publications and source records attributed to Shajahan, A..

8 recordsLinked to original sources

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↗

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↗

bioRxiv2020

SARS-CoV-2 Nucleocapsid protein is decorated with multiple N- and O-glycans.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19) started at the end of 2019 in Wuhan, China has spread rapidly and became a pandemic. Since there is no therapy available that is proven as fully protective against COVID-19, a vaccine to protect against deadly COVID-19 is urgently needed. Nucleocapsid protein (N protein), is one of the most abundant proteins in coronaviruses and is a potential target for both vaccine development and point of care diagnostics. The variable mass of N protein (45 to 60 kDa), suggests the presence of post-translational modifications (PTMs), and it is critical to clearly define these PTMs to gain the structural understanding necessary for further vaccine research. There have been several reports suggesting that the N protein is phosphorylated but lacks glycosylation. Our comprehensive glycomics and glycoproteomics experiments confirm that the N protein is highly O-glycosylated and also contains significant levels of N-glycosylation. We were able to confirm the presence of O-glycans on seven sites with substantial glycan occupancy, in addition to less abundant O-glycans on four sites. We also detected N-glycans on two out of five potential N-glycosylation sites. Moreover, we were able to confirm one phosphorylation site. Recent studies have indicated that the N protein can serve as an important diagnostic marker for coronavirus disease and a major immunogen by priming protective immune responses. Thus, detailed structural characterization of the N protein may provide useful insights for understanding the roles of glycosylation on viral pathogenesis and also in vaccine design and development.

biochemistry↗

bioRxiv2020

Mass spectrometric method for the unambiguous profiling of cellular dynamic glycosylation.

Various biological processes at the cellular level are regulated by glycosylation which is a highly micro-heterogeneous post-translational modification (PTM) on proteins and lipids. The dynamic nature of glycosylation can be studied through bio-orthogonal tagging of metabolically engineered non-natural sugars into glycan epitopes. However, this approach possesses a significant drawback due to non-specific background reactions and ambiguity of non-natural sugar metabolism. Here we report a tag-free strategy for their direct detection by glycoproteomics and glycomics using mass spectrometry. The method dramatically simplifies the detection of non-natural functional group bearing monosaccharides installed through promiscuous sialic acid, GalNAc, and GlcNAc biosynthetic pathways. Multistage enrichment of glycoproteins by cellular fractionation, subsequent ZIC-HILIC based glycopeptide enrichment, and a spectral enrichment algorithm for the MS data processing enabled direct detection of non-natural monosaccharides that are incorporated at low abundance on the N/O-glycopeptides along with their natural counterparts. Our approach allowed the detection of both natural and non-natural sugar bearing glycopeptides, N and O-glycopeptides, differentiation of non-natural monosaccharide types on the glycans and also their incorporation efficiency through quantitation. Through this we could deduce some interconversion of monosaccharides during their processing through glycan salvage pathway and subsequent incorporation into glycan chains. The study of glycosylation dynamics through this method can be conducted in high throughput as few sample processing steps are involved, enabling understanding of glycosylation dynamics under various external stimuli and thereby could bolster the use of metabolic glycan engineering in glycosylation functional studies.

biochemistry↗

bioRxiv2020

Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2.

The emergence of the COVID-19 pandemic caused by SARS-CoV-2 has created the need for development of new therapeutic strategies. Understanding the mode of viral attachment, entry and replication has become a key aspect of such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential for viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to human angiotensin converting enzyme 2 (hACE2) for entry. Herein, we describe glycomic and glycoproteomic analysis of hACE2 expressed in HEK293 human cells. We observed high glycan occupancy (73.2 to 100%) at all seven possible N-glycosylation sites and surprisingly detected one novel O-glycosylation site. To deduce the detailed structure of glycan epitopes on hACE2 that may be involved in viral binding, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc, and the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectrum, in addition to using bioinformatics tools to validate the hACE2 glycosylation. Our elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor, along with the modeling of hACE2 glycosylation sites can aid in understanding the intriguing virus-receptor interactions and assist in the development of novel therapeutics to prevent viral entry. The relevance of studying the role of ACE2 is further increased due to some recent reports about the varying ACE2 dependent complications with regard to age, sex, race, and pre-existing conditions of COVID-19 patients.

biochemistry↗

bioRxiv2020

Deducing the N- and O- glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2

The current emergence of the novel coronavirus pandemic caused by SARS-CoV-2 demands the development of new therapeutic strategies to prevent rapid progress of mortalities. The coronavirus spike (S) protein, which facilitates viral attachment, entry and membrane fusion is heavily glycosylated and plays a critical role in the elicitation of the host immune response. The spike protein is comprised of two protein subunits (S1 and S2), which together possess 22 potential N-glycosylation sites. Herein, we report the glycosylation mapping on spike protein subunits S1 and S2 expressed on human cells through high resolution mass spectrometry. We have characterized the quantitative N-glycosylation profile on spike protein and interestingly, observed unexpected O-glycosylation modifications on the receptor binding domain (RBD) of spike protein subunit S1. Even though O-glycosylation has been predicted on the spike protein of SARS-CoV-2, this is the first report of experimental data for both the site of O-glycosylation and identity of the O-glycans attached on the subunit S1. Our data on the N- and O-glycosylation is strengthened by extensive manual interpretation of each glycopeptide spectra in addition to using bioinformatics tools to confirm the complexity of glycosylation in the spike protein. The elucidation of the glycan repertoire on the spike protein provides insights into the viral binding studies and more importantly, propels research towards the development of a suitable vaccine candidate.

biochemistry↗

bioRxiv2019

Species-specific recognition of Sulfolobales mediated by UV inducible pili and S-layer glycosylation patterns

The UV-inducible pili system of Sulfolobales (Ups) mediates the formation of species-specific cellular aggregates. Within these aggregates, cells exchange DNA in order to repair DNA double strand breaks via homologous recombination. Substitution of the S. acidocaldarius pilin subunits UpsA and UpsB with their homologs from Sulfolobus tokodaii showed that these subunits facilitate species-specific aggregation. A region of low conservation within the UpsA homologs is primarily important for this specificity. Aggregation assays in the presence of different sugars showed the importance of N-glycosylation in the recognition process. In addition, the N-glycan decorating the S-layer of S. tokodaii is different from the one of S. acidocaldarius. Therefore, each Sulfolobus species seems to have developed a unique UpsA binding pocket and unique N-glycan composition to ensure aggregation and consequently also DNA exchange with cells from only the same species, which is essential for DNA repair by homologous recombination.\n\nImportanceType IV pili can be found on the cell surface of many archaea and bacteria where they play important roles in different processes. The Ups-pili from the crenarchaeal Sulfolobales species are essential in establishing species-specific mating partners, ensuring genome stability. With this work, we show that different Sulfolobus species have species-specific regions in their Ups-pilin subunits, which allow them to interact only with cells from the same species. Additionally, different Sulfolobus species all have unique S-layer N-glycosylation patterns. We propose that the unique features of each species allow the recognition of specific mating partners. This knowledge for the first time gives insights into the molecular basis of archaeal self-recognition.

microbiology↗

bioRxiv2017

Structure and function of accessory Sec proteins involved in the adhesin export pathway of Streptococcus gordonii

Many pathogenic bacteria, including Streptococcus gordonii, possess a pathway for the export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. These adhesins are O-glycosylated by several cytosolic glycosyltransferases and require three accessory Sec proteins (Asp1-3) for export, but how the adhesins are processed for secretion is not well defined. Here, we show that O-glycosylation of S. gordonii adhesin GspB occurs in a sequential manner by three enzymes (GtfA/B, Nss, Gly) that attach N-acetylglucosamine and glucose to Ser/Thr residues. The modified substrate is subsequently transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures show that both Asp1 and Asp3 are related to carbohydrate binding proteins. Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. These results suggest a mechanism for the modification of adhesin in the cytosol and its subsequent targeting to the export machinery.

biochemistry↗