Biology subjects

Mahal, L. K.

Publications and source records attributed to Mahal, L. K..

28 records · Page 2Linked to original sources

bioRxiv2022

Reduced inflammatory response and promoted multiciliated cell differentiation in mice protected by defective interfering influenza virus

Influenza defective interfering (DI) viruses have long been considered promising antiviral candidates because of their ability to interfere with replication-competent viruses and to induce antiviral immunity. However, the mechanisms underlying DI-mediated antiviral immunity have not been extensively explored. Here, we demonstrated interferon (IFN) independent protection conferred by influenza DI virus against homologous virus infection in mice deficient in type I and III IFN signaling. By integrating transcriptional and post-transcriptional regulatory data we identified unique host signatures in response to DI co-infection. DI-treated mice exhibited reduced viral transcription, less intense inflammatory and innate immune responses, and primed multiciliated cell differentiation in their lungs at an early stage of infection, even in the absence of type I or III IFNs. Overall, our study provides mechanistic insight into the protection mediated by DIs, implying a unifying theme involving inflammation and multiciliogenesis in maintaining respiratory homeostasis, and reveals their IFN-independent antiviral activity.

microbiology↗

bioRxiv2021

A Useful Guide to Lectin Binding: Machine-Learning Directed Annotation of 57 Unique Lectin Specificities

Glycans are critical to every facet of biology and medicine, from viral infections to embryogenesis. Tools to study glycans are rapidly evolving, however the majority of our knowledge is deeply dependent on binding by glycan binding proteins (e.g., lectins). The specificities of lectins, which are often naturally isolated proteins, have not been well- defined, making it difficult to leverage their full potential for glycan analysis. Herein, we use glycan microarray analysis of 116 commercially available lectins, including different preparations of the same lectin, to extract the specific glycan features required for lectin binding. Data was obtained using the Consortium for Functional Glycomics microarray (CFG v5.0) containing 611 glycans. We use a combination of machine learning algorithms to define lectin specificity, mapping inputs (glycan sequences) to outputs (lectin-glycan binding) for a large-scale evaluation of lectin-glycan binding behaviours. Our motif analysis was performed by integrating 68 manually defined glycan features with systematic probing of computational rules for significant binding motifs using mono- and disaccharides- and linkages. Using a combination of machine learning and manual annotation of the data, we created a detailed interpretation of glycan-binding specificity for 57 unique lectins, categorized by their major binding motifs: mannose, complex-type N-glycan, O-glycan, fucose, sialic acid and sulfate, GlcNAc and chitin, Gal and LacNAc, and GalNAc. Our work provides fresh insights into the complex binding features of commercially available lectins in current use, providing a critical guide to these important reagents.

biochemistry↗

bioRxiv2021

Carbohydrate sulfation as a mechanism for fine-tuning Siglec ligands

The immunomodulatory family of Siglecs recognize sialic acid-containing glycans as self, which is exploited in cancer for immune-evasion. The biochemical nature of Siglec ligands remains incompletely understood with emerging evidence suggesting the importance of carbohydrate sulfation. Here, we investigate how specific sulfate modifications affect Siglec ligands by overexpressing eight carbohydrate sulfotransferases (CHSTs) in five cell lines. Overexpression of three CHSTs (CHST1, CHST2, or CHST4) significantly enhances the binding of numerous Siglecs. Unexpectedly, two other CHSTs (Gal3ST2 and Gal3ST3) diminish Siglec binding, suggesting a new mode to modulate Siglec ligands via sulfation. Results are cell type dependent, indicating that the context in which sulfated glycans are presented is important. Moreover, pharmacological blockade of N- and O-glycan maturation reveals a cell type-specific pattern of importance for either class of glycan. Production of a highly homogenous CD33 (Siglec-3) fragment enabled a mass spectrometry-based binding assay to determine 10-fold and 3-fold enhanced affinity for Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4GlcNAc and Neu5Ac2-3Gal{beta}1-4(6-O- sulfo)GlcNAc, respectively, over Neu5Ac2-3Gal{beta}1-4GlcNAc. CD33 showed significant additivity in affinity (36-fold) for the disulfated ligand, Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4(6-O-sulfo)GlcNAc. Moreover, overexpression of both CHST1 and CHST2 in cells greatly enhanced the binding of several Siglecs, including CD33. Finally, we reveal that CHST1 is upregulated in numerous cancers, correlating with poorer survival rates and sodium chlorate sensitivity for the binding of Siglecs to cancer cell lines. These results provide new insights into carbohydrate sulfation as a modification that is a general mechanism for tuning Siglec ligands on cells, including in cancer.

immunology↗

bioRxiv2021

High-throughput analysis of B3GLCT regulation predicts phenotype of Peters' Plus Syndrome in line with the miRNA Proxy Hypothesis

MicroRNAs (miRNAs, miRs) finely tune protein expression and target networks of 100s-1000s of genes that control specific biological processes. They are critical regulators of glycosylation, one of the most diverse and abundant posttranslational modifications. In recent work, miRs have been shown to predict the biological functions of glycosylation enzymes, leading to the "miRNA proxy hypothesis" which states, "if a miR drives a specific biological phenotype..., the targets of that miR will drive the same biological phenotype." Testing of this powerful hypothesis is hampered by our lack of knowledge about miR targets. Target prediction suffers from low accuracy and a high false prediction rate. Herein, we develop a high-throughput experimental platform to analyze miR:target interactions, miRFluR. We utilize this system to analyze the interactions of the entire human miRome with beta-3-glucosyltransferase (B3GLCT), a glycosylation enzyme whose loss underpins the congenital disorder Peters Plus Syndrome. Although this enzyme is predicted by multiple algorithms to be highly targeted by miRs, we identify only 27 miRs that downregulate B3GLCT, a >96% false positive rate for prediction. Functional enrichment analysis of these validated miRs predict phenotypes associated with Peters Plus Syndrome, although B3GLCT is not in their known target network. Thus, biological phenotypes driven by B3GLCT may be driven by the target networks of miRs that regulate this enzyme, providing additional evidence for the miRNA Proxy Hypothesis.

biochemistry↗

bioRxiv2021

Integrated Systems-Analysis of the Human and Murine Pancreatic Cancer Glycomes Reveal a Tumor Promoting Role for ST6GAL1

Pancreatic ductal adenocarcinoma (PDA) is the 3rd leading cause of cancer-death in the U.S.. Glycans, such as CA-19-9, are biomarkers of PDA and are emerging as important modulators of cancer phenotypes. Herein, we utilized a systems-based approach integrating glycomic analysis of human PDA and the well-established KC mouse model, with transcriptomic data to identify and probe the functional significance of aberrant glycosylation in pancreatic cancer. We observed both common and distinct patterns of glycosylation in pancreatic cancer across species. Common alterations included increased levels of -2,3- and -2,6-sialic acids, bisecting GlcNAc and poly-LacNAc. However, core fucose, which was increased in human PDAC, was not seen in the mouse, indicating that not all human glycomic changes can be modeled in the KC mouse. In silico a nalysis of bulk and single cell sequencing data identified ST6GAL1, which underlies -2,6-sialic acid, as overexpressed in human PDA, concordant with histological data. Enzymes levels correlated with the stage of clinical disease. To test whether ST6GAL1 promotes pancreatic cancer we created a novel mouse in which a pancreas-specific genetic deletion of this enzyme overlays the KC mouse model. Analysis of our new model showed delayed cancer formation and a significant reduction in fibrosis. Our results highlight the importance of a strategic systems-approach to identifying glycans whose functions can be modeled in mouse, a crucial step in the development of therapeutics targeting glycosylation in pancreatic cancer. SIGNIFICANCEPancreatic ductal adenocarcinoma (PDA) is the 3rd leading cause of cancer-death in the U.S.. Glycosylation is emerging as an important modulator of cancer phenotype. Herein we use a systems-approach integrating glycomics of human PDA and a well-established PDA mouse model with transcriptomic data to identify ST6GAL1, the enzyme underlying -2,6-sialic acid, as a potential cancer promoter. A pancreatic specific ST6GAL1 knockout in the KC mouse showed delayed cancer formation and a reduction in fibrosis. Our results highlight the importance of a strategic systems-approach to identifying glycans whose functions can be modeled in mouse, a crucial step in the development of therapeutics targeting glycosylation in pancreatic cancer.

cancer biology↗

bioRxiv2021

Sex-specific glycosylation of secreted immunomodulatory proteins in the filarial nematode Brugia malayi

The extended persistence of filarial nematodes within a host suggests immunomodulatory mechanisms that allow the parasites to resist or evade the host immune response. There is increasing evidence for immunomodulatory glycans expressed by a diversity of parasitic worms. In this study, we integrate multiple layers of the host-parasite interface to investigate the glycome of a model filarial parasite, Brugia malayi. We report a significant overrepresentation of terminal GalNAc moieties in adult female worms coupled with an overall upregulation in O-glycosylation, T-antigen expression, and a bias for galactose containing glycans. Adult males preferentially displayed a bias for terminal GlcNAc containing glycans, and fucosylated epitopes. Subsequent proteomic analysis confirmed sex-biases in protein glycosylation and highlighted the sex-specific glycosylation of well characterized immunomodulators expressed and secreted by B. malayi. We identify sex-specific effectors at that interface and suggest approaches to selectively interfere with the parasitic life cycle and potentially control transmission.

microbiology↗

bioRxiv2020

Glycomic analysis reveals a conserved response to bacterial sepsis induced by different bacterial pathogens.

Sepsis is an extreme inflammatory response to infection (bacterial, viral, fungal) that occurs in the bloodstream and causes damage throughout the body. Currently, there are few diagnostic biomarkers of sepsis and new effective treatments have not been developed. There is a clear need to study the molecular underpinnings of this disease. Glycosylation is known to play a role in immunity and inflammation, but the role of glycans in sepsis is not well defined. Herein, we profiled the serum glycomes of experimental mouse sepsis models to identify changes induced by 4 different clinical bacterial pathogens (Gram-positive: Streptococcus pneumoniae, Staphylococcus aureus, Gram-negative: Escherichia coli and Salmonella Typhimurium) using our lectin microarray technology. We observed global shifts in the blood sera glycome that were conserved across all four species, regardless of whether they were Gram positive or negative. Bisecting GlcNAc was decreased upon sepsis and a strong increase in core 1/3 O-glycans was observed. Lectin blot analysis revealed a high molecular weight protein induced in sepsis by all four bacteria as the major cause of the core 1/3 O-glycan shift. While the identity of this protein remains to be elucidated, its presence indicates a common feature of bacterial sepsis associated with this glycomic signature.

biochemistry↗

bioRxiv2020

Age-dependent glycomic response to the 2009 pandemic H1N1 influenza virus and its association with disease severity

Influenza A viruses cause a spectrum of responses, from mild cold-like symptoms to severe respiratory illness and death. Viral strains and intrinsic host factors, such as age, can influence the severity of the disease. Glycosylation plays a critical role in influenza pathogenesis, however the molecular drivers of influenza outcomes remain unknown. In this work, we characterized the glycomic response to the H1N1 2009 pandemic influenza A virus in age-dependent severity. Using a ferret model and a lectin microarray technology we have developed, we compared responses in newly weaned and aged animals, a model for young children and the elderly, respectively. Glycomic analysis revealed changes in glycosylation over the course of the infection, that were associated with severity in an age-dependent manner. These responses may help explain the differential susceptibility to influenza A virus infection of young children and the elderly. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/165613v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1c5aebdorg.highwire.dtl.DTLVardef@15316e6org.highwire.dtl.DTLVardef@45ff3org.highwire.dtl.DTLVardef@1376533_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Glycomic analysis of host-response reveals high mannose as a key mediator of influenza severity

Influenza virus infections cause a wide variety of outcomes, from mild disease to 3-5 million cases of severe illness and ~290,000-645,000 deaths annually worldwide. The molecular mechanisms underlying these disparate outcomes are currently unknown. Glycosylation within the human host plays a critical role in influenza virus biology. However, the impact these modifications have on the severity of influenza disease has not been examined. Herein, we profile the glycomic host responses to influenza virus infection as a function of disease severity using a ferret model and our lectin microarray technology. We identify the glycan epitope high mannose as a marker of influenza virus-induced pathogenesis and severity of disease outcome. Induction of high mannose is dependent upon the unfolded protein response (UPR) pathway, a pathway previously shown to associate with lung damage and severity of influenza virus infection. Also, the mannan-binding lectin (MBL2), an innate immune lectin that negatively impacts influenza outcomes, recognizes influenza virus-infected cells in a high mannose dependent manner. Together, our data argue that the high mannose motif is an infection-associated molecular pattern on host cells that may guide immune responses leading to the concomitant damage associated with severity. SIGNIFICANCEInfluenza virus infection causes a range of outcomes from mild illness to death. The molecular mechanisms leading to these differential host responses are currently unknown. Herein, we identify the induction of high mannose, a glycan epitope, as a key mediator of severe disease outcome. We propose a mechanism in which activation of the unfolded protein response (UPR) upon influenza virus infection turns on expression of high mannose, which is then recognized by the innate immune lectin MBL2, activating the complement cascade and leading to subsequent inflammation. This work is the first to systematically study host glycomic changes in response to influenza virus infection, identifying high mannose as a key feature of differential host response.

microbiology↗

bioRxiv2018

O-fucosylation of thrombospondin-like repeats is required for processing of MIC2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites

Toxoplasma gondii is an intracellular parasite that causes disseminated infections which can lead to neurological damage in fetuses and immunocompromised individuals. Microneme protein 2 (MIC2)2, a member of the thrombospondin-related anonymous protein (TRAP) family, is a secreted protein important for motility, host cell attachment, invasion, and egress. MIC2 contains six thrombospondin type I repeats (TSRs) that are modified by C-mannose and O-fucose in Plasmodium spp. and mammals.\n\nHere we used mass spectrometry to show that the four TSRs in T. gondii MIC2 with protein O-fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, while Trp residues within three TSRs are also modified with C-mannose. Disruption of genes encoding either pofut2 or nucleotide sugar transporter 2 (nst2), the putative GDP-fucose transporter, results in loss of MIC2 O-fucosylation, as detected by an antibody against the GlcFuc disaccharide, and markedly reduced cellular levels of MIC2. Furthermore, in 10-15% of the{Delta} pofut2 or{Delta} nst2 vacuoles, MIC2 accumulates earlier in the secretory pathway rather than localizing to micronemes. Dissemination of tachyzoites in human foreskin fibroblasts is reduced in these knockouts, which both show defects in attachment to and invasion of host cells comparable to the phenotype observed in the Bmic2.\n\nThese results, which show O-fucosylation of TSRs is required for efficient processing of MIC2 and for normal parasite invasion, are consistent with the recent demonstration that P. falciparum {Delta}pofut2 has decreased virulence and support a conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes.

microbiology↗