Lara K. Mahal’s research while affiliated with University of Alberta and other places

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Publications (182)


Construction of Chimeric HIV-1
(A) Characteristics of Transmitted/Founder (T/F) and chronic HIV-1 viruses. N/A, not available. The table is colour-coded by risk group (pink: HET, brown: PWID, blue: MSM, grey: chronic viruses). (B) Schematic representation of chimeric virus construction (created with BioRender.com). The gp140 of env was cloned into pREC_nfl_NL4-3_Δenv (ecto-MSD) using yeast-based recombination followed by transfection into HEK 293T cells to generate functional chimeric viruses expressing different envelope (Env) gp140 proteins. Each chimeric virus was propagated and titrated on U87.CD4.CCR5 using 50% tissue culture infective dose (TCID50) assay. (C) A phylogenetic tree generated from DNA sequences of gp140 from chimeric viruses and reference sequences from different HIV subtypes using the neighbour-joining method. (D) Evaluation of Env expression levels by Western blotting. SDS-PAGE and Western blot probing p24 (D45F) and gp120 (B13) were performed on chimeric viruses and the signal intensity was quantified by ImageJ. Based on the uniformity of the p24 expression, the Env/p24 ratio was then calculated to compare the Env expression level. (E) Relative Env expression level comparison between T/F and chronic viruses. The student t-test was performed. (F) Relative Env expression levels of viruses from different risk groups. One-way ANOVA followed by Tukey’s multiple comparisons was performed.
HIV-1 multi-virus competitions in human genital tissues
(A) Schematic representation of multi-virus competitions (created with BioRender.com). A mix of 6 T/F viruses from different risk groups, each at 3750 IUs, was added to human explant genital tissue pieces for 6 hours. The tissue pieces were then washed with PBS and cultured in fresh medium for 10 days. Migratory cells (MCs) leaving the tissue were collected 48 hours post-infection and co-cultured with human Th1 or Th17 cell lines for 10 days. Concurrently, the mixture of T/F viruses was also added to Th1 or Th17 cell lines directly to compare the effect of tissue on virus competition. DNA was extracted from tissue, MC and Th1 co-cultures (MC + Th1), MC and Th1 co-cultures (MC + Th17), Th1, and Th17. The C2-V3-C3 region of env was PCR amplified for next-generation sequencing using Illumina. (B) The percent of replication fitness for the individual virus in each competition on cervical tissue (i), MC + Th1 (ii), MC + Th17 (iii), Th1 (iv) and Th17 (v). Eight competitions were designed, each involving 6 different chimeric T/F viruses, allowing each virus to compete with every other virus at least once. The viruses that participated in each competition were coloured by transmission group. Each competition was performed in tissue from 3 donors (normally in triplicate) or on Th1/Th17 cells 3 times in triplicate. The results were generated from SeekDeep analysis on Illumina sequencing results. (C) Total production of individual virus in each competition on cervical tissue (vi), MC + Th1 (vii), MC + Th17 (viii), Th1 (ix) and Th17 (x). Total production was calculated by multiplying the relative copy number per mitochondria DNA (from S12 Fig qPCR results) by the average percentage fitness in competitions. (D) Pearson correlation analysis between the average percent of fitness (data from B) and the total production (data from C) of each HIV-1 T/F in mixed infections.
Average percent of fitness of chimeric T/F HIV-1 from different risk groups
(A-E) The average percent of fitness of each T/F chimera in cervical tissue, migratory cells and Th1 co-culture (MC + Th1), migratory cells and Th17 co-culture (MC + Th17), Th1 and Th17. The average percent of fitness was calculated based on the virus content in all replicates of all donors. Different shapes represent data different donors. (F-H) The average percent of fitness in cervical tissue, MC + Th1, MC + Th17, Th1 and Th17 grouped by different risk groups. The Kruskal-Wallis test followed by Dunn’s multiple comparisons test was performed. (I-J) Comparison of average percent of fitness of T/F viruses on Th1 and Th17 with and without MCs. Mann-Whitney tests were performed.
Correlation analysis on the average percent of fitness of chimeric T/F viruses
(A-F) Two-tailed Spearman correlation analysis on the average percent of fitness of chimeric T/F viruses in cervical tissue, migratory cells and Th1 co-culture (MC + Th1), migratory cells and Th17 co-culture (MC + Th17), Th1, and Th17.
Evaluation of virus CD4/CCR5 utilization using Affinofile cells
Affinofile cells were induced with varying concentrations of minocycline (ranging from 0 to 20 ng/mL) and ponasterone A (ranging from 0 to 1 µM) to express varying levels of CD4 (approximately 850 to 125000 molecules per cell) and CCR5 (approximately 1274 to 15830 molecules per cell) independently and simultaneously. The infectivity was evaluated by luciferase activity expressed in Affinofile cells upon infection and the CD4/CCR5 response curves were characterized. (A-D) The mean area under the curve was calculated for each virus and grouped by transmission modes under low and high CD4 (approximately 100000 molecules per cell for low level; approximately 125000 molecules per cell for high level) and CCR5 (approximately 1296 molecules per cell for low level; approximately 15830 molecules per cell for high level) expression levels. Statistical analysis was performed using one-way ANOVA. LD, limit of detection.

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Transmitted/founder (T/F) HIV-1 derived from sexual contact exhibits greater transmission fitness in human cervical tissue than T/F HIV-1 from blood-to-blood contact: Unique glycan profiles on T/F envelopes associated with transmission phenotypes
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May 2025

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11 Reads

Yiying Zhang

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Annette Ratcliff

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Human immunodeficiency virus 1 (HIV-1) risk groups include, but are not limited to, heterosexual individuals (HET), men-who-have-sex-with-men (MSM), and people who inject drugs (PWID). Although genetically diverse HIV-1 populations are transferred from donor to recipient, systemic infection is often established by a single clone, the transmitted/founder (T/F) virus. This phenomenon is especially prevalent in sexual transmission, but less stringent in blood-to-blood contact transmission. Specific traits that permit successful transmission have not been well characterized. Thus, HIV-1 containing the chimeric T/F envelope (Env) from different transmission routes was assessed for ex vivo transmission fitness by performing mixed competition assays (also referred to as mixed competitions) on human cervical tissues. We found that chimeric T/F viruses isolated from the PWID exhibit limited replication capacity in cervical tissues when compared to those from MSM and HET, diminishing their chances of transmission to T helper type 1 (Th1) and Th17 cells. This reduced transmission fitness of T/F HIV-1 from PWID was not observed when infecting Th1 and Th17 cells directly, bypassing cervical tissues. Phenotypic assays showed that the chimeric T/F viruses from PWID differed from other groups by having an enhanced ability to utilize diverse CCR5 conformations, while Env expression level, CD4/CCR5 utilization, and entry speed did not differ. Different glycosylation profiles were detected on T/F compared to chronic Env with increased complex, fucosylated N- and O-glycans found more frequently on the T/F Env. Furthermore, the increased presence of these fucosylated glycans correlated with replication fitness in cervical tissues. In contrast, bisecting branched N-glycan found more frequently on chronic Env was associated with decreased entry efficiency and more stringent usage of CCR5. These findings suggest that glycosylation patterns/levels and/or Env structure greatly impact the differences in transmission fitness of T/F HIV-1.

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The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting lectin microarray data

February 2025

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14 Reads

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1 Citation

Glycobiology

The MIRAGE (Minimum Information Required for a Glycomics Experiment) project has been established by experts in glycobiology, glycoanalytics, and glycoinformatics under the auspieces of the Beilstein-Institut. The working group aims to develop guidelines for reporting results from various experiments and analyses conducted in structural and functional studies of glycans in the scientific literature. Previous guidelines have been established for glycomic analytics, including mass spectrometry and glycan microarrays. Lectin microarrays are used worldwide for glycan profiling of various biological samples, but there are often insufficient reports on information about experimental methods such as sample preparation and fluorescence labeling. Here, we propose guidelines specifically designed to improve the standards for reporting data from lectin microarray analyses. For each of the seven areas in the workflow of a lectin microarray experiment, we provide recommendations for the minimum information that should be included when reporting results. When adopted by the scientific community the MIRAGE lectin microarray guidelines are expected to enhance data interpretation, facilitate comparison of data between laboratories and encourage the deposition of lectin microarray data in international databases.


Profiling the Regulatory Landscape of Sialylation through miRNA Targeting of CMP- Sialic Acid Synthetase

February 2025

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20 Reads

Journal of Biological Chemistry

Cell surface sialic acid is an important glycan modification that contributes to both normal and pathological physiology. The enzyme cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) biosynthesizes the activated sugar donor cytidine monophosphate (CMP) sialic acid, which is required for all sialylation. CMAS levels impact sialylation with corresponding biological effects. The mechanisms that regulate CMAS are relatively uncharacterized. Herein, we use a high throughput genetically encoded fluorescence assay (miRFluR) to comprehensively profile the posttranscriptional regulation of CMAS by miRNA. These small non-coding RNAs have been found to impact glycosylation. Mapping the interactions of the human miRNAome with the 3′-untranslated region of CMAS, we identified miRNA whose impact on CMAS expression was either downregulatory or upregulatory. This follows previous work from our laboratory and others showing that miRNA regulation is bidirectional. Validation of the high-throughput results confirmed our findings. We also identified the direct binding sites for two upregulatory and two downregulatory miRNAs. Functional enrichment analysis for miRNAs upregulating CMAS revealed associations with pancreatic cancer, where sialic acid metabolism and the α-2,6-sialyltransferase ST6GAL1 have been found to be important. We found that miRNA associated with the enriched signature enhanced pancreatic cell-surface α-2,6-sialylation via CMAS expression in the absence of effects on ST6GAL1. We also find overlap between the miRNA regulation of CMAS and that of previously analyzed sialyltransferases. Overall, our work points to the importance of miRNA in regulating sialylation levels in disease and add further evidence to the bidirectional nature of miRNA regulation.


Fig. 1. Comprehensive map of CD98hc regulation by miRNA. (A) α-2,3-Sialylation of Cd98hc by St3GAl1 and St3GAl2 stabilizes this essential protein in melanoma (11). (B) miRFluR assay: the 3′UtR of gene of interest (Cd98hc) is cloned downstream of Cerulean in the pFmiR construct (pFmiR-Cd98hc). the plasmid is then screened with the human miRnAome library (dharmacon, miRbase v. 21) in heK-293t cells. Ratio of Cerulean: the mCherry signal normalized by median ratio indicates miRnA impact. (C) Scatterplot of miRFluR data for 3′UtR of Cd98hc. miRnAs in the 95% confidence interval by z-score are indicated (down-miRs: red; up-miRs: blue), and validated miRnAs are shown. (D) Pie chart showing % miRnA hits compared to the total library after QC. (E) Pie chart indicates % down-miR (48%) versus up-miR (52%) in the Cd98hc hit list. Additional data shown in figs. S1 to S3.
Fig. 3. Mutational analysis identifies the miR-155-5p binding site on CD98hc 3′UTR. (A) Comparison of wild-type (Wt) and mutant (MUt) pFmiR-3′UtR interactions with miRnA. (B) Alignment of miR-155-5p with the predicted Cd98hc-3′UtR site and corresponding mutant. Mutated residues are shown in red, and wobble interactions (G:U) are shown in gray. (C) Bar graph of data from wild-type and mutant miRFluR sensors. data were normalized over ntC in each sensor. the experiment was performed in n ≥ 3 biological replicates. errors shown are standard deviations. Statistical analysis using the paired t test compared the impact of miR-155-5p in the Wt sensor with the corresponding mutant (the P value is indicated on the graph).
Fig. 6. Up-regulatory miRNAs co-regulate CD98hc/ST3GAL1 or CD98hc/ST3GAL2. Sialylation is required for the stability of Cd98hc in melanoma where St3GAl1, St3GAl2, and Cd98hc are all essential genes.
Fig. 8. Co-up-regulation by miRNAs requires direct interactions with 3′UTRs of CD98hc, ST3GAL1, and ST3GAL2. (A and B) Alignment of miR-1246 with predicted Cd98hc-3′UtR (A) or St3GAl1-3'UtR (B) sites and their corresponding mutants. Mutated residues are shown in red. Wobble interactions are shown in gray. (C) Bar graph of data from miRFluR sensors. For each sensor, data were normalized over ntC. (D and E) Alignment of miR-30b-5p with predicted Cd98hc-3′UtR (d) or St3GAl2-3′UtR (e) sites and their corresponding mutants. Mutated residues are shown in red. Wobble interactions are shown in gray. (F) Bar graph of data from miRFluR sensors. data were normalized over ntC in each sensor. All experiments were performed in n ≥ 3 biological replicates. errors shown are standard deviations. Statistical analysis using the paired t test compared the impact of co-up-miR in the Wt sensor with the corresponding mutant (P values are indicated on the graphs).
Screening the human miRNA interactome reveals coordinated up-regulation in melanoma, adding bidirectional regulation to miRNA networks

January 2025

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16 Reads

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2 Citations

Science Advances

Cellular protein expression is coordinated posttranscriptionally by an intricate regulatory network. The current presumption is that microRNAs (miRNAs) work by repression of functionally related targets within a system. In recent work, up-regulation of protein expression via direct interactions of messenger RNA with miRNA has been found in dividing cells, providing an additional mechanism of regulation. Herein, we demonstrate coordinated up-regulation of functionally coupled proteins by miRNA. We focused on CD98hc, the heavy chain of the amino acid transporter LAT-1, and α-2,3-sialyltransferases ST3GAL1 and ST3GAL2, which are critical for CD98hc stability in melanoma. Profiling miRNA regulation using our high-throughput miRFluR assay, we identified miRNA that up-regulated the expression of both CD98hc and either ST3GAL1 or ST3GAL2. These co–up-regulating miRNAs were enriched in melanoma datasets associated with transformation and progression. Our findings add co–up-regulation by miRNA into miRNA regulatory networks and add a bidirectional twist to the impact miRNAs have on protein regulation and glycosylation.


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Profiling the Regulatory Landscape of Sialylation through miRNA Targeting of CMP- Sialic Acid Synthetase

December 2024

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9 Reads

Cell surface sialic acid is an important glycan modification that contributes to both normal and pathological physiology. The enzyme cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) biosynthesizes the activated sugar donor cytidine monophosphate (CMP) sialic acid, which is required for all sialylation. CMAS levels impact sialylation with corresponding biological effects. The mechanisms that regulate CMAS are relatively uncharacterized. Herein, we use a high throughput genetically encoded fluorescence assay (miRFluR) to comprehensively profile the posttranscriptional regulation of CMAS by miRNA. These small non-coding RNAs have been found to impact glycosylation. Mapping the interactions of the human miRNAome with the 3 prime-untranslated region of CMAS, we identified miRNA whose impact on CMAS expression was either downregulatory or upregulatory. This follows previous work from our laboratory and others showing that miRNA regulation is bidirectional. Validation of the high-throughput results confirmed our findings. We also identified the direct binding sites for 2 upregulatory and 2 downregulatory miRNAs. Functional enrichment analysis for miRNAs upregulating CMAS revealed associations with pancreatic cancer, where sialic acid metabolism and the alpha-2,6-sialyltransferase ST6GAL1 have been found to be important. We found that miRNA associated with the enriched signature enhanced pancreatic cell-surface alpha-2,6-sialylation via CMAS expression in the absence of effects on ST6GAL1. We also find overlap between the miRNA regulation of CMAS and that of previously analyzed sialyltransferases. Overall, our work points to the importance of miRNA in regulating sialylation levels in disease and add further evidence to the bidirectional nature of miRNA regulation.


Age-related remodeling of the glycocalyx drives T cell exhaustion

December 2024

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55 Reads

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1 Citation

Cell surface glycans, termed the glycocalyx, are essential regulators of cellular signaling and thus cellular development and functions, but how aging impacts the glycocalyx remains poorly understood. Here, using immune cells as a model system for studying the relationship between aging and glycocalyx remodeling, we show that α2,6-linked sialic acid — a terminal glycan epitope typically associated with inhibitory signaling — becomes downregulated in T cells from older animals. This downregulation is tightly correlated with age-associated accumulation of effector T cells, which are decorated with little to no α2,6-linked sialic acids. T cell aging renders older individuals more vulnerable to infections and cancers. To understand the role of α2,6-linked sialic acids in T cell physiology, we generated a mouse model with T cell-specific deletion of the sialyltransferase gene St6gal1. The chronic depletion of α2,6-linked sialic acids led to naive T (TN) cells expansion in the periphery and premature T cell exhaustion. As a result, these mice were less able to control acute Listeria infection and chronic tumor growth. Blockade of the PD-1 pathway can partially restore the ability of St6gal1-deficient T cells to control tumor growth. Together, these data suggest that α2,6-linked sialic acids are critical for maintaining long-term T cell responsiveness, and the loss of α2,6-linked sialic acids may directly contribute to age-related T cell exhaustion.



XBP1s-induction upregulates high mannose N-glycans on and within cells
a Schematic for dox-inducible XBP1s upregulating high mannose N-glycans. b RT-qPCR for targets of general UPRER and XBP1s. Gene expression is normalized to housekeeping genes GAPDH and HRPT1. c Fluorescent HHL and GRFT binding on A549 cells with or without dox-induction of XBP1s. Cells were treated with 2 µg/mL dox for 48 hours to overexpress XBP1s. d Fluorescent MBL2 binding on A549 cells with or without dox-induction of XBP1s. Cells were treated with 2 µg/mL dox for 48 hours to overexpress XBP1s. e UPLC quantification of high mannose N-glycan structures of A549 cells with or without XBP1s-induction. Levels of each high mannose structure are normalized to the protein amount of each replicate. f Schematic for lectin microarray analysis of A549s under basal or XBP1s-induced conditions. g Volcano plot of lectin microarray data. Median normalized log2 ratios (sample /reference) of the A549 samples are presented. Lectins are color-coded by their glycan-binding specificities. All Data are presented as mean ± s.e.m. unless otherwise indicated, and are representative of at least two independent experiments performed in triplicate with consistent results. p values were calculated from two-tailed Student’s t test.
Genome-wide CRISPR screen uncovers the expanded network of genes regulating high mannose
a Schematic for FACS-based CRISPR screen. Cas9-expressing A549s were lentivirally transduced with a genome-wide CRISPR-deletion sgRNA library. Resulting cells were dox-treated to induce XBP1s overexpression for 48 hours. Cells were then gently lifted with Accutase, fixed, and stained with FITC-labeled HHL. The top and bottom 25% of HHL stained cells were isolated by FACS. The resulting populations were subjected to deep sequencing and analysis. The screen was performed in duplicate. b Volcano plot of all genes indicating effect and confidence scores for the genome-wide screen performed in duplicate. Effect and P values were calculated by casTLE. c Schematic for initial steps of N-glycan mannose-trimming and remodeling. All three enzymes indicated are hits in genome-wide screen. d Disruption of tail-anchored protein insertion pathway by ASNA1 inhibitor Retro-2 in wild type A549s also upregulates cell surface high mannose glycan levels. A549s were treated with treated with 2 µg/mL dox, 100 µM of Retro-2, both, or left untreated for 48 hours. Resulting cells were lifted with Accutase and stained with FITC-labeled HHL, followed by flow cytometry analysis. Data are presented as mean ± s.e.m. of median of each replicate and are representative of two independent experiments performed in triplicate with consistent results. p values were calculated from two-tailed Student’s t test. e Schematic for competitive binding assays for measuring changes in high mannose levels. Cells expressing sgRNAs for CRISPRi-mediated knockdown (KD) and miRFP and cells expressing a control sgRNA and BFP were cocultured in 1:1 ratio. Cells were either treated with dox to induce XBP1s or left untreated for 48 hours. Resulting cells were lifted and stained with HHL-FITC, and log2 ratio of HHL intensity of KO: control was determined using flow cytometry. f Validation of hits in XBP1s-induced A549s using competitive HHL binding assays. Data are presented as mean ± s.e.m. and are representative of two independent experiments performed in triplicate with consistent results. p values were calculated from two-tailed Student’s t test.
Targeted CRISPRi screen uncovers additional regulators of high mannose glycans under basal and UPRER induced conditions
a Schematic for MACS-based CRISPR screen. A549 cells stably expressing CRISPRi machinery and the targeted sgRNA sublibrary were either dox-treated to induce XBP1s or left untreated for 48 hours. Cells were lifted and incubated with HHL coupled to magnetic beads. The cells were then placed on a magnet in which high HHL-binding cells would be retained on the magnet, whereas the low HHL-binding cells were removed from the population. This separation was repeated twice more on each high and low HHL binding cells to improve the purity of the populations. Finally, each resulting population were subjected to deep sequencing and analysis to identify hits. The screen was performed in duplicate. b The maximum effect size (center value) estimated by CasTLE from both basal and XBP1s-induced conditions with five independent sgRNA per gene. The bars represent the 95% credible interval, with red representing XBP1s and blue representing basal conditions. Only genes considered to be a hit in at least one condition are shown. Genes are ordered in descending order of estimated maximum effect size of XBP1s-induced condition. The top 30 positive and negative hits are shown in the expanded panels. c Top 30 regulators for high mannose N-glycans with their reported subcellular localization. d Validation of hits in A549 under basal conditions using competitive HHL binding assays. Each gene is knocked down by co-expression of two independent sgRNAs. Data are presented as mean ± s.e.m. and are representative of two independent experiments performed in triplicate with consistent results. e Validation of hits in A549 under XBP1s-induced conditions using competitive HHL binding assays. Each gene is knocked down by co-expression of two independent sgRNAs. Data are presented as mean ± s.e.m. and are representative of two independent experiments performed in triplicate with consistent results. p values were calculated from two-tailed Student’s t test.
TM9SF3 regulates the Golgi organization and is required for formation of complex N-glycans
a Competitive HHL binding assay in A549s with each TM9SF family member knocked down. b Flow cytometry quantification of intracellular staining of GM130 and TGN46 in A549s. c Representative confocal microscopy images of TM9SF3 knockdown and wildtype control cells, co-stained with cis-/medial-Golgi marker GM130 and TGN marker TGN46. Magnified views of the red boxed areas are shown in the right-most column. Scale bars, 10 μm. Images are representative of two independent experiments performed in triplicate. d Percent area of each Golgi compartment co-localized with the other compartment. Colocalized area is divided by total area of the indicated Golgi marker (GM130 or TGN46) to determine the percentage of each compartment that is colocalized with the other. Data are presented as mean ± s.e.m., from at least 12 images each from wildtype or TM9SF3 knockdown of two independent experiments, with > 20 cells per image. e Volcano plot for lectin microarray results of A549 cells with TM9SF3 knocked down compared to wildtype control. Lectins are color-coded by their glycan-binding specificities. f Competitive cell surface lectin binding assay for TM9SF3 knocked down A549s compared to wild type control under basal conditions. Lectin binding specificities and location of where the modification predominately occurs are indicated. Unless otherwise indicated, all Data are presented as mean ± s.e.m. and are representative of at least three independent experiments performed in triplicate with consistent results. p values were calculated from two-tailed Student’s t test.
CCDC22 regulates elongation and sialylation of glycans through modulating Golgi expansion
a Competitive HHL binding assays on A549s for all knock down of all members of the CCC complex. Each gene is knocked down by co-expression of two independent sgRNAs. b Flow cytometry quantification of intracellular staining of GM130 and TGN46 in A549s in CCDC22 knockdown and wild type control cells. c Representative confocal microscopy images of CCDC22 knockdown and wildtype control cells, co-stained with cis-/medial-Golgi marker GM130 and TGN marker TGN46. Magnified views of the red boxed areas are shown in the right-most column. Scale bars, 10 μm. Images are representative of three independent experiments performed in triplicate. d Percent area of each Golgi compartment co-localized with the other compartment. Colocalized area is divided by total area of the indicated Golgi marker (GM130 or TGN46) to determine the percentage of each compartment that is colocalized with the other. Data are presented as mean ± s.e.m., from > at least 12 images each from wildtype or CCDC22 knockdown of two independent experiments, with > 20 cells per image. e Volcano plot for lectin microarray results of basal A549 cells with CCDC22 knocked down compared to wildtype control. Lectins are color-coded by their glycan-binding specificities. f Competitive cell surface lectin binding assay for CCDC22 knocked down A549s compared to wildtype control under basal conditions. Lectin binding specificities and the location of where the modification predominately occurs are indicated. Unless otherwise indicated, all Data are presented as mean ± s.e.m. and are representative of at least three independent experiments performed in triplicate with consistent results. p values were calculated from two-tailed Student’s t test.
CRISPR screens and lectin microarrays identify high mannose N-glycan regulators

November 2024

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78 Reads

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5 Citations

Glycans play critical roles in cellular signaling and function. Unlike proteins, glycan structures are not templated from genetic sequences but synthesized by the concerted activity of many genes, making them historically challenging to study. Here, we present a strategy that utilizes CRISPR screens and lectin microarrays to uncover and characterize regulators of glycosylation. We applied this approach to study the regulation of high mannose glycans – the starting structure of all asparagine(N)-linked-glycans. We used CRISPR screens to uncover the expanded network of genes controlling high mannose levels, followed by lectin microarrays to fully measure the complex effect of select regulators on glycosylation globally. Through this, we elucidated how two high mannose regulators – TM9SF3 and the CCC complex – control complex N-glycosylation via regulating Golgi morphology and function. Notably, this allows us to interrogate Golgi function in-depth and reveals that similar disruption to Golgi morphology can lead to drastically different glycosylation outcomes. Collectively, this work demonstrates a generalizable approach for systematically dissecting the regulatory network underlying glycosylation.


Cracking the Glycome with the Sweet Tooth of Nature: Overview and Outlook of Lectin Microarray Technology

October 2024

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13 Reads

Glycoproteins are central to numerous cellular processes and are among the most structurally complex biomolecules in nature. This unique complexity stems from variability in complex oligosaccharides that are located throughout the protein, a feature that is profoundly important for regulating biomolecular interactions but also makes glycoproteins difficult to study. As such, glycoprotein analysis entails a range of techniques to bridge the knowledge gap between glycoprotein structure and biological function. This book serves as an authoritative guide to glycoprotein analysis, written by internationally recognised experts in the field and discussed in the context of real-world applications across the life sciences. It provides a wide-ranging assessment of the modern methods, from those used to characterise glycoprotein structure, to approaches proficient in uncovering the molecular mechanisms by which they function as well as those capable of measuring structural dynamics and macromolecular assembly. These methods differ to a large extent and include mass spectrometry, glycan/lectin arrays, nuclear magnetic resonance, infrared spectroscopy, scanning probe microscopy and high-performance liquid chromatography. Equally important are computational techniques, including molecular dynamics and bioinformatics, which are also covered and discussed in the wider context of glycoprotein analysis. Glycobiology is indeed a rapidly growing field and the development of advanced tools for glycoproteins analysis has been enabled by researchers from different backgrounds working to overcome long-standing analytical challenges and biological questions involving glycosylation. This book is intended to aid academic and professional researchers at various levels of their career to gain a deeper appreciation of cutting-edge methods in glycoprotein analysis and their applications in biomolecular research, biotherapeutic development, structural biology and biophysical chemistry.



Citations (58)


... As a feasibility study of the tissue glycome mapping approach, the LMD-assisted LMA method was applied to 14 tissues of normal mice, demonstrating its utility in evaluating and elucidating site-and tissue-specific glycosylation 9,14 . Currently, over 500 data of mouse tissue glycome mapping, along with relevant tissue histological images, are publicly available in the first LMAbased glycomics database, the LM-GlycomeAtlas [14][15][16] 26 , as LMA-based glycomics is technically mature and has become a well-accepted method for glycan analysis 7,8 . Although a repository system for protein microarrays is available for LMA data deposition 27 , it does not fully support data deposition or metadata description according to the MIRAGE guideline for LMA. ...

Reference:

LM-GlycoRepo Version 1.0: A novel repository system for mouse tissue glycome mapping data
The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting lectin microarray data
  • Citing Article
  • February 2025

Glycobiology

... Although not covered in detail in this review, an additional perspective involves miRNA-mediated translation activation [156][157][158][159][160][161][162][163][164]. The target mRNAs described thus far include mRNAs harboring AU-rich elements in their 3 ′ UTR (e.g., TNFα mRNA), 3 ′ poly(A)-less mRNA, hepatitis C virus RNA, and mitochondrial mRNA. ...

Screening the human miRNA interactome reveals coordinated up-regulation in melanoma, adding bidirectional regulation to miRNA networks

Science Advances

... Loss-of-function (LOF) genetic screening can be a powerful approach to identifying regulators of cell-surface glycosylation. Indeed, several other studies have since applied LOF CRISPR screening to study a variety of other significant processes in glycobiology 27,28 . Other studies have also addressed some of these same questions using carefully designed, focused arrays of sgRNAs against glycan biosynthesis genes 29,30 . ...

CRISPR screens and lectin microarrays identify high mannose N-glycan regulators

... These include insource dissociation of labile protein complexes, particularly those stabilized by hydrophobic interactions, 41,42 interference from nonspecic binding, 43,44 and non-uniform response factors between free and ligand-bound proteins. 45,46 While progress has been made to address these challenges-such as using chemical additives, 47,48 maintaining relatively low sampling temperatures, minimizing collisional activation to stabilize hydrophobically bound systems, 49,50 employing reference proteins to correct for nonspecic binding, 51,52 and adopting titration methods with parameters that account for dissociation or response factor discrepancies 33,52 -some bottlenecks remain unresolved. Despite the potential drawbacks, native MS measurements are independent of fast binding kinetics and limited ligand solubility, which hinder SPR and ITC, respectively. ...

Deciphering Pathways and Thermodynamics of Protein Assembly Using Native Mass Spectrometry
  • Citing Article
  • October 2024

Journal of the American Chemical Society

... As another approach, Meanwell and colleagues recently reported a fivesteps synthesis of 4ʹ-modified ribonucleosides from non-nucleosidic 2,2-dimethoxyacetoaldehyde. 5) The synthesis of 4ʹ-modified nucleosides using carbon radicals generated at the C4ʹ position is also an attractive strategy; however, preparing the radical precursors is generally tedious. [6][7][8][9][10] We also recently developed a novel method for the rapid and facile generation of 4ʹ-carbon radicals via the 1,5-hydrogen atom transfer (1,5-HAT) of iminyl radicals formed by the single-electron reduction of oxime imidates, which was applied to synthesis of 2ʹ-O,4ʹ-C-and 3ʹ-O,4ʹ-C-bridged nucleosides via intramolecular radical cyclization 11) (Chart 1c). ...

An enantioselective and modular platform for C4'-modified nucleoside analogue synthesis enabled by intramolecular trans-acetalizations

... Another study published by Sevim Bayrak et al. (24) analyzed pre-vaccination samples from recipients of the seasonal influenza vaccine and integrated transcriptom ics, proteomics, metabolomics, and glycomics data. Five clusters were identified and were associated with different levels of gene expression related to innate immune pathways or adaptive immune pathways. ...

Patient subtyping analysis of baseline multi-omic data reveals distinct pre-immune states associated with antibody response to seasonal influenza vaccination
  • Citing Article
  • July 2024

Clinical Immunology

... With this array, glycosylation profiles on different HIV-1 Env can be compared using panels of lectins and antibodies that bind to distinct glycan epitopes, including both N-and O-linked glycans [104][105][106]. These lectin arrays have been used in several previous studies to analyze HIV-1 glycosylation [107,108] and have the advantage of utilizing small amounts of material to test a multitude of lectins that bind to different glycan epitopes simultaneously. While this analysis does not identify specific glycan composition at each N-linked site in Env as can be obtained with mass spectrometry (MS), it will identify the relative amounts of glycans on the Env glycoproteins from multiple viral preparations ( Fig 7A). ...

HIV-1 interaction with an O-glycan-specific bacterial lectin enhances virus infectivity and resistance to neutralizing antibodies

iScience

... miRNA are known to impact expression of multiple proteins in a regulatory network. In recent work, we have shown that sialylation by ST3GAL1/2 controls the stability of CD98hc in melanoma (22) and that miRNA involved in melanoma upregulate both ST3GAL1/2 and CD98hc, providing evidence for bidirectional tuning in networks (17). ...

Integrated in vivo functional screens and multi-omics analyses identify α-2,3-sialylation as essential for melanoma maintenance

... (Figs 2A and S3). In fact, several groups have reported critical physical and functional interactions between SARS-CoV-2 and other viruses with the Wnt signaling machinery to promote viral survival [51], the role of glycosylation to enable S-mediated entry and stimulate innate immune activation [52], or the ability of SARS-CoV-2 to hijack MAPK11 to promote viral replication [53]. Less understood is the role of epigenetic regulation during SARS-CoV-2. ...

The Wnt/β-catenin pathway is important for replication of SARS-CoV-2 and other pathogenic RNA viruses

npj Viruses

... Our results on the nanomechanical changes in the tumor stroma underscore the importance of mechano surveillance by immune cells, including T cells. This concept, which refers to the ability of immune cells to sense and respond to the mechanical properties of their environment (41)(42)(43), plays a crucial role in their activation, motility, and overall function. ...

The microenvironment dictates glycocalyx construction and immune surveillance