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Open Access Publications from the University of California

Scholarly Works (20 results)

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UC Davis Electronic Theses and Dissertations (2021)

Microbe-host interactions are mediated by protein-carbohydrate binding processes. The microbial adherence to cellular targets is a crucial step in pathogenesis. N-Glycans are oligosaccharides attached to the polypeptide of proteins and can be found on the surface of mammalian cells. Within the respiratory and intestinal tracts, highly glycosylated epithelial cells represent the primary boundary separating embedded host tissues from pathogens. Currently, there are limited methods to comprehensively explore the roles of glycans involved in these infections, therefore both virus and bacterium were employed in this study to investigate this topic. SARS‑CoV‑2, the causative agent for the COVID-19 pandemic, reaches into the respiratory tract, and Salmonella. Typhi can infect the intestinal tract and cause typhoid fever.The cell surface glycome was manipulated via metabolic engineering. N-Glycans on the cell underwent cell membrane extraction, enzymatic release, enrichment and was eventually analyzed with an Agilent 6520 Accurate Mass Q-TOF LC/MS equipped with a PGC micro-fluidic chip. Peptides and glycopeptides from host cells were obtained using cell membrane extraction and trypsin digestion. Peptides were desalted by solid-phase extraction with C18 cartridges, whereas glycopeptides were enriched with HILIC. They were then both analyzed using an Orbitrap Fusion Lumos LC-MS/MS system. Viral infection assay and confocal microscopy were employed to explore the effect of glycome on binding with SARS-Cov-2. Bacterial adherence and invasion assays were used to assess bacterial infection capacity with different types of N-glycans dominating the host cell surfaces. We established a cell-based model that enabled us to perform reliable structure-phenotype correlative experiments and compare the effect of individual N-glycan types in microbial infection. Using specific inhibitors, we created host cell surfaces that were primarily fucosylated, sialylated, undecorated, or contained mainly oligomannose structures. All the glycomic cell profiles here were confirmed via Q-TOF LC/MS. Confocal microscopy helped to reinforce the notion that HMOs, such as sialylated structures, can function as decoys to prevent SARS-Cov2 infection. After modifying the host cell glycosylation, binding assays showed the spike protein had a strong affinity towards sialylated N-Glycans. Combined with molecular dynamics simulations, our data further demonstrated that the spike protein, which recognized with host receptors to initiate viral entry, preferentially bound to sialic acids in α2-3 linkage. Bacterial adherence assay illustrated that fucosylated N-glycans on HCT116 cell surface increased significantly in the number of adhered Salmonella. In HCT116 cells, fucosylation was tunable, providing variable amounts of exogenous fucose. We then found that adherence of Salmonella is associated with the abundance of host fucosylated N-glycans. Furthermore, adherence of Salmonella to cells could be blocked by co-incubation with fucose or pretreatment of cells with fucosidase. The results proved that fucose residues on host cells bind with Salmonella. We performed qualitative and quantitative analyses of membrane proteins from host cells. The proteomic analysis showed that the metabolic engineering did not change the abundance of membrane proteins, which indicated that host protein expression did not contribute to the altered adherence. Meanwhile, glycoproteomic analysis yielded site-specific glycopeptide information. The glycopeptides were identified and quantified using a standard glycoproteomic workflow. We found that the attached N-glycans rather than glycosylation sites were manipulated in host cells. These results highlighted the importance of glycans in host-microbe interactions. This study also developed a lectin proximity oxidative labeling (Lectin PROXL) method to identify lectin-binding glycoproteins. The lectin was modified with a probe to produce hydroxide radicals. Thus, the lectin-recognized glycoproteins are oxidized and identified using a conventional proteomic process. All the lectin probes oxidized around 70% of glycoproteins. The specificity and sensitivity of each lectin were assessed utilizing glycomic and glycoproteomic data. Furthermore, the sialic acid- and fucose-binding lectins have higher specificity and sensitivity than other lectins. This approach provides an unprecedented perspective of lectin- glycoprotein interactions and protein networks mediated by distinct glycan types on cell membranes.

    Cover page: Probing Host-Microbe Interactions Through Glycomic and Glycoproteomic Methods
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2020)
    The cell membrane is composed of a network of glycoconjugates including glycoproteins and glycolipids that presents a dense matrix of carbohydrates playing critical roles in many biological processes. Lectin-based technology has been widely used to characterize glycoconjugates in tissues and cell lines. However, their specificity toward their putative glycan ligand and sensitivity in situ have been technologically difficult to study. Additionally, because they recognize primarily glycans, the underlying glycoprotein targets are generally not known. In this study, we employed lectin proximity oxidative labeling (Lectin PROXL) to identify cell surface glycoproteins that contain glycans that are recognized by lectins. Commonly used lectins were modified with a probe to produce hydroxide radicals in the proximity of the labeled lectins. The underlying polypeptides of the glycoproteins recognized by the lectins are oxidized and identified by the standard proteomic workflow. As a result, approximately 70% of identified glycoproteins were oxidized in situ by all the lectin probes, while only 5% of the total proteins were oxidized. The correlation between the glycosites and oxidation sites demonstrated the effectiveness of the lectin probes. The specificity and sensitivity of each lectin were determined using site-specific glycan information obtained through glycomic and glycoproteomic analyses. Notably, the sialic acid-binding lectins and the fucose-binding lectins had higher specificity and sensitivity compared to other lectins, while those that were specific to high mannose glycans have poor sensitivity and specificity. This method offers an unprecedented view of the interactions of lectins with specific glycoproteins as well as protein networks that are mediated by specific glycan types on cell membranes.
      Cover page: Determination of the glycoprotein specificity of lectins on cell membranes through oxidative proteomics
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2020)
      Abstract: Large‐scale genome‐wide analyses scans on massive numbers of various cases and controls are archived in the genetic databases that are publically available, for example, the Database of Genotypes and Phenotypes (https://www.ncbi.nlm.nih.gov/gap/). These databases offer unprecscendented opportunity to study the genetic effects. Yet, the set of nongenetic variables in these databases is often brief. From the statistical literature, we know that omitting a continuous variable from a logistic regression model can result in biased estimates of odds ratios (OR), even when the omitted and the included variables are independent. We are interested in assessing what information is needed to recover the bias in the OR estimate of genotype due to omitting a continuous variable in settings when the actual values of the omitted variable are not available. We derive two estimating procedures that can recover the degree of bias based on a conditional density of the omitted variable given the disease status and the genotype or the known distribution of the omitted variable and frequency of the disease in the population. Importantly, our derivations show that omitting a continuous variable can result in either under‐ or over‐estimation of the genetic effects. We performed extensive simulation studies to examine bias, variability, false‐positive rate, and power in the model that omits a continuous variable. We show the application to two genome‐wide studies of Alzheimer's disease.
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2020)
        Genetic studies provide valuable information to assess if the effect of genetic variants varies by the nongenetic "environmental" variables, what is traditionally defined to be gene-environment interaction (GxE). A common complication is that multiple disease states present with the same set of symptoms, and hence share the clinical diagnosis. Because (a) disease states might have distinct genetic bases; and (b) frequencies of the disease states within the clinical diagnosis vary by the environmental variables, analyses of association with the clinical diagnosis as an outcome variable might result in false positive or false negative findings. We develop estimates for this setting to be able to assess GxE in a case-only study and we compare the case-control and case-only estimates. We report extensive simulation studies that evaluate empirical properties of the estimates and show the application to a study of Alzheimer's disease.
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2024)
          Cell membrane glycoproteins are generally highly fucosylated and sialylated, and post-translational modifications play important roles in the proteins' functions of signaling, binding and cellular processing. For these reasons, methods for measuring sialic acid-mediated protein-protein interactions have been developed. However, determining the role of fucose in these interactions has been limited by technological barriers that have thus far hindered the ability to characterize and observe fucose-mediated protein-protein interactions. Herein, we describe a method to metabolically label mammalian cells with modified fucose, which incorporates a bioorthogonal group into cell membrane glycoproteins thereby enabling the characterization of cell-surface fucose interactome. Copper-catalyzed click chemistry was used to conjugate a proximity labeling probe, azido-FeBABE. Following the addition of hydrogen peroxide (H2O2), the fucose-azido-FeBABE catalyzed the formation of hydroxyl radicals, which in turn oxidized the amino acids in the proximity of the labeled fucose residue. The oxidized peptides were identified using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Variations in degree of protein oxidation were obtained with different H2O2 reaction times yielding the acquisition of spatial information of the fucose-interacting proteins. In addition, specific glycoprotein-protein interactions were constructed for Galectin-3 (LEG3) and Galectin-3-binding protein (LG3BP) illustrating the further utility of the method. This method identifies new fucose binding partners thereby enhancing our understanding of the cell glycocalyx.
            Cover page: Protein oxidation of fucose environments (POFE) reveals fucose–protein interactions
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            • Peer Reviewed
            UC San Francisco Previously Published Works (2023)
            OBJECTIVES: Palpitations are common and have a negative impact on womens quality of life. While evidence suggests that inflammatory mechanisms may play a role in the development of palpitations, no studies have evaluated for this association in patients with breast cancer who report palpitations prior to surgery. The purpose of this pilot study was to evaluate for associations between the occurrence of palpitations and single nucleotide polymorphisms (SNPs) in genes for pro- and anti-inflammatory cytokines, their receptors, and transcriptional regulators. METHODS: Patients were recruited prior to surgery and completed a self-report questionnaire on the occurrence of palpitations. Genotyping of SNPs in cytokine genes was performed using a custom array. Multiple logistic regression analyses were done to identify associations between the occurrence of palpitations and SNPs in fifteen candidate genes. RESULTS: Of the 82 SNPs evaluated in the bivariate analyses, eleven SNPs in 6 genes were associated with the occurrence of palpitations. After controlling for functional status, the occurrence of back pain, and self-reported and genomic estimates of race/ethnicity, 3 SNPs in 3 different genes (i.e., interleukin (IL) 1-beta (IL1B) rs1143643, IL10 rs3024505, IL13 rs1295686) were associated with the occurrence of palpitations prior to surgery (all p ≤ .038). CONCLUSIONS: While these preliminary findings warrant replication, they suggest that inflammatory mechanisms may contribute to the subjective sensation of palpitations in women prior to breast cancer surgery.
              Cover page: A Pilot Study of Associations Between the Occurrence of Palpitations and Cytokine Gene Variations in Women Prior to Breast Cancer Surgery.
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2014)
              Sodium-glucose transporters (SGLTs) facilitate the movement of water across the cell membrane, playing a central role in cellular homeostasis. Here, we present a detailed analysis of the mechanism of water permeation through the inward-facing state of vSGLT based on nearly 10 μs of molecular dynamics simulations. These simulations reveal the transient formation of a continuous water channel through the transporter that permits water to permeate the protein. Trajectories in which spontaneous release of galactose is observed, as well as those in which galactose remains in the binding site, show that the permeation rate, although modulated by substrate occupancy, is not tightly coupled to substrate release. Using a, to our knowledge, novel channel-detection algorithm, we identify the key residues that control water flow through the transporter and show that solvent gating is regulated by side-chain motions in a small number of residues on the extracellular face. A sequence alignment reveals the presence of two insertion sites in mammalian SGLTs that flank these outer-gate residues. We hypothesize that the absence of these sites in vSGLT may account for the high water permeability values for vSGLT determined via simulation compared to the lower experimental estimates for mammalian SGLT1.
                Cover page: Structural determinants of water permeation through the sodium-galactose transporter vSGLT.
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2014)
                Sodium-glucose transporters (SGLTs) facilitate the movement of water across the cell membrane, playing a central role in cellular homeostasis. Here, we present a detailed analysis of the mechanism of water permeation through the inward-facing state of vSGLT based on nearly 10 μs of molecular dynamics simulations. These simulations reveal the transient formation of a continuous water channel through the transporter that permits water to permeate the protein. Trajectories in which spontaneous release of galactose is observed, as well as those in which galactose remains in the binding site, show that the permeation rate, although modulated by substrate occupancy, is not tightly coupled to substrate release. Using a, to our knowledge, novel channel-detection algorithm, we identify the key residues that control water flow through the transporter and show that solvent gating is regulated by side-chain motions in a small number of residues on the extracellular face. A sequence alignment reveals the presence of two insertion sites in mammalian SGLTs that flank these outer-gate residues. We hypothesize that the absence of these sites in vSGLT may account for the high water permeability values for vSGLT determined via simulation compared to the lower experimental estimates for mammalian SGLT1.
                  Cover page: Structural Determinants of Water Permeation through the Sodium-Galactose Transporter vSGLT
                  • Article
                  • Peer Reviewed
                  UC Davis Previously Published Works (2021)
                  A cross-linking method is developed to elucidate glycan-mediated interactions between membrane proteins through sialic acids. The method provides information on previously unknown extensive glycomic interactions on cell membranes. The vast majority of membrane proteins are glycosylated with complicated glycan structures attached to the polypeptide backbone. Glycan-protein interactions are fundamental elements in many cellular events. Although significant advances have been made to identify protein-protein interactions in living cells, only modest advances have been made on glycan-protein interactions. Mechanistic elucidation of glycan-protein interactions has thus far remained elusive. Therefore, we developed a cross-linking mass spectrometry (XL-MS) workflow to directly identify glycan-protein interactions on the cell membrane using liquid chromatography-mass spectrometry (LC-MS). This method involved incorporating azido groups on cell surface glycans through biosynthetic pathways, followed by treatment of cell cultures with a synthesized reagent, N-hydroxysuccinimide (NHS)-cyclooctyne, which allowed the cross-linking of the sialic acid azides on glycans with primary amines on polypeptide backbones. The coupled peptide-glycan-peptide pairs after cross-linking were identified using the latest techniques in glycoproteomic and glycomic analyses and bioinformatics software. With this approach, information on the site of glycosylation, the glycoform, the source protein, and the target protein of the cross-linked pair were obtained. Glycoprotein-protein interactions involving unique glycoforms on the PNT2 cell surface were identified using the optimized and validated method. We built the GPX network of the PNT2 cell line and further investigated the biological roles of different glycan structures within protein complexes. Furthermore, we were able to build glycoprotein-protein complex models for previously unexplored interactions. The method will advance our future understanding of the roles of glycans in protein complexes on the cell surface.
                    Cover page: Glycan–protein cross-linking mass spectrometry reveals sialic acid-mediated protein networks on cell surfaces
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                    • Peer Reviewed
                    UCLA Previously Published Works (2012)
                    Rab small G proteins control membrane trafficking events required for many processes including secretion, lipid metabolism, antigen presentation and growth factor signaling. Rabs recruit effectors that mediate diverse functions including vesicle tethering and fusion. However, many mechanistic questions about Rab-regulated vesicle tethering are unresolved. Using chemically defined reaction systems, we discovered that Vps21, a Saccharomyces cerevisiae ortholog of mammalian endosomal Rab5, functions in trans with itself and with at least two other endosomal Rabs to directly mediate GTP-dependent tethering. Vps21-mediated tethering was stringently and reversibly regulated by an upstream activator, Vps9, and an inhibitor, Gyp1, which were sufficient to drive dynamic cycles of tethering and detethering. These experiments reveal a previously undescribed mode of tethering by endocytic Rabs. In our working model, the intrinsic tethering capacity Vps21 operates in concert with conventional effectors and SNAREs to drive efficient docking and fusion.
                      Cover page: Intrinsic tethering activity of endosomal Rab proteins
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