Browsing by Subject "Glycomics"
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Item Advances in protein microarray technology for glycomic analysis(2011-08) Propheter, Daniel Champlin; Iverson, Brent L.; Mahal, Lara K.; Siegel, Dionicio R.; Keatinge-Clay, Adrian; Fast, Walter L.The cell surface is enveloped with a myriad of carbohydrates that form complex matrices of oligosaccharides. Carbohydrate recognition plays crucial and varying roles in cellular trafficking, differentiation, and bacterial pathogenesis. Lectin microarray technology presents a unique platform for the high-throughput analysis of these structurally diverse classes of biopolymers. One significant hinderance of this technology has been the limitation imposed by the set of commercially available plant lectins used in the array. To enhance the reproducibility and scope of the lectin panel, our lab generated a small set of bacteria-derived recombinant lectins. This dissertation describes the unique advantages that recombinant lectins have over traditional plant-derived lectins. The recombinant lectins are expressed with a common fusion tag, glutathione-S-transferase (GST), which can be used as an immobilization handle on glutathione (GSH)-modified substrates. Although protein immobilization via fusion tags in a microarray format is not novel, our work demonstrates that protein activity through site-specific immobilization is enhanced when the protein is properly oriented. Although orientation enhanced the activity of our GST-tagged recombinant lectins, the GSH-surface modification precluded the printing of non-GST-tagged lectins, such as the traditional plant lectins, thus limiting the structural resolution of our arrays. To solve this issue, we developed a novel print technique which allows the one-step deposition and orientation of GST-tagged proteins in a microarray format. To expand our view of the glycome, we further adapt this method for the in situ orientation of unmodified IgG and IgM antibodies using GST-tagged antibody-binding proteins. Another advantage of recombinant lectins is in the ease of genomic manipulation, wherein we could tailor the binding domain to bind a different antigen. We demonstrate this by producing non-binding variants of the recombinant lectins to act as negative controls in our microarrays. Along with the non-binding variants, we developed a lectin displayed on the surface of phage. In the hopes generating more novel lectins, I will describe our current efforts of lectin evolution using phage-displayed GafD. By generating novel tools in lectin microarray technology, we enhance our understanding of the role of carbohydrates on a global scale.Item Glycomic insights into microvesicle biogenesis(2011-08) Batista, Bianca Stella; Stein, David S.; Mahal, Lara K.; Iyer, Vishwanath R.; Sullivan, Christopher S.; Liu, Hung-WenCells can mediate intercellular communication by the secretion and uptake of microvesicles, nano-sized membranous particles that carry signaling molecules, antigens, lipids, mRNA and miRNA between cells. The biological function of these vesicles is dependent upon their composition and cellular origin which is regulated by mechanisms that are not well understood. Based on their molecular content, microvesicles may play a role in immune regulation, cancer progression, the spread of infectious agents and numerous other important normal and pathogenic processes. The proteomic content of microvesicles from diverse sources has been intensely studied. In contrast, little is known about their glycomic content. The glycosylation pattern of a protein or lipid plays a key role in determining its functional properties in several ways. Glycans can determine the trafficking of a protein to particular regions of the cell as well as the protein’s half life. In addition, the glycan-dervied oligomerization of glycolipids and glycoproteins is a known mechanism for the activation of receptors and recognition of ligands on the surface of the cell. Glycomic analysis may thus provide valuable insights into microvesicle function. I utilized lectin microarray technology to compare the glycosylation patterns of microvesicles derived from a variety of biological sources. When compared to cellular membranes, microvesicles were enriched in high mannose, polylactosamine, α2-6 sialic acid, and complex N-linked glycans but exclude terminal blood group A and B antigens. The polylactosamine signature in microvesicles from different cell lines derives from distinct glycoprotein cohorts. After treatment of Sk-Mel-5 cells with lactose to inhibit lectin-glycan interactions, secretion of microvesicle resident proteins was severely reduced. Taken together, this work provides evidence for a role of glycosylation in microvesicle-directed protein sorting.Item Glycomics : integration of lectin and gene expression microarray data(2011-08) Pilobello, Kanoelani Takaishi; Mahal, Lara K.; Anslyn, Eric V., 1960-Glycomics is the systematic study of glycosylation in the context of a whole cell or organism. Glycosylated proteins are estimated to make up 50% of all proteins and cover the outside of the cell. Functional roles in glycosylation have been noted in pathogenesis, metastasis, and embryogenesis. However, the structure of these carbohydrates has been difficult to study due to the chemical nature of carbohydrates. Lectins, carbohydrate binding proteins excluding antibodies and enzymes, can be utilized to study glycosylation in a high throughput manner using a microarray format. Glycans, the carbohydrates attached to a protein or lipid, are not synthesized from a template. They are added co- or post-translationally by a concerted set of enzymes in the secretory pathway. In addition, the glycan structures may be altered by metabolism or trafficking. Cell type specific glycosylation has long been hypothesized due to observations of bacteria homing to tissues. We use lectin microarray technology to define the glycosylation in a subset of the NCI-60, a set of cell lines from different tissues. Using a customized gene expression microarray, we identify cell type dependent glycosylation genes and observe evidence of cell type dependent spliceforms for an O-glycosylated mucin. Data from the lectin microarray and a published gene expression data set were integrated using Generalized Singular Value Decomposition (GSVD), a linear matrix decomposition method. We have successfully decomposed the data into 3 cell type dependent meta patterns that segregate by glycosylation family. Correlation projection of the genes and subsequent gene ontology enrichment suggests that genes in different pathways covary with the types of glycosylation. An inverse relationship was revealed for the N- glycosylation pattern between the SVD of the lectins and the GSVD of the genes and lectins together. Whereas, the relationship was correlative for O-glycosylation, which was clearly illustrated in biplots. This work argues that types of glycosylation are regulated by different mechanisms in different cell types.Item Qualitative and Quantitative Analysis of Glycans Released from Model Glycoproteins and Pooled Human Blood Serum using MRM Mode and ESI LC-MS/MS(2013-05) DeSantos Garcia, Janie; Mechref, Yehia; Thompson, Jonathan E.; Pare, PaulProtein glycosylation is one of the most common posttranslational modifications (PTM). Aberrant glycosylation has been correlated with various mammalian diseases. Thus, qualitative and quantitative strategies are necessary to monitor glycan changes correlated with different biological conditions. One of the most effective quantitative strategies for reliable and sensitive monitoring of glycan changes is mass spectrometry (MS). However, development of reliable sample sample preparation methods and optimization of analysis conditions is required to monitor the changes associated with disease states. The 2nd chapter describes an enhanced sensitivity through miniaturized solid-phase permethylation and online solid-phase permethylation. Solid-phase permethylation method was miniaturized by reducing the amount of sodium hydroxide beads (one-third the original amount) packed in microspin columns. The efficiency of glycan permethylation was not adversely affected by this reduction. Online solid-phase purification of permethylated N-glycans derived from model glycoproteins, such as fetuin, a-1 acid glycoprotein and ribonuclease B, offered more sensitive and reproducible results than offline liquid–liquid and solid-phase extractions. The online solid-phase purification method described here permitted a 75% increase in signal intensities of permethylated glycans relative to offline purification methods. This is mainly due to the minimized sample handling associated with an online cleaning procedure. The efficiency and utility of online solid-phase purification was also demonstrated here for N-glycans derived from human blood serum. Online solid-phase purification permitted the detection of 73 N-glycan structures, while only 63 glycan structures were detected in the case of samples purified through liquid–liquid extraction. The intensities of the 63 structures that were detected in both cases were 75% higher for samples that were purified through the online method. vii The 3rd chapter introduces the quantification of N-linked glycans using multiple reaction monitoring (MRM) on a triple quadrupole instrument. The optimized collision energy (CE) for sialylated and fucosylated N-glycans was 35 ev and the CE for mannose, and complex type N-glycans was 30 ev. In addition to the optimized CE, the selection of 3 transitions was also established for reliable quantification. A total of 88 N-glycans in human blood serum were quantified using a multiplexed MRM approach with these optimized conditions. Reliable detection of these N-glycans measured at 0.005 μL indicates that N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, yielding a dynamic concentration range of 3 orders of magnitude in a single experiment. Also, MRM was used to identify the N-glycans differentially expressed in brain-targeting breast carcinoma cells (MDA-MB-231-Br) and was compared with MDA-MB-231 breast cancer cell line. Thus, MRM measurements provide a rapid and reliable identification and quantification of glycans that could be applied in biomarker discovery studies.