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University dissertation from Stockholm. KTH
Abstract: Interactions between carbohydrates and proteins are increasingly being recognized as crucial in many biological processes, such as cellular adhesion and communication. In order to investigate the interactions of carbohydrates and proteins, the development of efficient analytic technologies, as well as novel strategies for the synthesis of carbohydrates, have to be explored. To date, several methods have been exploited to analyze interactions of carbohydrates and proteins, for example, biosensors, nuclear magnetic resonance (NMR); enzyme-linked immunosorbent assays (ELISA), X-ray crystallography and array technologies. This thesis describes the development of novel strategies for the synthesis of carbohydrates, as well as new efficient strategies to Quartz Crystal Microbalance- (QCM-) biosensors and carbohydrate microarrays technologies. These methodologies have been used to probe carbohydrate-lectin-interactions for a range of plant and animal lectins.
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Humana Press; 1st Edition. edition (March 3, 2011) 386 pages ISBN-10: 1617790427
Protein microarrays have been used for a wide variety of important tasks, such as identifying protein-protein interactions, discovering disease biomarkers, identifying DNA-binding specificity by protein variants, and for characterization of the humoral immune response. In Protein Microarray for Disease Analysis: Methods and Protocols, expert researchers provide concise descriptions of the methodologies currently used to fabricate microarrays for the comprehensive analysis of proteins or responses to proteins that can be used to dissect human disease. These methodologies are the toolbox for revolutionizing drug development and cell-level biochemical understanding of human disease processes. Beginning with a section on protein-detecting analytical microarrays, the volume continues with sections covering antigen microarrays for immunoprofiling, protein function microarrays, the validation of candidate targets, proteomic libraries, as well as signal detection strategies and data analysis techniques. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.
Practical and cutting-edge, Protein Microarray for Disease Analysis: Methods and Protocols serves as a solid framework to aid scientists in understanding how protein microarray technology is presently developing and how it can be applied to transform our analysis of human disease.
Protein-Detecting Analytical Microarrays
Detecting and Quantifying Multiple Proteins in Clinical Samples in High-Throughput Using Antibody Microarrays
Analysis of Serum Protein Glycosylation with Antibody-Lectin Microarray for High-Throughput Biomarker Screening
Antibody Suspension Bead Arrays
Reverse Protein Arrays Applied to Host-Pathogen Interaction Studies
Identification and Optimization of DNA Aptamer Binding Regions Using DNA Microarrays
Recombinant Lectin Microarrays for Glycomic Analysis
Antigen Microarrays for Immunoprofiling
Recombinant Antigen Microarrays for Serum/Plasma Antibody Detection
SPOT Synthesis as a Tool to Study Protein-Protein Interactions
Native Antigen Fractionation Protein Microarrays for Biomarker Discovery
Immunoprofiling Using NAPPA Protein Microarrays
Protein Function Microarrays
High-Throughput Mammalian Two-Hybrid Screening for Protein-Protein Interactions Using Transfected Cell Arrays (CAPPIA)
Protein-Protein Interactions: An Application of Tus-Ter Mediated Protein Microarray System
Kinase Substrate Interactions
A Functional Protein Microarray Approach to Characterizing Posttranslational Modifications on Lysine Residues
Strategies for Validation of Candidate Targets
Multiplexed Detection of Antibodies Using Programmable Bead Arrays
A Co-Precipitation-Based Validation Methodology for Interactions Identified Using Protein Microarrays
Generation of Proteomic Libraries
Development of Expression-Ready Constructs for Generation of Proteomic Libraries
Reverse Phase Protein Microarrays: Fluorometric and Colorimetric Detection
Förster Resonance Energy Transfer Methods for Quantification of Protein-Protein Interactions on Microarrays
Label-Free Detection with Surface Plasmon Resonance Imaging
Data Analysis Techniques for Protein Function Microarrays
Data Processing and Analysis for Protein Microarrays
Database Resources for Proteomics-Based Analysis of Cancer
The cell surface is covered with a myriad of carbohydrates that form a complex matrix of oligosaccharides. Carbohydrate recognition plays critical roles in pathogenesis, trafficking, and differentiation. Lectin microarray technology presents a novel platform for the high-throughput analysis of these structurally diverse biopolymers. One drawback of this technology has been limitations imposed by the commercially available plant lectins used in the array. Not only are a majority of these plant-derived proteins glycosylated, which can complicate glycomic analysis, but they also differ in activity and availability. Our lab has recently introduced recombinant lectins to enhance the stability and scope of our lectin panel. Herein, we provide a detailed procedure for the expression of bacterially-derived lectins and their application to a recombinant lectin microarray.Affiliation
Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX, USA.
1. Mahal, L.K. Catching Bacteria with Sugar. Chem. & Biol. 2004. 11, 1602-1604.
2. Pilobello, K.T.; Krishnamoorthy, L.; Slawek, D.; Mahal, L.K. Development of a Lectin Microarray for the Rapid Analysis of Protein Glycopatterns. ChemBioChem2005. 6, 985-989.
3. Hsu, K.-L.; Pilobello, K.T; Mahal, L.K. Analyzing the dynamic bacterial glycome with a lectinmicroarray approach. Nature Chem. Biol.2006. 2, 153-157.
4. Sanki, A.; Mahal, L.K. A One-Step Synthesis of Azide-Tagged Carbohydrates: VersatileIntermediates for Glycotechnology. Synlett2006. 3, 455-459.
5. Hsu, K.-L.; Mahal, L. K. Profiling the sweet structures of the bacterial glycome. Nature Protocols. 2006. 1, 543-549.
6. Carrillo, L.D.; Krishnamoorthy, L.; Mahal, L.K. A Cellular FRET Sensor for β-O-GlcNAc, a Dynamic Carbohydrate Modification Involved in Signaling, J. Am. Chem. Soc.. 2006. 128, 14768-14769.
7. Pilobello, K.T.; Mahal, L.K. Deciphering the glycocode: the complexity and analytical challenge of glycomics, Curr. Opin. Chem. Biol.. 2007 11, 300-305.
8. Pilobello, K.T.; Slawek, D.; Mahal, L.K. A ratiometric lectin microarray approach to analysis of the dynamic mammalian glycome, Proc. Natl. Acad. Sci. USA,2007. 104, 10534-10539.
9. Pilobello, K.T.; Mahal, L.K. Lectin Microarrays for Glycoprotein Analysis, Methods Mol. Biol.. 2008. 385, 193-203.
10.Mahal, L.K. Glycomics: Towards Bioinformatic Approaches to Understanding Glycosylation, Anti-Cancer Agents Med. Chem.2008. 8, 37-51.
11.Hsu, K.-L.; Gildersleeve, J.C.; Mahal, L.K. A simple strategy for the creation of a recombinant lectin microarray, Mol. BioSystems. 2008. 4, 654-662.
12. Krishnamoorthy, L.; Bess, J.W.; Preston, A.B.; Nagashima, K.; Mahal, L.K. HIV-1 and microvesicles from T cells share a common glycome, arguing for a common origin, Nature Chem. Biol.2009 5, 244-250.
13. Hsu K.L.; Mahal L.K. Sweet tasting chips: microarray-based analysis of glycans. Curr Opin Chem Biol.2009. 13, 427-432.
14. Lebrilla C.B.; Mahal L.K. Post-translation modifications. Curr Opin Chem Biol.2009 13, 373-374.
15. Krishnamoorthy L.; Mahal L.K. Glycomic analysis: an array of technologies. ACS Chem Biol.2009. 4, 715-732.
16. Propheter, D.C.; Hsu, K.-L.; Mahal, L.K. Fabrication of an Oriented Lectin Microarray, ChemBioChem. 2010. 11, 1203-1207.
17. Hsu, KL; Pilobello, K.; Krishnamoorthy, L; Mahal, L.K. Ratiometric lectin micorarray analysis of the mammalian cell surface glycome. Methods Mol. Biol.2011. 671, 117-31.
18. Carillo, L.D.; Froemming, J.A.; Mahal, L.K. Targeted in Vivo O-GlcNAc Sensor Reveals Discrete Compartment-specific Dynamics During Signal Transduction. J. Biol. Chem.2011. 286, 6650-6658.
19. Krishnamoorthy, L.K.; Mahal, L.K. Lectin Microarrays: Simple Tools for the Analysis of Complex Glycans, chapter in Functional and Structural Proteomics of Glycoproteins, eds. Owens, R.J; Nettleship, J.E. 2011. Springer Verlag.
20. Propheter, D.C.; Mahal, L.K. Orientation of GST-tagged lectins via in situ surface modification to create an expanded lectin microarray for glycomic analysis. Mol. Biosystems2011. 7, 2114-7.
21. Rakus, J.F.; Mahal, L.K. New Technologies for Glycomic Analysis: Toward a Systematic Understanding of the Glycome. Ann. Rev. Anal. Chem. 2011. 4, 367-92.
22. Propheter, D.C.; Hsu, K.-L.; Mahal, L.K. Recombinant lectin microarrays for glycomic analysis. Methods Mol. Biol.2011. 723, 67-77.
23. Gaziel-Sovran, A; Segura, M.F.; Di Micco, R; Collins, M.K.; Hanniford, D.; Vega-Saenz de Miera, E.; Rakus, J.F.; Dankert, J.F.; Shang, S.; Kerbel, R.S.; Bhardwaju, N.; Yongzhao, S.; Darvishan, F.; Zavadil, J.; Erlebacher, A.; Mahal, L.K.; Osman, I.; Hernando, E. MiR-30b/30d regulation of GalNAc transferases enhances invasion and immunosuppression during metastasis. Cancer Cell2011. 20, 104-18.
24. Batista, B.S.; Eng, W.S.; Pilobello, K.T.; Hendricks-Munoz, K.; Mahal, L.K. Identification of a Conserved Glycan SIgnature for Microvesicles, J. Proteome Res.2011. 10. 4624-33.
25. Reuel, N.F.; Ahn, J.-H.; Kim, J.-H.; Zhang, J.; Boghossian, A.A.; Mahal, L.K. ; Strano, M.S. Transduction of Glycan␣Lectin Binding Using Near-Infrared Fluorescent Single-Walled Carbon Nanotubes for Glycan Profiling. J. Am. Chem. Soc.,2011. 133. 17923-33.
26. Bird-Lieberman, E.L.; Neves, A.A.; Lao-Sirieix, P.; O’Donovan, M.; Novelli, M.; Lovat, L.B.; Eng, W.S.; Mahal, L.K.; Brindle, K.M.; Fitzgerald, R.C. Molecular imaging using fluorescent lectins permits rapid endoscopic identification of dysplasia in Barrett’s esophagus. Nat. Medicine,2012. 18, 315-21.
27. Pilobello, K.T; Agrawal, P.; Rouse, R.; Mahal, L.K. Advances in lectin microarray technology: Optimized protocols for piezoelectric print conditions. Curr. Prot. Chem. Biol.2013,5, 1-23.
28. Ribeiro, J.P.; Mahal, L.K. Dot by dot: analyzing the glycome using lectin microarrays. Curr. Opin. Chem. Biol.2013. 17. 827-31.
29. Kasper, B.T.; Koppolu, S.; Mahal, L.K. Insights into MiRNA Regulation of the Human Glycome. Biochem. Biophys. Res. Commun.2014, doi: 10.1016/j.bbrc.2014.01.034, in press.
30. Agrawal, P.; Kurcon, T.; Pilobello, K.T.; Rakus, J.F.; Koppolu, S.; Liu, Z.; Batista, B.S.; Eng, W.S. Hsu, K.-L.; Liang, Y.; Mahal, L.K. Mapping posttranscriptional regulation of the human glycome uncovers microRNA defining the glycocode. Proc. Natl. Acad. Sci. USA,2014. PMID 24591635 in press .
31. Wang, L.; Cummings, R.D.; Smith, D.F.; Huflejt, M.; Campbell, C.T.; Gildersleeve, J.D.; Gerlach, J.Q.; Kilcoyne, M.; Joshi, L.; Serna, S.; Reichardt, N.-C.; Pera, N.P.; Pieters, R.; Eng, W.S.; Mahal, L.K. Cross-Platform Comparison of Glycan Microarray Formats. 2014Glycobiology, in Press.
Markiv, Anatoliy. Peiris, Diluka. Curley, G. Paul. Odell, Mark and Dwek, Miriam (2011) Identification, cloning and characterization of two N-acetylgalactosamine binding lectins from the albumen gland of Helix pomatia. Journal of Biological Chemistry, 286 (23). pp. 20260-20266. ISSN 0021-9258
Full text not available from this repository.Abstract
Helix pomatia agglutinin (HPA), the lectin from the albumen gland of the Roman snail, has been used in histochemical studies relating glycosylation changes to the metastatic potential of solid tumors. To facilitate the use of HPA in a clinical (diagnostic) setting, detailed analysis of the lectin, including cloning and recombinant production of HPA is required. A combination of isoelectric focusing, amino acid sequence analysis and cloning revealed two polypeptides in native HPA preparations (HPAI and HPAII) both consistent with GalNAc binding lectins of the H-type family. Pairwise sequence alignment showed that HPAI and HPAII share 54% sequence identity while molecular modelling using SWISS-MODEL suggest they are likely to adopt similar tertiary structure. The inherent heterogeneity of native HPA highlighted the need for production of functional recombinant protein; this was addressed by preparing His-Trx tagged fusion products in Escherichia coli Rosetta-gami B (DE3) cells. The recombinant lectins agglutinated human blood group A erythrocytes while their oligosaccharide specificity, evaluated using glycan microarrays, showed they predominantly bind glycans with terminal alpha-GalNAc residues. Surface plasmon resonance with immobilized GalNAc-BSA confirmed that recombinant HPAI and HPAII bind strongly with this ligand (Kd = 0.60 nM and 2.00 nM, respectively) with a somewhat higher affinity to native HPA (Kd = 7.67 nM). Recombinant HPAII also bound the breast cancer cells of breast cancer tissue specimens in a similar manner to native lectin. The recombinant HPA described here shows important potential for future studies of cancer cell glycosylation and as a reagent for cancer prognostication.
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.Department URI Collections