Lecture Summaries

WEEK # TOPICS LECTURE SUMMARIES
1 Introduction
  • Introduction of instructors and students
  • Discuss course overview and objectives
  • Introduction to PubMed and other search engines
  • Overview of paper layouts
  • Introduction to next week's topic

Optional

You can familiarize yourself with some basic aspects of glycobiology by watching a couple of video's on iBiology.org.

Carolyn Bertozzi. "Chemical Glycobiology." iBiology. Accessed November 19, 2014. http://www.ibiology.org/ibioseminars/biophysics-chemical-biology/carolyn-bertozzi-part-1.html

TEDx. "Knowledge of glycobiology can improve your health: Geiske de Ruig at TEDxRoermond." April 3, 2013. TEDx. Accessed November 24, 2014. http://tedxtalks.ted.com/video/Knowledge-of-glycobiology-can-i
2 Aberrant protein glycosylation in human disease

Glycoproteins account for approximately 50% of the total proteins in a human cell and are critical for human health. Mutations in genes that encode the glycosylation machinery can lead to various congenital disorders of glycosylation (CDGs). This week students will be introduced to asparagine (N)-linked glycosylation, a protein modification in which asparagine residues within a protein are sitespecifically modified with a glycan. The paper by Losfeld et al. demonstrates the development and application of a glycosylation-sensitive reporter used to study basic molecular pathways involved in CDGs and that can be used to screen for drugs that could mitigate the negative consequences CDGs has on human development. Next we will discuss the paper by Chu et al., which examines the effects of CDGs on development phenotypes in zebrafish as a model organism. The authors also explore the efficacy of mannose treatment, which is currently used to treat humans with this form of CDGs, in rescuing abnormal phenotypes caused by the disease. We will learn about methods used to analyze the glycosylated state of proteins using western blot analysis and in vivo light and fluorescence microscopy, as well as enzymatic activity assays and genetic techniques. The goal of this class is to understand the basic process of N-linked glycosylation and learn about several methods used to analyze it in vitro and in vivo.

Optional

Visit Hudson Freeze's website to read about his passion for his research and watch a video about his work.

Learn about how morpholinos are injected into zebrafish embryos:

Yuan, S., Sun, Z. "Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos." Journal of Visualized Experiments (27), e1113, doi:10.3791/1113 (2009).
3 Imaging glycan patterns in developing embryos

Glycosylation during animal development is a complex process that occurs in a spatially- and temporally-controlled manner. Identifying changes in glycosylation patterns is important to determine how deviations from these patterns result in developmental abnormalities. Observing these changes in real time—in whole cells and whole animals—is possible through bioorthogonal (bio-compatible) metabolic labeling methods, which exploit unnatural sugar mimics that can be coupled to fluorescent probes. Major advantages of this technique are that it is noninvasive and has low cross-reactivity because of the use of non-biological chemical groups. Using the two papers this week, we will discuss the rationale for designing these unnatural sugars and new findings from their application in zebrafish. Baskin et al. demonstrate the incorporation of unnatural sugars into mucin-type O-glycans, which participate in key cellular processes, including adhesion and migration. Using time-lapse microscopy, the authors elegantly show the dynamic incorporation and turnover of these glycans during embryonic development. The second paper, by Beahm et al., demonstrates the application of unnatural glycans to study the biosynthesis of glycosaminoglycans, which has glycan modifications on the cellular matrix. The use of these powerful chemical biology approaches to study biosynthetic pathways in vivo complements classical genetic techniques and broadens our understanding of these processes.

Optional

Watch a video from Carolyn Bertozzi, the guru of glycan imaging and metabolic labeling, explaining the ins and outs of glycome imaging from iBiology.org.

Carolyn Bertozzi. "Imaging the Glycome." iBiology. Accessed November 19, 2014. http://www.ibiology.org/ibioseminars/biophysics-chemical-biology/carolyn-bertozzi-part-2.html
4 The role of protein glycosylation in bacterial motility

In addition to eukaryotes, bacteria are also capable of protein glycosylation. Bacterial protein modifications are found on only a specific set of proteins in the cell, many of which are cell-surface exposed. Campylobacter jejuni is a human pathogen that uses an O-linked glycosylation pathway in which serine or threonine are site-specifically modified with glycans. This modification is found on the flagellum—an extracellular tail-like structure involved in motility and required for pathogenesis. This week we will learn about how the role of O-linked glycosylation in C. jejuni has been explored using 5 various approaches. We will discuss the paper by Ewing et al. in which bacterial genetics and fluorescence microscopy were used to determine the function and cellular localization of enzymes that participate in O-linked glycosylation of flagella. The authors found that mutations of flagella glycosylation sites abolishes flagella assembly and impairs motility. Next we will examine work by Liu et al. wherein a metabolic labeling method was developed for bacteria using bioorthogonal chemistry techniques that were discussed in last week's class. This labeling method offers a new tool for studying various aspects of O-linked glycosylation of flagella, including biophysical properties, antigen presentation and in vivo visualization during infections.

Optional

Learn about bacterial motility by watching some videos of different motile species.

jameskguy. "Bacterial Flagellum - Evolution's Nightmare & Demise." October 8, 2006. YouTube. Accessed November 19, 2014.  https://www.youtube.com/watch?v=0N09BIEzDlI

microfetish's channel. "Bacterial flagellar motility." December 14, 2009. YouTube. Accessed November 19, 2014. https://www.youtube.com/watch?v=4hexn-DtSt4
5 Engineering bacteria for glycoprotein production

Escherichia coli is a bacterial species that has become an important tool not only in research but also in biotechnology, as it can be engineered for robust production of recombinant proteins. However, E. coli does not harbor a native protein glycosylation system, which has impeded its use in the production of many protein-based therapeutics that require glycan modifications to be fully active. Because of the ease of engineering E. coli, recent efforts have been made towards the production of glycoproteins in this bacterium. This week we will discuss the seminal paper by Wacker et al. that demonstrates, through simple genetic manipulation, the bioengineering of a C. jejuni glycosylation system in E. coli. This work formed the basis for the second paper by Valderrama-Rincon et al., who advanced the system to generate eukaryotic glycoproteins in E. coli. Biochemical techniques used to probe for glycosylation, including mass spectrometry and flow cytometry, will be discussed.
6 Chemical strategies for glycoprotein production

Post-translational glycosylation of proteins is not genetically encoded, and the attachment of glycans is driven by the dynamic interplay between the protein translation and glycosylation machineries. As a result, glycoproteins are highly heterogeneous with respect to site occupancy. The isolation of pure glycoprotein is severely hampered by this diversity, and this limitation, in turn, has inspired chemists to develop new methods to generate well-defined glycoproteins. This week we will discuss two examples of methods to produce pure glycoproteins and how the resulting material was used to study glycoprotein interactions in inflammation. In the first paper by Zhang and co-workers, a controversial method is described that tricks the translation machinery of E. coli to accept unnatural glycosyl-amino acids. 6 Although this paper has been retracted for suspected research misconduct, the brilliant design of the method warrants its discussion. The second paper by Van Kasteren et al. describes a reliable approach to produce homogenous glycoproteins using two different chemical reactions. In this way a synthetic Pselectin ligand was generated that was used to visualize sites of inflammation. We will learn about new biochemical techniques, such as the incorporation of unnatural amino acids using amber codon suppression and methionine-auxotrophic expression, while revisiting chemical ligation strategies discussed earlier.

Optional

Read a blog about the Science paper retraction, and the research paper that fueled the retraction.

Odyssey. "You lost WHAT?!?!?!?!" Pondering blather blog. December 1, 2009.

Antonczak, A.K., Z. Simova, and E.M. Tippmann. "A critical examination of Escherichia coli esterase activity." J. Biol. Chem. 284, no. 42(2009): 28795-800.
 
7 Development and application of glycan arrays

Array-based platforms have emerged as a quick and easy way to identify interactions between biomolecules on a surface and in solution in a high-throughput manner. In this way, hundreds of different glycans can simultaneously be profiled for their binding specificities to a diverse range of glycan-binding proteins. In the paper by Blixt and colleagues, the experimental development of a glycan array is described and optimized. Also, a proof-of-concept is provided for the detection of many specific interactions between glycans and lectins, antibodies and proteins. The second paper, by Hung et al., deals with the application of a glycan array to detect the unknown receptor of a common malignant tumor marker glycolipid. The receptor was successfully identified using affinity capture and mass spectrometry, and subsequently probed for its specificity to the tumor marker using a glycan array.

Optional Field Trip to the Boston Glycobiology Discussion Group: Students will have the opportunity to attend a seminar organized by the Boston Glycobiology Discussion Group. The group is situated at Boston College and is comprised of individuals interested in all areas of glycobiology, both from academia and industry. We will have the opportunity to choose from one of the following lectures: Bernd Stahl, an expert on human milk oligosaccharides and prebiotics, from Danone Research (food industry). Charles Dimitroff, an expert on glyco-immunology, from Harvard Medical School.

Optional

Watch a video from the Journal of Visualized Experiments (JoVE) on the application of a glycan array.

Moller, I. E., F.A. Pettolino, et al. "Glycan Profiling of Plant Cell Wall Polymers using Microarrays." J. Vis. Exp. (70), e4238, doi:10.3791/4238 (2012).
8 Metabolic glycan labeling in cancer

The glycosylation of cell-membrane proteins is important for cell communication, proliferation and migration and is often altered in diseases such as cancer. Understanding the glycosylation patterns of cell-membrane proteins both in healthy and diseased states might provide insight into developing new ways to combat cancer by targeting tumor-specific glycans. Using the first paper by Neves et al., we will discuss the application of two consecutive chemical labeling methods to visualize cell-membrane glycoproteins expressed by tumor cells. The novelty of this two-step method lies in the reduction of background labeling to improve the specific detection of a tumor in a live mouse model. The second paper, by Furumoto et al., describes the advancement of a hallmark method to detect tumors based on radiolabeled glucose using Positron Emission Tomography (PET). These authors generated radiolabeled mannose and investigated its uptake, metabolism and biodistribution as compared to radiolabeled glucose using tumor models in vitro and in rats.

Optional

Watch a 40–min lecture on the use of 18F-radiolabeled glucose (FDG) to increase your understanding of the use of FDG in the clinic.

Ken Rikard-Bell. "November 8, 2012: FDG PET - A new paradigm for therapy monitoring in Oncology." February 6, 2013. YouTube. Accessed November 19, 2014. https://www.youtube.com/watch?v=Q6W-XI95Vmw
9 Glycan-based synthetic vaccines

The glycans displayed by pathogens and the abnormal glycosylation patterns of cancer cells form the basis of this week's topic. Ever since it was discovered that non-human glycans from bacteria and viruses can trigger the immune system and cause disease, researchers have tried to use glycans as antigenic epitopes in vaccine development. From the first paper this week, by Broecker et al., we will learn about some important steps in the development of a vaccine against Yersinia pestis, the causative agent of the plague. Topics will include the optimization of glycan-antibody interactions and how to determine the specific epitope from a bacterial antigen. Using the paper by Lakshminarayanan et al., we will discuss the development of a glycan-based vaccine by combining three critical components: an adjuvant to stimulate the immune system, a peptide Thelper epitope to facilitate antigen presentation, and a glycosylated peptide as an antigen. Interestingly, the authors found that the components had to be chemically linked together to elicit an immune response. In this class, we will revisit glycan arrays and learn about surface plasmon resonance, nuclear magnetic resonance and mice immunization studies.
10 A close look at influenza–host interactions

One of the major targets for vaccine development against influenza viruses is hemagglutanin (HA), a surface-exposed viral envelope protein that is prone to high antigenic variation. HA interacts directly with sialic acid-containing receptors present on the surfaces of host cells. As such, it is involved in the specificity of influenza for different hosts (such as humans, birds, or swines). Interestingly, point mutations in HA can alter binding preferences for sialic acid, and thus alter host specificity. This week we will go over the basics of X-ray crystallography and how these data have been useful for understanding influenza host specificity through our examination of the paper by Zhang et al. This work is based on a previous highly controversial study in which the authors mutated an avian influenza virus strain that ultimately resulted in mammal-to-mammal transmission. A debate was ignited because of concerns over the possibility that a highly transmissible strain could escape from the lab and cause a pandemic. We will consider both sides of this controversial research in a brief conversation. Next we will review one of the many efforts that have been made toward the development of anti-viral therapies, reported in a paper by Connaris et al. The authors use protein engineering to develop protein-based therapeutics that contain multiple sialic acid binding sites and test their abilities to inhibit viral infection using a mouse model.

Optional

Read more about the biosafety ethical discussion on a science blog.

Nature News Special: Mutant flu. Nature.com
11 Synthetic antigens to generate HIV vaccines

Glycoproteins found on the surface of HIV are densely glycosylated with high-mannose glycans. Since the isolation of broadly-neutralizing antibodies against these glycoproteins, it has been a scientific quest to make a synthetic vaccine that can generate equally potent antibodies. The high number of glycans on the viral envelope glycoprotein has especially attracted a lot of interest, and the paper by Wang and co-workers describes an effort to mimic the dense glycan shield. The authors create multivalent structures called dendrimers and investigate their abilities to interact with lectins found on dendritic cells, antigen-presenting cells that are important gatekeepers for infection. The second paper, by Doores et al., describes the development of synthetic antigens to generate more powerful vaccines. Inspiration is taken from the natural glycans found on the viral envelope, and they are modified in such a way that stronger binding of the antibody is obtained. During this class we will discuss chemical and biological strategies to generate HIV-inspired antigenic molecules and their effectiveness in generating an immune response. Other techniques used in these papers include X-ray crystallography, immunoprecipitation, and rabbit immunization studies.

Optional

Watch a video on the HIV life cycle:

kleptoplast. "HIV life cycle: How HIV infects a cell and replicates itself using reverse transcriptase." January 6, 2012. YouTube. Accessed November 19, 2014. https://www.youtube.com/watch?v=odRyv7V8LAE
12 The role of sugar metabolites in diabetes

The attachment and removal of N-acetylglucosamine (GlcNAc) to serine or threonine is a highly dynamic process, and this modification is found on numerous proteins both in the nucleus and in the cytosol. O-GlcNAc-glycosylation is shown to be sensitive to nutrients such as glucose, and it has been linked to the development of diabetes. This week we will use the paper by Clark and colleagues to discuss an advanced method to detect O-GlcNAc-glycosylation using fluorescent tags. The authors invented a transferase enzyme to specifically label O-GlcNAc and thereby facilitate the facile identification of modified proteins, which would otherwise have remained unknown. In the second paper by Wang et al., this method is applied to study and compare O-GlcNAc levels on proteins in both healthy and diabetic individuals. This research tries to identify glycoproteins that are specifically found in diabetes so that they can be used as biomarkers for disease.

Optional

Watch a TED talk video by Peter Attia, a former general surgeon that recently took an interest in obesity-related diseases, in which he shares his opinion on diabetes.

TED. "Peter Attia: What if we're wrong about diabetes?" June 25, 2013. YouTube. Accessed November 19, 2014. https://www.youtube.com/watch?v=UMhLBPPtlrY
13 Presentations This week will be devoted to the students' presentations. Each student will make a 15–minute presentation based on a preselected paper. The students should be prepared to answer questions during the presentation, and the entire group will engage in a discussion based on the presentations. See p. 2 for more details. During this class we will reflect on key topics we learned about over the semester. Finally, we will ask for your impressions of the course and hand out course evaluation forms.