Transformative Technologies in Glycomics Workshop

In November 2013, the Consortium for Functional Glycomics (CFG), an international body of researchers seeking to understand the functions of glycans and glycan-binding proteins (GBPs) that impact human health and disease, held a workshop entitled “Development and Applications of Transformative Technologies in Glycobiology” immediately preceding the Annual Meeting of the Society for Glycobiology. The workshop was organized by Christine Szymanski and Brian Cobb and was funded by the National Institute of General Medical Sciences (NIGMS) through a U13 grant (Michael Tiemeyer and James Prestegard, Principal Investigators) that provides support to the CFG working groups for identifying and addressing cross cutting issues facing the field, in order to “promote problem solving,” and develop approaches/solutions that move the field forward. Richard Cummings, the current CFG Director, opened the workshop with a status report. The CFG continues to grow, even after the termination of the original NIGMS glue grant program that it was developed under. Now numbering over 600 members, the CFG is funded through R24 and P41 grants from NIGMS, and remains active through the work of its steering committee, the glycan array core at Emory, the informatics core at MIT, and through the NIGMS U13 meeting grant which supports its subgroup activities and fosters growth in the field of glycomics and glycosciences in general. The workshop focused on the continued evolution of technologies such as glycan arrays, for deciphering the role of glycans, and their binding partners in normal and disease processes, and attracted 88 attendees representing 11 countries, including 8 invited speakers, 4 speakers selected from submitted abstracts, and 27 poster presenters. Two highlighted presentations were provided by glycoscience trainees. Overall, the workshop was a successful exposition of the current state of the art in glycomics research and transformative technologies and it illuminated the needs for additional technology development. The morning session primarily focused on the development and use of glycan arrays, pioneered by the CFG, to interrogate the glycan binding properties of viruses, bacteria, and antibodies that arise in response to infection, inflammation, or autoimmunity. Jim Paulson (CFG Director Emeritus) began the workshop by highlighting several arrays now either under development or in use at the Scripps Research Institute. These include sialoside and sialoside-analog arrays which have been used as a platform to screen influenza virus binding specificities – a key to understanding viral tropism. Paulson also highlighted the expansion of a microbial polysaccharide array that is now offered as a resource through the CFG portal. He then moved into a discussion about the history and his current work on the importance of the hemagglutinin (HA) and neuraminidase classification in viral transmission, and how adaptation from avian sources to humans involves the transition from preferentially binding α2,3-linked to α2,6-linked sialic acids. Following this discussion, Gillian Air presented her work on the antigenic drift seen in flu viruses over the last several decades, particularly in the H3 family. One key change has been the gradual but consistent increase in the number of glycans that decorate the HA protein. Using glycan array technology, it was also found that the binding of HA to host glycans is directly impacted by the length of N-acetyllactosamine (poly-LacNAc) repeats. More specifically, most of the viruses tested bound to long poly-LacNAc containing glycans, whereas only subsets of viruses bound short poly-LacNAc structures. Over time, the genetic drift in HA has yielded a greater tendency to preferentially bind long poly-LacNAc, a tendency that may reflect selective pressure on the virus to increase binding to human airway tissue. Evidence also supports the notion that the humoral response to flu inflection is the driving evolutionary force behind the observed changes in binding specificity. Completing the viral discussion, Sasirekha Ramani presented findings on the role of host glycans as attachment factors for rotoviruses (RV) and noroviruses. The traditional view is that rotavirus binds terminal or internal sialic acids, thereby resulting in classification of strains as sialidase sensitive or sialidase insensitive. However, recent studies have challenged this notion. Ramani used an RV strain with an unusual tropism for neonates and demonstrated that RVs exhibit strain-dependent glycan specificity. Indeed, glycan array experiments revealed significant binding to LacNAc repeats, a hitherto unsuspected class of non-sialoglycans. Binding was also seen to certain human milk oligosaccharides (HMOs) suggesting a role for HMOs in susceptibility to neonatal rotavirus infections. Stem cell adherence and differentiation has historically been a significant barrier to the development of replacement tissues for the clinic. Kevin Yarema discussed the use of glycoengineering to solve this problem by taking advantage of the adhesive potential of glycans at the cell surface. More specifically, using the lessons learned through the Click Chemistry platform, unnatural saccharides carrying thiol groups were introduced into stem cell surfaces to provide binding to gold-based scaffolds. Not only can this greatly enhance cell adherence, but it also lead to the discovery that attachment chemistry directly impacts cell fate such that stem cells develop into the target tissue with a vastly greater efficiency. Specific examples of applying this technology included adipogenic and neuronal differentiation of stem cells, highlighting the potential behind using cellular glycans for manipulating stem cell fate in tissue engineering. The two abstract presentations in the first session included a study described by Sean Ryan in which a conditional knockout of the mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat2) locus in mice lead to an immunodeficiency through the generation of autoantibodies in response to abnormally glycosylated erythrocytes. CFG glycan array analysis of these antibodies revealed that the change in erythrocyte glycosylation seems to uncover a cryptic non-glycan epitope that is normally present on lymphocytes. The second short presentation by Jamie Heimburg-Molinaro described the development of a glycopeptide array carrying divergent peptides decorated with the Tn antigen (GalNAc-alpha-1-O-serine/threonine-), an epitope present on the immunodominant glycoprotein from Cryptosporidium parvum, an important parasite causing diarrheal disease in humans. Using this array, patient sera was screened for anti-Tn antigen reactivity, revealing that parasite carriers generate anti-Tn antibodies, which could enable the use of this novel array as a diagnostic test for infection. The afternoon session had a greater focus on technology development. Todd Lowary described studies on the immunoactive lipoarabinomannan (LAM) of Mycobacteria. Antibodies generated against this glycolipid have been shown to be protective in patients infected with M. tuberculosis. Using synthetic LAM fragments, Lowary examined interactions of the carbohydrates with monoclonal antibodies through X-ray crystallography, mutations in the putative binding site, and new mass spectrometry methods developed by John Klassen to identify key features of the epitopes that could be used for the development of anti-mycobacterial vaccines. Geert-Jan Boons described a novel bio-orthogonal chemical reporter strategy taking advantage of the specificity of beta-galactoside alpha 2,6-sialyltransferase (ST6GalI), an enzyme that adds sialic acids to N-linked oligosaccharides of glycoproteins, to specifically label N-glycoproteins with azido-modified sialic acid on living cells. He called this method SEEL, or Selective Exo-Enzymatic Labeling, and described its application in studying trafficking and storage diseases. The next four talks described exciting new mass spectrometry-based approaches for examining glycoproteins. Bernd Meyer coupled NMR and MS in a 3DCC (cross-correlation) technique showing that NMR spectra of pure compounds can be obtained following liquid chromatography coupled to electrospray ionization mass spectrometry without ever isolating a pure compound. This top-down approach allowed them to characterize pmol quantities of isobaric mixtures. They also compared prostrate specific antigen from several donors and showed that protein glycosylation and phosphorylation patterns are correlated with age and occupation. Ron Orlando also described LC-MS/MS methods for resolving linkage isomers by improving the chromatographic separation and using selected reaction monitoring (SRM) to examine samples in the lower fmol limit of detection. Orlando uses hydrophilic interaction liquid chromatography (HILIC), but with superficially porous particles with fused cores. He then links several columns in tandem to provide greater separation with less back pressure. The two abstract talks in the second session were given by Thomas Powers and Christina Dobson (the latter presented by Lance Wells) and described two very different mass spectrometry methods for glycoprotein analyses. Powers described the use of matrix-assisted laser desorption ionization IMAGING mass spectrometry (MALDI-IMS) to profile N-glycosylation patterns directly from tissues. When this method was combined with off-tissue extraction and analysis, the group could provide a snapshot of the major glycans, their localization and distribution, structure and relative abundance. This method identified glycans specific to cancer tissue types and could be used for tumor diagnosis. Lance Wells described an intelligent consecutive reaction monitoring (iCRM) technique for profiling O-linked glycoproteins. This technique could be used to develop a blood test for dystroglycanopathies, which currently require muscle biopsies, useful for diagnosis or for assessing therapeutic efficacy. The speakers and poster presenters in the CFG workshop highlighted the use of several exciting glycomics technologies that have been developed in recent years. These technologies have direct and indirect applications for human health and to the understanding of key biological processes. Emphasized in the closing remarks by Pamela Marino, from the NIGMS, the workshop highlighted new opportunities for the advancement and application of glycomics research to science and health. The availability of these methods will not only advance the glycosciences, but has the potential to advance all areas of life sciences where glycans are likely involved. The abstracts for all the workshop talks and posters are posted on the CFG website at: www.functionalglycomics.org.

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