Chemical Glyco-Biology Laboratory Carbohydrates (glycans) are one of the major post-translational modifications of proteins (>80% of all proteins) and they are involved in a broad range of biological processes, including intercellular adhesion, signaling and microbial attachment. Glycans are directly involved in the pathophysiology of every major disease and are increasingly important in pharmaceutical developments. The Human Genome Project and the transformation of genomics was initiated by the research community beyond any technical capability available at the time, but succeeded largely due to the tools that were developed. Today, the glycoscience field is at a similar place. Therefore, development of new glycotechnology tools for detection, imaging, and biosynthesis of glycans is a prime interest in our laboratory.

We are very inspired by many of our collaborators who are the real experts within their fields (eg. Virology, Oncology, Epidemiology) and large part of our synthetic and method developments are done in consensus with them. The sugars around you……

http://www.youtube.com/watch?v=DHCpbEZ3kEg&feature=youtu.be

Several MSc projects are available related to activities presented below.

If you are interested in any of these activities, please contact olablixt@chem.ku.dk

RESEARCH ACTIVITIES Viral and bacterial receptor host interactions. Most viral and bacterial interactions with their hosts are influenced by glycans and glycan-binding receptors. A strategy targeting these interactions could provide efficient ways to detect, prevent and treat for example viral infections. Whereas close to all bacterial infections can be treated with common antibiotics, only a very few infections can be treated with antiviral drugs. So far, no antiviral drugs are available for treatment of common viral infections such as adeno- or enteroviruses. Very few drugs (neuraminidase inhibitors such as Tamiflu and Relenza) are available for treatment of infections caused by the globally widespread influenza A, B and C viruses but with limited efficacy.

We are developing tools to study viral and bacterial glycan binding receptors such as:

Identification of virus-binding glycans via glycan-, glycolipid-, and glyco-peptide/mucin microarrays (GlycoChip).

Synthetic analogs for identification of high affinity ligands.

Development of multivalent probes for detection and inhibition.

Avianadapted Humanadapted

SA2,3 SA2,6

UniversalSA2,3/6Inhibitorsandprobes

Glyco-Mucin

Glyconano-probesandar ficialRBCs

Au

HiaffinityGlyco-analoginhibitors

Influenza(Avian/Human)EnterovirusesAdenoviruses

Virustargets

RBC

Relevant publications: o Blixt, O., Head, S. et al (2004) Printed covalent glycan array for ligand

profiling of diverse glycan binding proteins. PNAS 101(49): 17033-17038. o Stevens J., Blixt O, et al. (2006) Glycan microarray technologies: tools to

survey host specificity of influenza viruses. Nat Rev. (2006) Microbiol. 4, 857-64.

o Blixt O., Razi N. Chemoenzymatic synthesis of glycan libraries. Methods Enzymol. (2006) 415, 137-53.

o Blixt, O., Han, S. et al. (2008) Sialoside analog arrays for rapid identification of high affinity siglec ligands J Am Chem Soc. 130, 21, 6680-1.

o Chen LM., Blixt O., et al. (2012) In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity. Virology. 422,105-13.

o Park, S., Gildersleeve et al, (2013) Carbohydrate Microarrays, Chemical Society Reviews 42, 4310-26.

Serological Fingerprinting of the Viral O-glycoproteome Viral envelope proteins and bacterial outer-membrane proteins are extensively glycosylated and known to serve important functions such as in host-cell interactions and in masking host immunity to these proteins. However, the viral infectious cycle in cells may also lead to aberrant glycosylation that may elicit immunity. Our knowledge of immunity to aberrant viral glycoproteins is limited, potentially due to technical limitations in identifying immunogenic O-glycopeptide epitopes (OGEs). We recently demonstrated in a proof-of-principle study that serum IgG reactivity to OGEs may be a highly significant phenomenon. We are currently expanding this concept by investigating other important viruses such as Herpes Simplex, Varicella, Tick-borne encephalitis and many more.

The phenomenon of viral OGE, as presented here, may have several important medical implications, of which two appear to be of immediate significance. (i) Firstly, recombinant or synthetic vaccines are widely acclaimed, not least because of the success of the rotavirus and human papilloma virus vaccines, based on virus-like particles. When it comes to enveloped viruses, the recombinant approach may be hampered by the fact that the glycosylation status

of the cell line used for vaccine production restricts the range and pattern of glycan structures associated with a viral glycoprotein. By applying our approach for vOGE identification and characterization in analysis of a large number of sera from patients that have recovered from a specific virus or bacterial disease, it should be possible not only to map but also structurally characterize important neutralizing targets of vOGE nature. (ii) Secondly, there is now a growing insight that viral infection of a human in most cases involves several types of cells and that the pattern of cell types addressed by a virus within certain limits may vary from individual to individual. The balance of particular GalNAc transferases in any tissue infected by a virus determines the pattern of viral vOGE presented to the immune response in the infected individual, likely to result in unique “fingerprint” IgG responses in patient sera. It is therefore possible that vOGE serology may be a complement to traditional serology. OGE-specific cancer biomarkers and antibodies

Aberrant glycosylation of proteins in cancer is a relative well-known phenomenon. However, identification of these changes is not trivial especially as glycosylation is very widespread and present on nearly all proteins produced by the cell including normal healthy tissues. Nevertheless, there are different strategies to detect such changes (i) by measuring the immune response to specific glyco-peptide epitopes (OGEs) or by (ii) qualitative or quantitative measurement of the disease specific protein harboring the specific glycan of interests. Measuring the immune response in terms of autoantibodies may enable detection of disease at an early stage. In contrast, detection of the antigens often require significant tumor burden (advanced stage) to accumulate sufficient material for analysis. We are working on both strategies. Along the lines of mapping viral OEGs, we can also target specific tumor proteins searching for autoantibodies to human OGEs. One prominent glycoprotein, Mucin-1 (MUC1, CA15-3) is over expressed by many different cancers. Previously we identified several hOGEs to MUC1 and collectively they were shown to be associated with a better prognosis in early stage breast cancer patients. We also developed an antibody capture microarray to identify glycosylation changes on the tumor antigens CA15-3 and CA125 (MUC16) in ovarian cancer patients.

A third strategy (iii) to detect cancer specific OGEs is with monoclonal antibodies. It is possible to create a mAb specific to almost any extracellular/ cell surface target. However, it is not trivial to make them disease specific differentiating ‘self’ from ‘non-self’. Thus, targeting aberrant OGEs with a plethora

self or non-self protein

Autoantibodies

Normal PTG Disease PTG

of disease specific mAb binders specifically targeting the disease cells and not the healthy cells expressing normal glycosylation, better diagnostic and prophylactic therapies should be possible. A powerful approach towards therapeutic antibodies is to create a library of antibody-genes. This library can be expressed as scFv antibody fragments on phagemids and be selected onto any format including cells, proteins, peptides or glycans.

The origin of natural anti-carbohydrate antibody repertoire It is well known that blood of humans and mammals contains natural antibodies and many of them directed to the glycan epitope without any influence from the adjacent peptide. This appearance is not associated with previous immunization. Being a part of innate immunity natural antibodies are the first defense against bacteria and viruses as well as constantly occurring transformed cells. Extreme diversity in specificity of human natural anti-carbohydrate antibodies (nAbs) was shown earlier using GlycoChip [Blixt et al 2004, PNAS]. Nevertheless, origin and functions and principle of pattern formation of these Abs are still unclear. “Bacterial paradigm” proposed by Springer [2] postulating that presence of nAbs is a result of immune system stimulation by bacterial O-polysaccharides during first months of life is also disputable. We are also interested in understanding the origin of such serum antibodies and are utilizing various glycan and bacterial polysaccharide arrays to understand this phenomena.

Related OGE activities includes:

Chemical synthesis of peptide and glycopeptides using automated Solid-Phase-Peptide-Synthesis (SPPS).

Peptide and glycopeptide bead libraries.

Enzymatic synthesis of glycopeptides using recombinant glycosyltransferases.

Microarray printing and serum analysis (infectious diseases or cancer).

Antibody microarrays for glycoprofiling

ScFv expressing Phage-display libraries.

Characterization and epitope mapping of monoclonal antibodies.

Luminex based assays.

Relevant references:

o Blixt O, Cló E, et al. (2010) A high-throughput O-glycopeptide discovery platform for seromic profiling. J Proteome Res. 9, 5250-61.

o Kracun S, Cló E, et al. (2010) Random Glycopeptide Bead Libraries for Seromic Biomarker Discovery. J. Proteome Res. 9, 6705-14.

o Cló, E., Kracun, S. et al. (2012) Characterization of the viral O-glycopeptidome: a novel tool of relevance for vaccine design and serodiagnosis. Journal of Virology 86, 6268-78).

o D’Arrigo, I., Cló, E. et al (2013) Diverse IgG serum response to Epstein-Barr virus glycoprotein 350/220 revealed with O-glycopeptide microarray Glycoconjugate Journal (in press).

o Pincus SH., Moran E. et al. (2012) Fine specificity and cross-reactions of monoclonal antibodies to group B streptococcal capsular polysaccharide type III. Vaccine 30, 4849-4858.

o Blixt O, Boos I., Mandell U. (2012) Glycan Microarray Analysis of Tumor-Associated Antibodies; in Anticarbohydrate Antibodies. Editor P. Kosma, Springer Verlag [in press].

o Blixt, O., Lavrova, OI. et al. (2012) Analysis of Tn-antigenicity with a panel of new IgM and IgG1 monoclonal antibodies raised against leukemic cells. Glycobiology. 22, 529-42.

o Welinder, C., Baldetorp, B. et al. (2013) Primary Breast Cancer Tumours Contain High Amounts of IgA1 Immunoglobulin: An immunohistochemical analysis of a possible carrier of the tumour-associated Tn antigen, PLoS in press

Important collaborators: Peptide synthesis and nano-technology

Prof. KJ Jensen (University of Copenhagen) Dr. Kasper Kilderaard Sorensen (University of Copenhagen) Prof. Mikkel Boas Thygesen (University of Copenhagen) Dr. Ulrika Westerlind (Dortmund)

Virology Prof. Tomas Bergstrom (Gothenburg University) Prof. Sigvard Olofsson (Gothenburg University) Prof. Ali Mirazimi (SMI, KI, Stockholm) Dr. Thea Kolsen-Fischer (SSI, Copenhagen) Prof. John Skehel (Mill Hill, London) Prof. Niklas Arnberg (Umea University)

Glycan microarrays Prof. Nicolai Bovin (Shemakyin Institute, Moscow) Prof. Yuriy Knirel (Moscow) Prof. Christoff Wagener (Hamburg University) Prof. Carlos Unverzagt (Beyruth University) Prof. William Willats (University of Copenhagen) Prof. Reinhard Schwartz-Albeiz (Heidelberg Cancer Center) Prof. Monical Palcic (Carlsberg Laboratory) Prof. Ole Hindsgaul (Carlsberg Laboratory)

Prof. Sabine Flitsch (Manchester University) Prof. Ricardo U. Sorensen (Louisiana State University) Prof. Jaime Inostroza (University de la Frontera, Chile) Prof. Fredrik Backhed (Gothenburg University)

Monoclonal antibodies and tumor biology Prof. Joyce Taylor-Papadimitriou Prof. Joy Burchell (Kings College, London) Dr. Bo Jansson (Lund University) Dr. Charlotta Welinder (Lund University) Dr. Lena Danielsson (Lund University) Dr. Romain Micol (Transgene, France)

Bacterial pathogens

Prof. Thomas Boren (Umea) Prof. Joergen Leisner (UCPH) Prof. Dlawer AlaÁldeen (Univ of Nothingham) Prof. Jafar Mahdavi (Univ of Nothingham) Prof. Karen Krogfelt (SSI, Copenhagen)

Resources and Technologies SYRO Wave, SYRO-II MultiSyntech / Biotage automated peptide synthesizers Integrated SPPS and microarray Technologies. Tip-synthesis of 288 peptides per run with Syro-II. Difficult synthesis with Automated Microwave Initiator+ Alstra. Analytical and preparative HPLC purification stations

Microarrayer: A robotic array-spotting device (MicroGrid II, Genomic Solutions) is available to print microarrays. This instrument will print microarrays with a spotting density of >1000 spots per cm2. We can print arrays from our glycan library, glycopeptide, glycoprotein, glycolipid and antibody collections or other materials. The print table holds up to 120 slides, which can be printed in one print run. All slide surfaces and buffers can be used. Most print conditions, such as temperature and humidity can be met. Slide Scanning: Our biomarker discovery lab is equipped with a ProScanArray HT confocal laser scanner with a 20-slide autoloader (Perkin Elmer, Waltham, MA). Configured with red (632 nm), green (543 nm),

and HeNe lasers and a blue (488 nm) Argon laser and the professional ProScanArray Express software, this system is very sophisticated and meets all of our needs. There are 10 emission filters (20nm band pass) at 508nm, 522nm, 530nm, 570nm, 578nm, 592nm, 614nm, 660nm, 670nm, and 694nm. This instrument will scan 25mm x 75mm microscope slides with 5, 10, 20, 30, and 50 micron pixel resolution. Data Analysis: The Tif image created by the scanner is processed by imaging software, such as Imagene and ProScanArray Express. These applications are capable of spot-finding, background intensity determination, and signal intensity generation. The output is a tab-delimited text file, which has each spot’s intensity. The process of analyzing the text file output is the most non-standardized aspect of data analysis. The group collaborates with bioinformatics expertise from various project partners to process and evaluate data.

Funding sources

www.gastricglycoexplorer.eu

www.glycobiom.eu

www.immunocan.org