Glycosylation and disease mechanisms

Group leader: Göran Larson (PhD, MD, Prof. Laboratory medicine and Glycobiology) 

Members: Ammi Grahn (PhD), Jonas Nilsson (PhD), Camilla Hesse (PhD), Inger Johansson (PhD), Gustaf Rydell (PhD), Adnan Halim (PhD student), Waqas Nasir (PhD student).

There are four major lines of research projects that have been lately developing in the group; all projects relate to biomedical impact of variations of glycosylation being involved in infectious diseases, in congenital muscular dystrophies, in transfusion medicine and finally in developing novel methods for characterization of glycoproteins in biological fluids, cells and tissues.

Winter vomiting disease and its agent Norovirus (NoV family of Calicivirus), is nowadays recognized as the dominating cause of non bacterial gastroenteritis worldwide. NoV, a small single stranded RNA virus, is an increasing threat to individuals in both developing countries (200,000 deaths/year) and in closed settings throughout the world (hospitals, nursing homes, day-care centres, cruise ships etc.). Within the last five years our group has, in collaboration with prof. Lennart Svensson Linköping, substantially contributed to the understanding of the carbohydrate binding specificities of NoV virus-like particles to human histo-blood group antigens giving explanations to outbreak variations in individual´s susceptibility to NoV, both at the molecular and at the genetic level (allelic variations in glycosyltransferase genes) [1, 5, 6, 8, 13, 14]. In addition we have identified novel, NoV strain specific, binding specificities to both glycoproteins and to glycosphingolipids [10, 12] and are now, in close collaboration with prof. Fredrik Höök, Chalmers, studying the dynamic mechanisms of virus binding to glycosphingolipids in bilayer membranes [16]. By homology modelling and molecular dynamic simulations [, together with Per-Georg Nyholm at Biognos AB, were are also in the process of screening for and designing appropriate inhibitors of virus adhesion, using binding techniques that are now well established in our laboratory.

A major concern in viral diseases is how pathogens manage to evade the immune response after propagation and subsequent release from the host cell. In collaboration with prof. Sigvard Olofsson, Inst of Biomedicine, Göteborg, we have studied the effect of the latent virus Herpes simplex type 1 (HSV1) on glycosyltransferase expression in human cells. Interestingly, there is an early gene induction of specific fucosyltransferases [2] leading to expression of the Sialyl Lewis x antigen [2, 7, 11], a common mammalian receptor structure of the selectin family used by leukocytes to evade the blood circulation and get into sites of inflammation (through E-selectin binding) or into lymph nodes (L-selectin binding). The biosynthesis of this carbohydrate based receptor structure has been the object of several studies from our own group some of which during the last five years [3, 4]. We are now collaborating with prof. Olofsson on the structurally characterization of specific glycosylation sites of glycoprotein gC1 of HSV-1.

Glycosylation is generally recognized as the most complex post translational modification of proteins and due to its structural variability two separate methodologies, essentially based on mass spectrometry, for characterizing glycoproteins have evolved; glycomics for released glycans and proteomics for proteins. A few years ago we set out to fuse these two techniques in order to map the specific glycans while still attached to the amino acid residues of the protein backbone. A first publication came out 2009 in Nature Methods [17] where we introduced a capture-and-release technology to specifically enrich and characterize sialylated glycoproteins. The technique allowed us to define 36 N-linked and 44 O-linked glycosylation sites on glycoproteins of human cerebrospinal fluid. In parallel we have employed the technique for mapping unique O-glycosylation sites of a-dystroglycan prepared from human skeletal muscle, of platelet surface glycoproteins [15] released during storage in medium, and of glycoproteins prepared from human serum [9] and urine samples. The technology is now being refined for higher sensitivity, better primary and secondary ion fragmentation and applicability to characterize also membrane bound glycoproteins. Structural information on glycosylation and attachment sites of defined glycoproteins are continuously being added to the SwissProt database and thus made publicly searchable through Mascot database searches.

Important publications:

1) Nilsson M, Hedlund KO, Thorhagen M, Larson G, Johansen K, Ekspong A, Svensson L (2003) Evolution of Human Calicivirus RNA in vivo: accumulation of mutations in the protruding P2 domain of the capsid leads to structural changes and possibly a new phenotype. J Virol 77 (24), 13117-13124.

2) Nyström K, Biller M, Grahn A, Lindh M, Larson G, Olofsson S (2004) Real time PCR for monitoring of viral influences on transcription of host cellular genes in herpes simplex virus type-1 infected human diploid cells. J Virol Methods, 118, 83-94.

3) Grahn A, Shah Barkhordar G, Larson G. (2004) Identification of seven new a2,3- sialyltransferase III, ST3GalIII, transcripts from human foetal brain. Glycoconj J 20, 493-500.

4) Bengtson P, Zetterberg H, Mellberg T, Påhlsson P, Larson G. (2005) Characterization of EBV-transformed B-cells established from an individual homozygously mutated (G329A) in the FUT7 a1,3-fucosyltransferase gene. Scand J Immunol 62, 251-258.

5) Thorven M, Grahn A, Hedlund K-O, Johansson H, Wahlfrid C, Larson G, Svensson L. (2005) A homozygous nonsense mutation (428G>A) in the human secretor (FUT2) gene provides resistance to symptomatic norovirus (GGII) infections. J Virol 79, 15351-15355.

6) Modin Larson M, Rydell GEP, Grahn A, Rodriguez-Díaz J, Åkerlind B, Hutson AM, Estes MK, Larson G, Svensson L (2006) Antibody prevalence and titer to norovirus (genogroup II) correlate with secretor (FUT2) but not with ABO phenotype or Lewis (FUT3) genotype. J Infectious Diseases 194, 1422-1427.