Research

Research context Interspecies transmission of influenza viruses may require viral adaptation to cellular sialic acid (Sia)-containing receptors in the target tissues of a new host. Some subtypes of avian influenza viruses (AIVs), such as H5, are able to propagate in a wide range of hosts, while other subtypes, such as H16 (and to a lesser extent H13), have only been isolated from a narrow range of gull species. Hosts also have different levels of susceptibility to AIVs. Some species of wild birds, such as ducks, have been infected with nearly every subtype of AIV (except H16), while some species, such as gulls, are considered as exclusive reservoirs of the H16 subtype. The structure of the receptor binding domain of the AIV hemagglutinin protein and the cellular receptors of hosts are probably instrumental in the virus-host attachment and infection process. We hypothesize that: 1) the HA protein of H16 subtype AIVs has a unique 3D structure that restricts the viruses to a limited range of cell receptors, 2) gull species become more infected with H16 (and H13) subtype AIVs due to the presence of specific types of receptors on the surface of their respiratory and intestinal cells (which are different from the common receptors that are abundant in duck species) and 3) H16 subtype AIVs can propagate more efficiently in gull tissues and gull-derived cells than in duck tissues and duck-derived cells. Our approach: 1) At AIV level: We will study a wide range of H16, H13 and H5 AIVs, their hemagglutinin sequences and 3D structures and their affinity to a range of glycans using sialylglycopolymers and glycan microarray chips to correlate virus structural and genetic properties to glycan (indirectly representing receptor) affinity. 2) At cell and tissue level: We will study the distribution of Sia receptors in the intestine/respiratory tissues and cell cultures of duck and gulls using single specific lectins and lectin microarray chips. We will study the expression of the genes of the enzymes that produce Sias in the cells as an indirect indicator of the presence of the receptors. The results will provide a tool to evaluate any bird species as a potential host for H16 AIVs. 3) At AIV-host interaction level: We will study the affinity of FITC-labeled AIVs to tissue sections; and the replication rate of AIVs by cultivating a variety of H16 and H13 viruses in duck-/gull-derived cell lines; and we will perform animal experiments using selected H16 AIVs and bird species. This will enable us to validate the in vitro and in vivo results in a real virus-host replication and interaction study. Level of originality Our study is novel in that it incorporates a range of conventional and modern techniques to address an important yet neglected question about the role of receptors in the susceptibility of wild birds to infection with two groups of AIVs.

Glycans cover the surfaces of all cells and are biosynthesised by specific biochemical pathways in intracellular compartments known as the endoplasmic reticulum and Golgi apparatus prior to transport to the cell surface. Genetic defects in the trafficking between and within these compartments result in altered cell surface glycosylation resulting often in neurological and other phenotypes in human patients. In this project we will investigate trafficking in a model nematode (Caenorhabditis elegans) and in insect cell lines. Mutant strains will be generated and examined in terms of altered glycosylation and altered localisation of key biosynthetic enzymes using modern mass spectrometric, array-based and microscopic methods; in the case of mutant nematode lines, also their behaviour will be analysed using a high-throughput video system. Particularly the data from C. elegans will yield a basic understanding of trafficking in a model organism with relevance to human disease. Wider research context / theoretical framework: Glycans cover the surfaces of all cells and the Golgi apparatus is key to their biosynthesis in eukaryotes. The intracellular trafficking of Golgi resident enzymes ensures that they act on glycan substrates in the correct order. Alterations to trafficking caused by mutations in the Conserved Oligomeric Golgi (COG) and the Trafficking Protein Particle (TRAPP) complexes thereby result in shifted glycomes in human, which can result in biological phenotypes as observed in some trafficking-dependent congenital disorders of glycosylation. Hypotheses/research questions /objectives: Based on previous work by us and others, we hypothesise that knocking-out genes encoding components of the COG and TRAPP complexes has variable impacts on N- and O-glycosylation in Caenorhabditis elegans and in insect cell lines due to alterations in the localisation of key glycan-modifying enzymes. This affects also their interactions with lectins and the behaviour of the mutants. Approach/methods The proposed methods fall into foue parts: (i) identification or design of COG and TRAPP mutants using C. elegans or Sf9 cells together with glycomic analyses; (ii) glycan array experiments focussed on addressing lectin specificity towards wild-type and mutant N-glycans; (iii) examination of altered Golgi localisation of key enzymes by microscopic and proteomic techniques; (iv) behavioural analyses of C. elegans COG and TRAPP mutants. Level of originality / innovation To date, there have been few studies on the impact of mutations affecting COG and TRAPP complexes in either nematodes or insects. The glycomic studies will be combined uniquely with behavioural studies on mutant C. elegans as well as determining the impact on recombinant protein glycosylation in Sf9 cells. Particularly the data from C. elegans will yield a basic understanding of trafficking in a model organism with relevance to human disease. Primary researchers involved The project will be led by Dr. Iain Wilson with Prof. Dominique Glauser as international partner.

Our long-term vision is to routinely produce recombinant proteins as single defined glycoforms without the need for genetic manipulation. This will functionally be the equivalent for glycans as site-directed mutagenesis. We will showcase this by the production of therapeutically relevant proteins with homogenous pre-defined N-linked glycoforms. Our approach is a radically new approach to the manipulation of cell function; we will use the delivery of computationally defined mixtures of enzyme-specific inhibitors to tune the activity of glycosyltransferases and thereby the glycans displayed on secreted proteins such as antibodies, hormones and other therapeutic modalities. The untemplated nature of glycan synthesis leads to inherent heterogeneity in these glycans. This variation can compromise the behaviour of these complex molecules which are increasingly important in modern disease treatments. The complexity of glycan biosynthesis is staggering; in mammals there are up to 200 different glycosyltransferases (GTs) that compete for substrates within the Golgi, the organelle in which most glycans are synthesized. Generation of a given glycan structure depends on the activities and levels of a suite of glycosylation enzymes in each of the Golgi’s sub-compartments (cisternae). In order to manipulate glycans, we will build an understanding of how the arrangement of key biosynthetic enzymes, their activities and their locations specify glycan structures. Our approach, termed Inhibitor-Mediated Programming of Glycoforms (IMProGlyco) will provide an effective strategy to manipulate these enzymes and thereby deliver defined mixtures of glycosylated proteins. This strategy will be adaptable and expandable into other cell types and organisms and thereby enable scalable production of highly specified recombinant biomolecules. Supply of these will enable both production of high performance biotherapeutics and modulation of cell function together with fundamental studies of the consequence of variation in glycosylation patterns in laboratory studies.