Rare diseases have a significant impact on Canadian families affecting 1 in 12 Canadians with great cost to our society. Ninety percent of rare diseases are life-limiting and have no treatment, and 50% affect children (http://igniteproject.ca/). Our group is actively engaged in work focusing on rare disease research, with specific attention to congenital disorders of glycosylation (CDG) which results from faulty N-linked glycosylation.
N-linked glycosylation is a critical process involved in the post-translational modification of newly synthesized proteins. This process is responsible for the formation of glycoproteins by the attachment of a sugar molecule (known as a glycan) to proteins. The resulting linkage influences protein structure and function. Glycosylated proteins play a vital role in essential cellular processes and regulate developmental pathways through embryogenesis and beyond.
The biosynthesis pathway of N-linked glycoproteins begins in the endoplasmic reticulum (ER) of the cell and ends in the Golgi apparatus. As shown below in Figure 1 from Hum Mutat 2009;30:1628-41, the pathway begins on the cytoplasmic side of the ER and is catalysed by glycosyltransferase enzymes that utilize nucleotide activated sugars (UDP-GlcNAc and UDP-Glc) to form Man5GlcNAc2-PP-Dol. The evolving glycan is then translocated to the luminal side of the ER membrane where it is further processed by luminally-oriented glucosyl and mannosyltransferases to form the fourteen sugar precursor, Glc3Man9GlcNAc2. Once the lipid-linked oligosaccharide (LLO) is assembled, it is then transferred to an asparagine side chain of a nascent growing protein, which bears a consensus sequence for glycosylation (Asn-X-Ser/Thr). Once the glycoproteins have folded properly, they move to the Golgi complex where they are subjected to further trimming and modification to form mature glycoproteins.
N-linked glycosylation is an essential process for maintaining normal biological activity of proteins. Impairment in this process may lead to formation of proteins with reduced or lost function. In general, disturbances of glycosylation may be either congenital or acquired. Disorders of glycosylation are rare though identified sub-types and cases of CDG are growing rapidly. CDG is a devastating disorder impacting numerous organ systems in developing children resulting in severe cognitive and physical developmental delay and early death in 20% of cases. (Acta Biochimica Polonica 2013;60:151-161.)
There is currently no treatment for CDG; however, with a growing understanding of the cellular pathways and the associated genetic mutations that result in CDG sub-types, the avenue for therapeutic research has now been opened widely. The approach to therapeutic research involves the development of a disease model with critical measurable biomarkers. Glycosylation studies are currently underway at the Dalhousie Medicine Research Laboratory in Saint John, New Brunswick. Current disease models being used and developed include the yeast Saccharmomyces cerevisiae, the Zebrafish Danio rerio, and human fibroblasts.
The Zebrafish have become an invaluable tool for studying developmental human disorders. This vertebrate organism develops from embryonic stages to a fully grown adult within days and its transparent nature allows for clear observation of cellular functions and organogenesis. Through CRISPR/Cas9 technology, the zebrafish genetic structure may be manipulated and organisms with CDG constructed. Subsequently, these constructs may be utilized as disease models and potential therapeutic strategies studied. With assistance from Prof Aiebi's lab in Zurich, our group has successfully completed preliminary work utilizing transformed yeast strains, which serve as disease models for CDG. With this background work we are now prepared to develop a vertebrate disease model.
Saccharomyces cerevisiae, also known as yeast, is a common model organism utilized by researchers. The cellular process of glycosylation is similar in both yeast and humans as the pathway is highly conserved among eukaryotes. Thus, studying a yeast disease model is an effective and ethical way to investigate CDG and potential therapeutics. With the assistance of Professor Markus Aebi's lab at the Swiss Federal Institute of Technology, we have transformed mutant S. cerevisiae strains to establish a disease model for studying ALG9-CDG and ALG12-CDG. In future research projects we plan to experimentally manipulate these disease models in an attempt to rescue the mutant phenotype.
Transformation of mutant Saccharomyces cerevisiae strains and establishing a disease model for ALG9-CDG
Rattina Dasse Nadaradjan was was first author of the poster presentation "Transformation of mutant Saccharomyces cerevisiae strains and establishing a disease model for ALG12-CDG". He presented this work at the New Brunswick Health Research Foundation Annual Gala in November 2015 in Fredericton, New Brunswick. For his exceptional work he was awarded second place in the Health Professionals Category.
Danio rerio, commonly referred to as the zebrafish, is an aquatic vertebrate that can be used to examine human glycosylation disorders. Zebrafish are especially powerful in studying developmental disorders and screening for therapeutics as their proteins and genes are similar to that of humans. By fusing a protein of interest with a florescent protein, researchers are able to identify when and in what cell types a specific protein is expressed. The FoG and associated researchers are currently working on developing a zebrafish disease model of ALG9-CDG and ALG12-CDG by utilizing Crisper/Cas9 genome editing technology. Other prospective research plans include developing endoplasmic reticulum and lysosome isolation techniques in zebrafish and establishing a morpholino target for ALG9 and ALG12 for the purpose of monitoring gene expression in disease models.
This photo shows Dr. Webster and Dr. Perez in our Zebrafish lab. The following video demonstrates how zebrafish are used to study developmental and neuromotor diseases.
Human fibroblast cells function to maintain cell structural integrity by producing collagens, fibres and glycoproteins. Fibroblast cells, which are deficient in the glycosylation enzyme ALG9, are valuable for studying glycosylation disorders. Tess Robart and Alyson Zwicker, were hired by the FoG as undergraduate researchers in 2014. They worked at the Dalhousie Medicine New Brunswick Research Laboratory under the supervision of Dr. Thomas Pulinilkunnil and Dr. Petra Kienesberger. Tess and Alyson presented their work at the 6th Annual New Brunswick Health Research Conference in Moncton, New Brunswick on November 14, 2014. Their research focus was developing a subcellular fractionation methodology for isolation of enriched endoplasmic reticulum from ALG9 deficient human dermal fibroblasts. For their work they were awarded 1st place in the Student-Health Professionals category at the 6th Annual Conference. Next steps include exposing fibroblasts to cellular stress in order to identify deficient cellular responses. Subsequently, our goal is to modify ALG9 in an attempt to improve or rescue the diseased phenotype.