Oral Presentation 23rd Annual Lorne Proteomics Symposium 2018

A Genetic Dissection Approach to Functional Glycomics (#50)

Henrik Clausen 1 , Yoshiki Narimatsu 1 , Yen-His Chen 1 , Weihua Tian 1 , Hiren Joshi 1 , Katrine Schjoldager 1 , Zhang Yang 1 , Adnan Halim 1 , Eric Bennett 1 , Sergey Vakhrushev 1
  1. Copenhagen Center for Glycomics, Depart. of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

Glycosylation is one of the most abundant and diverse posttranslational modifications of proteins, but arguably also one of the most difficult to study with respect to occurrence, structure, and biological functions. Heterogeneity in occupancy and structures of glycans on proteins is the major obstacle for analytic strategies, and this is also the major obstacle for exploiting biological functions of glycans in biotechnology. We have therefore taken genetic deconstruction & reconstruction approaches to dissect the glycosylation and modification processes of proteins and proteoglycans. We use rational combinatorial knockout/in of relevant genes with precise gene editing tools (DNA nuclease “scissors” ZFNs and CRISPR/Cas9) to simplify1, dissect2, and design3 glycosylation more or less at will. The strategy has led to increased knowledge of different glycoproteomes as well as discovery of new types of protein glycosylation4, 5. Genetic dissection of glycosylation in isogenic cells enables exploration of biological functions of specific glycosylation features, and the generated libraries of cells may be used to develop cell-based glycan arrays for interrogation of interactions with glycans. We will present illustrative examples of the power of gene editing in the glycosylation field that is rapidly turning into “Lego-Toying”.

 

  1. Steentoft, C. et al. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines. Nature Methods 8, 977-982 (2011).
  2. Schjoldager, K.T. et al. Deconstruction of O-glycosylation-GalNAc-T isoforms direct distinct subsets of the O-glycoproteome. EMBO Rep 16, 1713-1722 (2015).
  3. Yang, Z. et al. Engineered CHO cells for production of diverse, homogeneous glycoproteins. Nature Biotech 33, 842-844 (2015).
  4. Halim, A. et al. Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast. Proc Natl Acad Sci USA 112, 15648-15653 (2015).
  5. Larsen, I.S.B. et al. Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins. Proc Natl Acad Sci USA 114, 11163-11168 (2017).