Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Protein glycosylation – an overlooked feature impacting Stem cell factor and Stem cell factor receptor function (#49)

Tiago Oliveira 1 , Francis Jacob 2 , Chi-Hung Lin 1 , Natalie Rimmer 2 , Andreia Almeida 1 , Michaela Mischak 1 , Kathirvel Alagesan 1 , Mark von Itzstein 1 , Tiffany Walsh 3 , Daniel Kolarich 1 4
  1. Griffith University, Southport, QLD, Australia
  2. Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland
  3. Institute for Frontier Materials, Deakin University, Waurn Ponds, VIC, Australia
  4. ARC Centre for Nanoscale BioPhotonics, Australia, Southport, QLD, Australia

c-KIT (also known as Mast/stem cell growth factor receptor Kit, tyrosine-protein kinase KIT and CD117) plays an essential role in the regulation of cell survival and proliferation, haematopoiesis, stem cell maintenance, gametogenesis, mast cell development, migration and function, and in melanogenesis. It is a class III receptor tyrosine kinase (RTK) that contains 10 known sites of N‑glycosylation that are overall highly conserved across most mammals, as well as some predicted sites of O‑glycosylation. Our knowledge on c-KIT and SCF glycosylation and its effect on their function, however, has to date still not been answered.

Recombinant c-KIT and SCF were expressed in mammalian cells, affinity-purified and subjected to an in-depth glycomics and glycoproteomics characterisation using porous graphitized carbon LC ESI MS/MS glycomics and Orbitrap Fusion Reversed Phase LC glycoproteomics to guide in silico structure modelling and function impact prediction.

HEK293 cell expressed c-KIT is glycosylated at all 10 sites with largely complex type N-glycans. O‑glycosylation prediction by NetOGlyc 4.0 indicated that up to four Ser residues (Ser28, 30, 35, 38) are potentially O‑glycosylated. Glycoproteomics analyses confirmed that various Ser-residues are O-glycosylation with mostly core 1 type O-glycans. On the basis of available c‑KIT/SCF protein structure models we have in silico modelled how the presence of N-glycans impacts the c‑KIT/SCF interaction and dimerization. Our modelling data clearly shows that due to the extensive glycosylation in the vicinity of the c-KIT/SCF interface and the c-KIT extracellular dimerisation interface it is highly likely that both glycan-glycan and glycan-protein interactions will play a key role in the c-KIT/SCF interaction, and in the dimerisation of c-KIT receptors.

This is the first in-depth study of c-KIT and SCF glycosylation that shows that protein glycosylation plays a yet not satisfactorily understood role in the interaction of SCF with c-KIT, and hence, its activation.