Nico CALLEWAERT - Rewiring the synthesis of protein-linked glycosylation...
Channel: Institut des Hautes Études Scientifiques (IHÉS)
Date: 21/12/2015

Rewiring the synthesis of protein-linked glycosylation in eukaryotic biopharmaceutical expression hosts

Medical Biotechnology Center, VIB and UGhent, Ghent, Belgium.
Correspondence: nico.callewaert@vib-ugent.be

Glycosylation is the enzymatically catalysed modification of biomolecules with carbohydrate structures.  For  secreted  and  membrane-integral  proteins,  it  is  the  most  common  post-
translational   modification.   Glycan   structures   characterize   the   molecular   environment immediately  outside  of  all  cell  types  and  hence  have  critical  functions  in  interactions  of  any 
cell    with    its    environment    (cell-cell,    cell-pathogen,    cell-
molecule).    The    field    of glycobiotechnology    is    concerned    with    understanding    and    re-engineering    of    these glycosylation-dominated   interactions.   In   particular,   the   understanding   of   the   synthetic pathways  and  functions  for  eukaryotic  N-and  O-glycosylation,  gained  over  the  past  few 
decades,  has  enabled  the  rewiring  of  these  pathways  for  the  benefit  of  pharmaceutical applications. Based on the conservation of the core pathways between eukaryotes, it has been possible  to  transfer  the  efficient  synthesis  of  particular  human-specific  glycan  structures  to other  eukaryotes  such  as  yeasts  and  plants.  This  is  enabling  the  cost-effective production  of biopharmaceutical  proteins  with  glycosylation  patterns  customized  to  particular  therapeutic functionality  (e.g.  targeting  to  particular  glycan  receptors,  or  customized  for  particular pharmacokinetic behaviour). I will illustrate our work with regard to the production of human IgG-like   glycosylation   patterns   in   yeast1,   and   the   production   of   mannose-6-phosphate modified   lysosomal   enzymes   for   the  treatment   of   human   inherited   lysosomal   storage diseases2. 
Whereas these earlier synthetic biology endeavours were geared towards efficiently synthesising  proteins  with  complex  mammalian  glycan  structures  in  other  eukaryotes,  more 
recently we  have  generated  mammalian  cells  and  plants in  which  glycosylation  complexity has been reduced to the bare minimum, while still being compatible with eukaryotic cell life 
and  protein  productivity.  This  ‘GlycoDelete’  technology3,4 opens  up  many  new  structural biology   and  biopharmaceutical   applications   that   are   currently   being   explored   in   our laboratory.

1.Jacobs, P. P., Geysens, S., Vervecken, W., Contreras, R. & Callewaert, N. Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology. 
Nat. Protoc.4,58–70 (2009).
2.Tiels, P. et al.A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-
produced recombinant human lysosomal enzymes. 
Nat. Biotechnol.30,1225–1231 (2012).
3.Meuris, L. et al.GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins. 
Nat. Biotechnol.32,485–489 (2014).
4.Piron, R., Santens, F., De Paepe, A., Depicker, A. & Callewaert, N. Using GlycoDelete to produce proteins lacking plant-specific N-glycan modification in seeds. 
Nat. Biotechnol.33,1135–1137 (2015).