Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Abstract

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

Structural characterization of the ICOS/ICOS-L immune complex reveals high molecular mimicry by therapeutic antibodies

“…Genes were synthesized at GeneArt (Life Technologies) and cloned into the pHLsec expression vector containing a C-terminal His 6x tag for downstream purification. Proteins were expressed in HEK 293F cells (ThermoFisher Scientific) following standard protocols 59 : about 200 mL of cells were seeded at a density of 0.8 × 10 6 cells/mL and incubated with 125 rpm oscillation at 37°C, 8% CO 2 , and 70% Similarly, the ICOS-L/prezalumab complex was produced in the presence of VNAR by co-transfecting ICOS-L:Fab HC : Fab LC :VNAR in a 2:2:1:4 ratio. The ICOS/ STIM003 complex was obtained by co-transfection of ICOS C136AC137A N23Q (residues 21-138):Fab HC :Fab LC in a 2:2:1 ratio.…”

“…Genes were synthesized at GeneArt (Life Technologies) and cloned into the pHLsec expression vector containing a C-terminal His 6x tag for downstream purification. Proteins were expressed in HEK 293F cells (ThermoFisher Scientific) following standard protocols 59 : about 200 mL of cells were seeded at a density of 0.8 × 10 6 cells/mL and incubated with 125 rpm oscillation at 37 °C, 8% CO 2 , and 70% humidity in a Multitron Pro shaker (Infors HT). Twenty-four hours after seeding, cells were transiently transfected using 50 μg of filtered DNA preincubated for 10 min at room temperature (RT) with the transfection reagent FectoPRO (Polyplus Transfections) in a 1:1 ratio.…”

Cholesterol Interaction Directly Enhances Intrinsic Activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

“…The FLAG-CFTR-His construct was transiently transfected in HEK293F (Thermo Fisher Scientific) suspension cell line capable of complex N-glycosylation. The DNA at 50 μg was mixed in a 1:1 ratio with transfection reagent FectoPRO ® (Polyplus, Berkeley, CA, USA) and added to each 200 mL suspension culture at 0.8 × 10 6 cells per mL, as previously described [63,64]. The cells were grown at 37 °C shaking at 180 rpm with 8% CO 2 for 24 h. The next day, the cells were treated with 10 mM sodium butyrate (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C shaking for 24 h. The HEK293F cells transfected with FLAG-CFTR-His were collected and spun down at 6000 rpm at 4 °C for 30 min.…”