Leader Signal Peptides

Proteins used for binding studies (Supplementary Table 2) were expressed in E. coli. cDNA sequences without the leader peptide were codon optimized and cloned into a pET28-N-His₆-SUMO vector. IFNγ^ΔKRKR and IFNγ^ΔKRKR–GFP mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene). The extracellular domain (amino acids 26–254) of the IFNγR1 protein was cloned based on mouse cDNA into a pET26b-N-His₆-SUMO vector, resulting in periplasmic expression of an N-terminal His₆-SUMO-tagged protein bearing an additional C-terminal His₆ tag. All proteins were produced using T7 express competent E. coli (New England Biolabs) cotransformed with the pRARE plasmid. For purification of IFNγ proteins, bacteria were collected and resuspended in lysis buffer (1× PBS (pH 7.4), 0.2 M NaCl, 5% glycerol supplemented with cOmplete EDTA-free protease inhibitor cocktail (Roche), 0.25% (wt/vol) 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate, 1 mM phenylmethylsulfonyl fluoride, 100 µl of lysozyme (100 mg ml^–1) and 1 µl of benzonase (250 U µl^–1; Merck)) and lysed by two freeze–thaw cycles. Proteins were purified on a HisTrap FF crude column (Cytavia), followed by size-exclusion chromatography on a Superdex 75 column (XK 26 × 60, Cytavia) or Superdex 200 column (XK 26 × 60, Cytavia) for C-terminal GFP fusion proteins, respectively. The N-terminal His₆-SUMO tag was cleaved with yeast Ulp1p SUMO protease (produced in-house) followed by a gel filtration step and reapplication of the cleaved protein on the Ni²⁺ affinity column. For purification of IFNγR1, bacteria were lysed by osmotic shock using 0.2 M Tris-HCl (pH 8.0), 0.5 mM EDTA (pH 8.0), 0.5 M sucrose and 1 mM phenylmethylsulfonyl fluoride as lysis buffer. Purification, as described above, was followed by cleavage of the N-terminal His₆-SUMO tag and a final gel filtration step on a 10/300 Superdex 200 GL increase column (Cytavia). Recombinant proteins were stored in 20 mM HEPES (pH 7.5) and 0.2 M NaCl at –80 °C until further use. Binding kinetics of IFNγ variants to HS and the recombinant IFNγR1 were determined by SPR on a Biacore T200 (GE Healthcare) using a dextran Series S Sensor Chip CM4 (GE Healthcare). For analysis of HS binding, commercial HS derived from porcine intestinal mucosa has been used (Celsus Laboratories), and this preparation was previously characterized^{33 (link)}. The average molecular weight of HS was determined to be 12 kDa with a polydispersity of 1.59, and its sulfation degree, evaluated by S and N elemental analysis, was ~1.4 sulfate groups per disaccharide, on average. For IFNγR1, the Sensor Chip was coated with anti-His₅ (Qiagen), onto which the recombinant IFNγR1 proteins were immobilized.

Firstly, the eukaryotic expression vector CAGs-GFP-T2A-Luciferase-Enhanced Episomal Vector (EEV) (SBI system biosciences, Palo Alto, CA, USA) was simultaneously digested by EcoRI and XhoI. The cDNA sequence encoding IgK leader peptide and PK5, Gal-3C, or PK5-RL-Gal-3C as well as ligating EcoRI and XhoI cutting sites were synthesized in GENEWIZ (Suzhou, China). Then, all cDNA fragments were respectively inserted into EEV vector to generate plasmids EEV-PK5, EEV-Gal-3C, and EEV-PK5-RL-Gla-3C. All the plasmids were sequenced and then purified with PureLink™ Hipure plasmid maxiprep kit (Invitrogen, CA, USA) for further used.