Protein purification, de-glycosylation, and fluorescence labeling
The coding sequence of Orysata (NCBI: XM_015766617) was amplified from the pPICZα::Orysata vector6 with primers KpnI-Ory and Ory-XbaI (Supplementary Table 1), containing the KpnI and XbaI restriction sites at their respective 5′ ends. The fragment was blunt cloned into the pJet vector (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions and sequenced. Subsequently, the Orysata fragment was ligated into the pColdII (Takara bio, Kusatsu, Shiga, Japan) vector generating an in-frame fusion with an N-terminal poly-histidine tag. This recombinant vector, pColdII::Orysata, is transformed into competent E. coli strain BL21 cells. Transformed bacteria were selected on LB plates containing 100 µg/ml ampicillin.
For the bacterial produced Orysata (BOry), the recombinant BL21::pColdII::Orysata bacteria were grown to an OD600 of 0.5 and induced with 400 µM IPTG for 48 h. After harvesting the cells by centrifugation (7000g for 10 min at room temperature), the cells were resuspended in 20 mM of 1,3-Diaminopropane and homogenized by ultrasonication. The cell lysate was then centrifuged to collect the soluble protein fraction, which, after adjusting the pH to10, was applied to a QFF ion exchange column (DEAE Sepharose Fast Flow, Cytiva, Marlborough, MA, USA) and eluted by 0.1 M Tris–HCl pH 7.5 + 0.5 M NaCl. The pH of the eluted protein solution was adjusted to 7.5 prior to being applied to the Ni–NTA matrix column (Invitrogen, Carlsbad, CA, USA). After elution with 0.1 M Tris–HCl pH 7.5 + 250 mM imidazole, the buffer is exchanged to PBS using Amicon Ultra filters (Sigma-Aldrich, Saint Louis, MO, USA) with a molecular weight cut-off of 3 kDa. The protein sample was lyophilized and stored at − 20 °C until further use. For the production of the rOrysata in P. pastoris (YOry), the protocol was followed as described before6.
PNGase F was used to digest the N-glycan from both native YOry and heat denatured YOry. For the denaturing, 4 µg of YOry was diluted into PBS (pH 7.5) and heated at 98 °C for 1 h. 100 U of PNGase F (Promega, Madison, WI, USA) were added to the native and denatured samples and the samples were incubated for 12 h at 37 °C. As controls, native and denatured YOry without PNGase F treatment were included in the assay. Afterwards, all samples were analyzed by SDS-PAGE.
To verify the N-glycan of YOry can be bound by another Orysata, the glycosylated and non-glycosylated forms of YOry were first separated by SDS-PAGE, after which they were blotted to a membrane (FluoroTrans PVDF Transfer Membranes). Subsequently, the membranes were incubated with FITC labeled BOry for 20 min in the dark. After a gentle wash by PBS, FITC-fluorescence was detected using the Image Lab system (Bio-Rad, Hercules, CA, USA). To confirm the carbohydrate-lectin dependency, FITC-BOry was incubated with 20 mM mannose in PBS for 20 min, before being added to the YOry blot. The blots were finally stained by diluted Coomassie Blue and de-stained. BOry was labeled with FITC according to the product manual. Briefly, 2 mg of BOry was dissolved in 0.5 mL of 0.1 M sodium carbonate pH = 10. 50 µL of fresh 4 mg/mL fluorescein isothiocyanate (FITC, Sigma, Saint Louis, MO, USA) was added to the BOry solution in 10 steps of 5 µL each and, in between, the mixture was kept at 4 °C and mixed gently. After 8 h incubation while gently shaking at 4 °C in the dark, NH4Cl was added to a final concentration of 50 mM, and subsequently incubated for another 2 h at 4 °C. Finally, the free dye was removed by overnight dialysis in PBS pH = 7.4 at 4 °C.
Glycan array analysis
The glycan array preparation and analysis was based on the method previously published18. Briefly, BOry was labeled with Alexa Fluor 555 NHS ester (Thermo Fischer Scientific) following manufacturer instructions and YOry binding was determined after incubation with rabbit-anti-His antibody (GenScript, Piscataway, NJ, USA) and Alexa Fluor-555 goat anti-rabbit IgG (Thermo Fischer Scientific). rOrysata (0.5 nM) was incubated in the microarray chip printed with 144 different glycans (Fig. S1). After washing, the fluorescence signal was analyzed on an Agilent G265BA microarray scanner system (Agilent Technologies, Santa Clara, CA, USA). Quantification of the signals was performed with ProScanArray Express (Perkin Elmer, Waltham, MA, USA) and Microsoft Excel software. The average of mean RFU values after background subtraction and standard deviation for four replicate spots was calculated. Average RFU values for each data set were normalized to the highest RFU value and represented as histograms employing Prism 6 software v6.07 (GraphPad, San Diego, CA, USA) (https://www.graphpad.com/).
Agglutination assay
Rabbit red blood cells (RBCs) (purchased from Fiebig-Nährstofftechnik, Idstein, Germany) were prepared for agglutination assays as described before6. In brief, RBCs were rinsed twice with PBS before adding about 2 mg of trypsin and incubating for 10 min at 37 °C. Afterwards, the RBCs were rinsed three times to remove excessive trypsin and finally the RBCs were resuspended in PBS. For the lectin agglutination assays, 10 µl of 1 M ammonium sulfate was mixed with 10 µl of YOry or BOry lectin at different concentrations in a clean test tube. Subsequently, 30 µl of RBCs suspension was added and incubated for 30 min. The lectin Hippeastrum hybrid lectin (HHA) was added as a positive control (PC), and PBS was used as negative control (NC).
Insect cell culture, aggregation assay, cell spreading and AMP expression
S2 cells from Drosophila melanogaster19 were purchased from the Bloomington Drosophila stock center (Indiana University, Bloomington, IN, USA) and are maintained under standard growing conditions in Sf-900 III SFM medium (Thermo Fisher Scientific) supplemented with 10% Gibco fetal bovine serum (FBS) (Thermo Fisher Scientific).
For the agarose beads-mediated aggregation assays, agarose beads were coated with rOrysata first. 10 µl of Ni–NTA agarose beads suspension were washed twice with PBS prior to adding 30 µg of YOry or BOry, PBS was supplemented to a final volume of 50 µl. After mixing at 4 °C for 1 h, excessive lectin was removed by washing the coated beads twice with PBS. Finally, the beads were equilibrated in 200 µl of PBS. In each well of a sterile CELLSTAR Cell-Repellent Surface 96-wells plate (Greiner Bio-One, Vilvoorde, Belgium), 60 µl of a D. melanogaster S2 cell suspension at a density of 106 cells/ml in FBS-free medium was added. For the phosphatase inhibition assay, cells were treated with 20 µl of phosphatase inhibitor (PPI) for 10 min before adding 20 µl of Orysata-coated beads. For the PPI solution, one tablet of Pierce Phosphatase Inhibitor Mini Tablets (Thermo Fisher) was dissolved in 5 ml of PBS to make a stock solution for direct use.
To measure the cell spreading, 105 cells were seeded in each well of a 24-wells plate in a total volume of 500 µL of cell culture medium supplemented with 10% FBS. After overnight incubation, 100 µL of YOry (YO) or BOry (BO) was added to a final concentration of 3, 0.6, 0.3 and 0.03 µM. PBS was used as control. After incubation for 3 h at 27 °C, cells were observed under the microscope and maximum cell length was measured using the image J software20 v2 (https://imagej.net/software/fiji/). In total 120 cells were measured from 4 independent replicates. Data was analyzed by one-way ANOVA with post-hoc Tukey HSD (Honestly Significant Difference) Test Calculator for comparing multiple treatments.
To quantify mRNA expression for AMPs, 100,000 cells were seeded in each well of a 24-wells plate (VWR, Leuven, Belgium) and incubated overnight as mentioned above. Afterwards, YOry or BOry were added to a final concentration of 3 µM. Cells were harvested after 24 h and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. About 1 µg of purified RNA was used for cDNA synthesis with SuperScript IV Reverse Transcriptase (Invitrogen) in a 20 µL reaction system with polyT primers. After diluting the cDNA samples 20-fold, they were used as template for quantitative PCR (qPCR). For qPCR, every 20 µl of reaction system included 8.0 µl of cDNA template, 1.0 µl of primer F, 1.0 µl of primer R and 10 µl of GoTaq qPCR Master Mix (Promega). Reaction cycles are: 3 min at 95 °C, then 39 cycles of 10 s at 95 °C and 30 s at 55 °C. Relative expression was analyzed in qBase (Biogazelle, Gent, Belgium). The Ct values are normalized using two internal references, SdhA and RPL32. All primers are listed in Supplementary Table S2. Statistical differences were calculated in the qBase software.
