Ac4ManNAz was synthesized following the protocol described by Srinivasa-Gopalan Sampathkumar . In this study, biotin-LC-alkyne analogs were synthesized using the modified commercialized reagent sulfo-NHS-LC-biotin (Pierce Biotechnology, Rockford, IL, USA), which underwent reaction with propargylamine in borate buffer (0.2 M, pH 8) to modify one end of the long chain with an alkyne. HeLa, SW1990, A549 cells MCF-7 and SK-BR-3 cells were obtained from ATCC (USA).
Biotinylation of azido-labeled glycoproteins
HeLa, SW1990, and A549 cells were cultured for 48 h, at 37 °C and 5 % CO2 in DMEM medium (10 % FBS, 100 units·mL−1 penicillin, and 100 ng mL−1 streptomycin) supplemented with 0.2 mM Ac4ManNAz. As a control, an identical volume of PBS was added to the culture medium. Cells were washed three times with ice-cold 0.1 M PBS (phosphate buffer and 0.15 M NaCl, pH 7.4) and then treated with 1 mL of reaction solution (0.2 mM biotin-LC-alkyne, 0.2 mM Tris-triazoleamine catalyst, 1.0 mM CuSO4, and 2.0 mM sodium ascorbate in ice-cold PBS). After incubation for 1 h at 4 °C, the cells were fixed in 4 % paraformaldehyde for 30 min and then washed three times with PBS. Cells were stained using 1 mL of a 1:1000 dilution of avidin-Cy3 (Boster, Wuhan, China) at room temperature for 30 min. After washing samples with PBS carefully, fluorescence images were obtained using laser scanning confocal microscopy.
Purification of biotinylated glycoproteins
Cells were cultured in Ac4ManNAz for 48 h as previously described and treated with biotin-LC-alkyne at 4 °C. After incubation for 1 h, the reaction solution was removed, and the cells were carefully washed with ice-cold PBS. The cells were scraped into ice-cold hypotonic buffer (10 mM HEPES, pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 1× protease inhibitor cocktail, 1 mM NaF, and 1 mM Na3VO4), incubated on ice for 15 min, and then lysed by ultrasonication. After centrifugation at 1000g and 4 °C for 10 min, the supernatant was collected. Next, KCl was added to a concentration of 150 mM. Protein concentrations were quantified and diluted to 1 mg mL−1. SMMs were added at a volume that was 1/10 of the volume of the supernatant, and the samples were incubated at 4 °C for 1 h. Finally, SMMs were carefully and separately washed with ice-cold PBS and 1 M KCl, suspended in 1× SDS-PAGE loading buffer, and boiled for 10 min, resulting in the purified glycoprotein samples.
The purified glycoproteins were separated using SDS-PAGE and transferred onto a PVDF membrane. Membranes were blocked in 4 % nonfat dry milk for 1 h at room temperature (RT). A 1:1000 dilution of HRP-labeled streptavidin (Boster, Wuhan, China) was added, and the membrane was incubated at RT for 1 h. Biotinylated glycoproteins were detected using electrochemiluminescence (ECL). To detect gp96 protein expression, the membrane was incubated with a 1:1,000 dilution of an anti-gp96 antibody (Santa Cruz, Dallas, USA) overnight at 4 °C followed by incubation with an HRP-labeled secondary antibody for 1 h. Sialyltransferase was analyzed using 1:1,000 dilution of anti-ST3Gal (Santa Cruz) and 1:1000 dilution of anti-ST6Gal1 (Immunoway, Newark, USA) as primary antibodies. Protein expressions were detected using ECL. β-actin served as an internal control and a 1:1000 dilution of anti-β-actin antibody was used.
HPLC–MS/MS and the analysis were performed by Shanghai Applied Protein Technology Co., Ltd.
The mRNA of the different cell lines was isolated using a PolyATtract System 1000 (Promega, Madison, USA) and reverse transcribed to cDNA using a PrimeScript RT reagent kit (Takara, Dalian, China). Real-time PCR was performed using an SYBR Premix EX Taq II kit (Takara). The primers used in this study were as follows: gp96 forward primer, 5′-TGT TTC CCG CGA GAC TCT TC-3′; gp96 reverse primer, 5′-CAC ACC AGC ACT GCT GTG CAC CAC AGT GG-3′; GAPDH forward primer, 5′-GTG AAG GTC GGA GTC AAC G-3′; and GAPDH reverse primer, 5′-TGA GGT CAA TGA AGG GGT C-3′.
A 96-well plate was coated with a 1:1000 dilution of an anti-gp96 antibody at 37 °C for 4 h and subsequently blocked with 1 % normal goat serum at 4 °C overnight. After careful washing with PBS, purified glycoproteins (0.5 mg mL−1) were added and incubated at 37 °C for 1 h. After washing with PBS, a 1:1000 dilution of HRP-streptavidin or a 1:1000 dilution of HRP-WGA (Sigma, St. Louis, MO, USA) was added and incubated at RT for 1 h. Finally, the signal was visualized using TMB as a chromogen. To detect sialyation of gp96 in MCF-7 and SK-BR-3 cells, we performed ELISA using HRP-WGA.