β galactosidase from bovine testes

Five to fifty units (determined empirically for each enzyme) of exo-glycosidase (Jack bean α-mannosidase, Sigma-Aldrich; β-galactosidase from bovine testes, Sigma-Aldrich), endo-glycosidase (endo-β-N-acetylglucosaminidases Endo Tv [21 ] and Endo H, Roche), and glyco-amidase (N-glycoamidase F, PNGase F) were used to modify the surface glycosylation of the EVs. Each enzyme was added to 1 μg of PKH26-labelled EVs (total protein content) in 200 mM sodium phosphate buffer including protease inhibitors (Roche) at pH 4.5 for α-mannosidase and β-galactosidase, pH 5.5 for endoglycosidases and pH 7.4 for glyco-amidase treatment. The reactions, and enzyme-free negative controls, were incubated in the dark for 14 h at 37°C. In parallel, the activity of β-galactosidase and PNGase F were verified by digestion of bovine fetuin under the same conditions used for the EVs. Similarly, the activity of Endo H, Endo Tv and α-mannosidase were verified by digestion with bovine RNAse B.

Purified IgA1 (5 µg) was digested sequentially with exoglycosidases at 37°C under the following conditions: neuraminidase from Arthrobacter ureafaciens (Nacalai Tesque, Kyoto, Japan), 5 mU in 25 µL of 80 mM sodium acetate buffer (pH 5.0) for 18 h; β-galactosidase from bovine testes (Sigma), 2.2 mU in 5 µL of 80 mM sodium acetate buffer (pH 5.0) overnight; β1,4-galactosidase from Streptococcus pneumoniae (Calbiochem, La Jolla, CA), 3 mU in 5 µL of 80 mM sodium acetate buffer (pH 5.0) overnight.

For desialylation, 10 µg of AK23 mAb was incubated with 52.5 mU of neuraminidase from Vibrio cholerae (Sigma) for 120 h at 37 °C in a 50 mM sodium acetate buffer (pH 5). Enzyme and buffer were removed using Amicon Ultra 0.5 mL centrifugal filters with cut-off of 100 kDa (Merck Millipore, Burlington, MA, USA). Fifty micrograms of patient’s IgG was incubated with 37.5 mU for 120 h at 37 °C in a 50 mM sodium acetate buffer (pH 5). Enzymatic digestion efficiency was tested by Eastern blot using biotinylated conjugated Maackia amurensis lectin II (MAL II, Bioworld, Dublin, OH, USA) or Sambucus nigra lectin (SNA, EBL, Vector Laboratories, Inc, Burlingame, CA, USA).
For degalactosylation, 10 µg of AK23 mAb was incubated with 28 mU of β-galactosidase from bovine testes (Sigma) for 72 h at 37 °C in a 50 mM sodium acetate buffer (pH 5) (complete digestion). Enzyme and buffer were then removed using Amicon Ultra 0.5 mL centrifugal filters with cut-off of 100 kDa (Merck Milipore, Burlington, MA, USA). Enzymatic digestion efficiency was tested by Eastern blot using biotinylated conjugated Erythrina cristagalli lectin (ECA, Vector Laboratories, Inc, Burlingame, CA, USA).