O glycosidase

Five μg of purified human lubricin was diluted in 100 μl reaction buffer (50 mM NaHPO₃, pH 7.2, 0.01% w/v BSA) and treated with 80 mU of neuraminidase (ProZyme, Hayward, CA) and/or 4 mU of O-glycosidase (Roche, Germany) at 37°C overnight. The digests were mixed with 100 μl of 2X SDS-PAGE loading buffer containing 2% v/v BME and boiled for 5 min. Two μl were then electrophoresed in 4–20% gradient gels (Bio-Rad, Hercules, CA) and transferred to Immobilon P-PVDF (Millipore, Bedford, MA).
Twenty μl of serum-free conditioned medium from HEK-293T cells containing rhPRG4 were treated with 30 mU of neuraminidase and/or 2 mU of O-glycosidase in a 50 μl reaction at 37°C overnight. The digested samples were then mixed with 50 μl of 2X SDS-PAGE loading buffer and boiled for 5 min. Five μl of sample was electrophoresed and transferred to PVDF.

Human recombinant full-length MR with His-tag, expressed in mouse myeloma cell line NS0, was purchased from R&D systems, anti-human IgG Alexa-Fluor 488-conjugated from Jackson ImmunoResearch, and anti–penta-His Alexa-Fluor 488 conjugate from Qiagen. Biotinylated Concanavalin A, R. communis agglutinin-I, Maackia amurensis lectin-I, Sambucus nigra lectin, fluorescein Concanavalin A, fluorescein Wheat germ agglutinin, and Man-BSA were purchased from Vector Laboratories; neuraminidase and Protein A-agarose beads from Roche; O-glycosidase, PNGase F, α-galactosidase, β1,3-galactosidase, and β1,4-galactosidase from New England Biolabs; sequencing grade trypsin from Promega; Glucitol-polyacrylamide (PAA), Man₃-PAA, and Glucitol-PAA biotin from Lectinity; GlcNAc and GlcNAc-BSA from Carbosynth. All other chemicals were purchased from Sigma-Aldrich if not indicated differently.

Two μl of MCF-7 protein extract (7.5 μg protein) were mixed with 3 μl 16x deglycosylation buffer (400 mM sodium phosphate, pH 7.4), 0.5 μl protease inhibitor (Sigma-Aldrich) and 9 μl water. The deglycosylation reaction was started by adding either 0.5 μl each of N-glycosidase (PNGase F, New England Laboratories), O-glycosidase (Roche Applied Sciences) and sialidase (Roche Applied Sciences) or by adding 1.5 μl PNGase F alone. For mock-treatment, 1.5 μl water was added to the reaction mix instead of the enzymes. The mixtures were incubated at 37°C for 24 hours and analyzed by Western blot analysis for TMEM26 protein expression. To inhibit N-glycosylation, MCF-7 cells were treated with tunicamycin (Merck Chemicals) at a final concentration of 5 μg/ml for up to five days.

ProBNP is post-translationally glycosylated to varying degrees in its N-terminal region, at Thr36, Ser37, Ser44, Thr48, Ser53, Thr58 and Thr71. [10] (link) NT-proBNP is similarly glycosylated. The Elecsys proBNP II system (Roche Diagnostics, Germany) is comprised of a capture monoclonal antibody that recognizes NT-proBNP[27–31] and a monoclonal signal antibody that recognizes NT-proBNP[42–46], which contains a glycosylation site at amino acid residue 44. [14] (link) Notably, O-linked oligosaccharide attachment inhibits the binding of the signal antibody to NT-proBNP. [15] (link), [16] (link) We therefore postulated that NT-proBNP measured using Elecsys proBNP II is actually only nonglycoNT-proBNP. To measure total NT-proBNP, plasma samples were incubated for 24 h at 37°C with or without a cocktail of deglycosylating enzymes, included O-glycosidase (Roche Diagnostics) and neuraminidase (Roche Diagnostics) at final concentrations of 4.25 and 42.5 mU/ml, respectively, as described previously. [10] (link)
[14] (link) NT-proBNP levels were then measured using Elecsys proBNP II, after which the glycoNT-proBNP level was calculated as: total NT-proBNP – nonglycoNT-proBNP.

For PNGase F treatment, proteins (50 μg) were denatured by boiling in 20 μl of PBS containing 0.5% SDS, 1% 2-mercaptoethanol, and 4 mM EDTA. After the solution had been diluted with four volumes of PBS containing Nonidet P-40 (NP-40, final concentration 0.5%), PNGase F (1,000 units, New England Biolabs) was added, and the solution was incubated for 3 h at 37°C. For sialidase and O-glycosidase treatment, APP was first immunoprecipitated from mouse brain membrane fraction. After washing the beads with TBS/0.1% NP-40, immobilized APP was incubated with 10 mU sialidase (Arthrobacter ureafaciens, Nacalai tesque) with or without 1.5 mU O-glycosidase (Roche) in acetate buffer (100 mM sodium acetate pH 4.5, 50 mM NaCl, 0.1% NP-40, 1 mM EDTA, protease inhibitor cocktail) for 12 h at 37°C.