Arthrobacter ureafaciens sialidase

Sialidase treatment or mild periodate/borohydride oxidation/reduction was used to eliminate homotypic or heterotypic CD33 cis interactions as described [8 (link), 23 (link)]. Briefly, NK cells were treated with Arthrobacter ureafaciens sialidase (Roche, Mannheim, Germany) at 10-20 mU/10⁶ cells in RPMI 1640 pH 6.9 for 1.5 h at 37°C and then extensively washed with 2% FCS in PBS. For mild periodate oxidation, cells (1 × 10⁶ cells/mL) were incubated with sodium periodate (1 mM, Sigma-Aldrich) in PBS for 30 min on ice and then quenched with 1 mM glycerol and washed twice with cold PBS. Cells were then treated with freshly prepared sodium borohydride (20 mM) for 10 min to reduce the aldehydes generated by periodate. They were washed again with PBS and culture medium. Finally, cells were counted and used for functional assays or stained with the corresponding mAb or with biotinylated Sambucus nigra lectin (SNA, Vector Laboratories, Burlingame, CA, USA) to test the effectiveness of the treatment. Cell viability after treatment was determined by using the MTT assay [24 (link)].

The N-glycans present on the serum proteins were analysed using DSA-FACE technology as described previously [12 (link), 24 (link)]. Briefly, the N-linked glycans were denatured and released from serum glycoproteins by adding the peptide N-glycosidase-F (PNGaseF) (New England Biolabs, Boston, MA) in 2 μl of serum. Thereafter, N-glycans were labelled with APTS (8-aminonaphtalene-1, 3, 6-trisulphonic acid) (Invitrogen, Carlsbad, CA). Sialic acid was removed using Arthrobacter ureafaciens sialidase (Roche Bioscience, Palo Alto, CA) and the processed samples were analysed using a capillary electrophoresis-based ABI3500 Genetic Analyzer (Applied Biosystems, Foster city, CA). Twelve obvious N-glycan peaks detected in all serum samples were analysed using the GeneMapper v4.1 software (Applied Biosystems). The abundance of each N-glycan peak was described by normalizing its height to the sum of the heights of all 12 peaks. Before applying DSA-FACE method to discover N-glycan biomarkers for IgD myeloma, we have evaluated the feasibility of this method, such as test reproducibility. The coefficient of variation (CV) value of run-to-run was less than 15% for each N-glycan marker. In addition, a pooled serum was aliquoted as the standard sample, and it was analyzed in each experiment to ensure the stability of the system and the reliability of the results (Additional file 1: Table S1).

Native acutobin was purified from D. acutus venom (Hunan province, China) [7] (link). Restriction enzymes were from Promega (Madison, WI, USA). N-Tosyl-Gly-Pro-Arg- p-nitroanilide was from Sigma Aldrich (St Louis, MO, USA). Recombinant PNGase F of Flavobacterium meningosepticnm and Arthrobacter ureafaciens sialidase (neuraminidase) were purchased from Roche (Mannheim, Germany) and Bio-Rad Laboratories (Hercules, CA, USA), respectively. Pre-cast NuPAGE Novex Bis-Tris mini gels and buffers were obtained from Invitrogen Inc. (Carlsbad, CA, USA).

Serum protein N-glycan analyses were performed as described previously [11] (link). Briefly, the N-glycans present on the proteins in 2 µl of serum were released with peptide N-glycosidase-F (PNGaseF) (New England Biolabs, Boston, Mass) and then labeled with 8-aminonaphtalene-1, 3, 6-trisulphonic acid (APTS) (Invitrogen, Carlsbad, Calif).Sialic acid was removed with arthrobacter ureafaciens sialidase (Roche Bioscience, Palo Alto, Calif), and the processed samples were analyzed with DSA-FACE technology using a capillary electrophoresis-based ABI3500 Genetic Analyzer (Applied Biosystems, Foster City, Calif). The 9 most intense peaks that were detected in all samples (together, these peaks accounted for >90% of total serum N-glycans) were analyzed using GeneMapper software (Applied Biosystems). Each structure of N-glycan was described numerically by normalizing its height to the sum of the heights of all peaks.

HEK-CCR5 cells (1 × 10⁷) were detached with 0.5 mM EDTA in PBS, washed once with medium, and treated for 1.5 h at 37 °C with 200 μL DMEM supplemented with 0.3 U Arthrobacter ureafaciens sialidase from (Roche) or with 200 μL unsupplemented DMEM. Cells were then washed twice with FACS buffer (1 × PBS, 1 mM EDTA, 1% BSA) prior to analysis by flow cytometry.