α1 2 3 mannosidase

The following enzymes were used for confirming the glycan linkages; sialidase (Neuraminidase, Arthrobacter ureafaciens) (Roche Applied Science) specific for α2-3,6,8,9 sialic acid; α2-3-specific sialidase (GLYKO Sialidase S from Streptococcus pneumoniae, ProZyme); β1-3,4 galactosidase (bovine) (ProZyme); β 1 to 4,6 galactosidase (jack bean; Canavalia ensiformis) (Seikagaku Biobusiness Corporation); α1-2,3,6 mannosidase (C. ensiformis) (Seikagaku Biobusiness Corporation); α1-2 mannosidase (Aspergillus saitoi) (Agilent Technologies); α1-6 mannosidase (Xanthomonas manihotis) (New England Biolabs,); α1-2,3 mannosidase (X. manihotis) (New England Biolabs); β-N-acetylhexosaminidase (C. ensiformis)(Seikagaku Biobusiness Corporation); and α1-2,3,4,6 Fucosidase (bovine) (New England Biolabs).
The colorimetric glycosidase substrates p-nitrophenyl-α-D-mannopyranoside (pNP-α-Man), p-nitrophenyl N-acetyl-β-D-glucosaminide (pNP-β-HexNAc), and p-nitrophenyl-ß-D-galactopyranoside (pNP-β-Gal) were from Tokyo Chemical Industry Co., Ltd. The fluorescence-based sialidase substrate 4-Methylumbelliferyl-α-D-N-acetylneuraminic acid sodium (4-MU-NANA) was from Sigma-Aldrich.

A 0.5 × 20 cm column was packaged with 6 ml of the BioGel P4 resin (Bio-Rad) and conditioned with a 25 mM NH₄CH₃COO (Sigma–Aldrich) buffer. Ten nanomoles of the total fOS were loaded into the column and 24 fractions (500 ul per fraction) were collected. Sample were dried in vacuum and later resuspended in water and desalted by ion exchange chromatography. Ten percent of each fraction was collected for FACE analysis, and the remaining 90% was divided in half and used for macrophage stimulation in biological duplicates. For the mannosidase digestion, 10 nmol of fOS or the synthetic ManGlcNAc disaccharide were treated with 40 U of α1-6 mannosidase (New England Biolabs) and 32 U of α1-2, 3 mannosidase (New England Biolabs). Samples were then mixed with 3 volumes of cold ethanol and centrifuged at 20,000 × g for 20 min at 4 °C. The supernatant was dried in vacuum and later resuspended in water and desalted by ion exchange chromatography. Ten percent of the digested product was collected for FACE analysis and the remaining 90% was divided in half and used for macrophage stimulation in biological duplicates.

Purified monogalactosylated complex truncated glycans 7 and 9 were first treated with β-N-acetylglucosaminidase (New England Biolabs, catalog no.: #P0744) to cleave the nonreducing terminal GlcNAc residue of glycans 1 and 2. Reactions were monitored by HPAEC-PAD, and glycans were purified using HPLC as described previously with the following gradient: 4.0% to 12.0% MP-A:MP-B (MP-A: 95% acetonitrile in MQ H₂O containing 0.1% TFA; MP-B: MQ H₂O containing 0.1% TFA) at 2 ml/min over 34 min, followed by an increase 50% MP-A for the next 1.0 min, then maintained at 50% MP-A for another 3 min, at 40 °C. As aforementioned, purified fractions were confirmed by HPAEC-PAD, combined, and lyophilized. To pseudohybrid glycan 1, α1-2,3-mannosidase (New England Biolabs, catalog no.: #P0729) was added to cleave the branching monomannose residue on the α3 antenna to form linear pentasaccharide 5. The resulting product was then purified by HPLC using the same method. The β- and α-anomers of glycan retention times are as follows: pseudohybrid glycan 1, 38.8 min and 38.9 min; hybrid glycan 2, 26.8 min and 31.5 min; linear glycan 5, 28.7 min and 33.6 min.

Pre-formed fixed Detroit 562 pharyngeal cell monolayers of the 96-well microtiter plate were treated with 30 µL/well reaction volumes of each of the exoglycosidases. For mannosidases, the reaction volume comprises 3 µL 1 × GlycoBuffer 1 (NEB, Notting Hill, Australia), 0.3 µL 100 µg/mL purified BSA (NEB, Notting Hill, Australia), 0.2 µL α1-6 mannosidase (8 U) or α1-2,3 mannosidase (8 U) (NEB, Notting Hill, Australia), and 26.5 µL PBS. For Sialidase A, 6 µL 5 × Reaction Buffer B (250 mM sodium phosphate pH 6.0) (Prozyme, Hayward, CA, USA), 0.2 µL Sialidase A (1 × 10⁻³ U) (Prozyme, Hayward, CA, USA), and 23.8 µL PBS. Untreated PBS wells representing the intact surface glycome of the fixed Detroit 562 pharyngeal cell monolayers were also included as a control. The plate was incubated for 2 h, 37 °C. Once incubated, the wells were washed once with PBS. Glycan removal was confirmed via lectin binding assay as per [25 (link)], with exoglycosidase pre-treated pharyngeal cell monolayers incubated with either biotinylated Hippeastrum hybrid lectin (binding mannose residues) (Vector Laboratories, Burlingame, CA, USA) or biotinylated Sambucus nigra lectin (binding sialic acid residues) (Vector Laboratories, Burlingame, CA, USA) (Supplementary Material 5, Figure S2).

A reporter assay was performed as described previously (64 (link)). Briefly, the 96-well flat-bottom ELISA plate (MaxiSorp, Thermo Scientific Nunc) was coated with 100 μl containing 40 ng of LPS or polysaccharides in 100 mm sodium bicarbonate buffer, pH 9.5, for 16 h at 4 °C. Dectin-2 or mock BWZ cells were then cultured in the well, and β-galactosidase activity was monitored as described previously (64 (link)). For the mannosidase treatment, a 96-well plate was coated with 1.6 ng of Man-LPS. After discarding the solution, wells were washed once with 200 μl of PBS and blocked with 100 μl of 4% BSA in PBS for 1 h at 25 °C. Blocked wells were washed, and 50 units of α1–2,3 mannosidase (New England BioLabs) was added to the wells and incubated at 37 °C for 14 h. The reporter cells were added to test the binding. For the reporter assay with bacteria, 1.0 × 10⁶ of PFA-fixed H. alvei were immobilized on the 96-well plate as described above.

Human fibronectin and vitronectin were purchased from R&D Systems (Minneapolis, MN), human laminin from Millipore (Temecula, CA), β-1,6-glucanase from Takara Bio Inc. (Otsu, Shiga, Japan), β-1,3-glucanase (lyticase) from Sigma and α1-2,3 mannosidase and α1-6 mannosidase from New England Biolabs (Ipswich, MA). Proteinase K and bovine serum albumin (BSA) were obtained from BioShop Canada Inc. (Burlington, Ontario, Canada), trypsin from Promega (Madison, WI) or Biocentrum (Krakow, Poland) and horseradish peroxidase-conjugated streptavidin solution (SA-HRP) from MP Biomedicals (Solon, OH).