**3. Materials and Methods**

#### *3.1. Cloning, Expression and Purification the Glycosyl Hydrolase Domain of Emn*

*B. circulans* TN-31 (ATCC®290101™) was obtained from the American Type Culture Collection and grown on agar medium at 30 ◦C as recommended by the ATCC. Genomic DNA was isolated using a standard extraction protocol. The coding sequence of the full-length mature protein (3150 bp) was amplified using primers Emn1 (5 -CTCGAGTATACCGCATCAGATGGGG-3 ) and Emn2 (5 -CTCGAGTTACTCCAAGCCATCCTGCC-3 ). The glycosyl hydrolase domain sequence of *emn* (GH-emn; 1092 bp) was PCR-amplified using primers GH-emn1 (5 -CATATGTATACCGCATCAGAT GGGG-3 ) and GH-emn2 (5 -CTCGAGTTAATTGTAGCGTTCCGCTTCGA-3 ). Both PCR fragments

were cloned into the *Nde*/ and *Xho*I sites of the pET14b expression plasmid and transformed into *E. coli* BL21 DE3 pLysS. For GH-emn production, freshly prepared LB broth containing 100 μg/mL ampicillin was inoculated with a preculture of pET14b-GH-emn transformant and incubated at 30 ◦C until optical density measured at 600 nm (OD600) reached 0.2. Gene expression was induced by adding isopropyl-β-thio-galactopyranoside (1 mM final concentration) to the culture medium. After 4 h of induction at 30 ◦C, cells were collected by centrifugation and resuspended in lysis buffer consisting of Tris-HCl pH 7.5 containing 150 mM NaCl, 1 mM MgCl2, 1 μg/mL DNase and protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Cells were broken using a French press (1500 psi; 3 passages) and cell debris were removed by ultracentrifugation at 4 ◦C for 30 min at 110,000 × g. The supernatant was then applied to a His-trap column (GE Healthcare, Pittsburgh, PA, USA) and the protein eluted in a gradient of 10–500 mM imidazole, prior to gel filtration using a Superdex 200 column (GE Healthcare). Aliquots of the purified GH-emn protein were stored at –80 ◦C in 20 mM Tris pH 7.5 buffer containing 150 mM NaCl and 20% glycerol until further use. Protein concentrations were determined using the Pierce BCA protein assay kit as recommended by the manufacturer.

#### *3.2. Assays Using Synthetic Mannosides Substrates*

<sup>α</sup>*-*d*-*Man*p*-(1→6)-α-d-Man-(1→6)-α-d-Man-(1→octyl] (octyl trimannoside-) and <sup>α</sup>-d-Man*p*-(1→6) -α-d-Man-(1→6)-α-d-Man-(1→6)-α-d-Man-(1→6)-α-d-Man-(1→octyl (octyl pentamannoside) were chemically synthesized as described [12,13]. Endomannanase reaction mixtures contained the synthetic mannosides (100 μM) and purified GH-emn (0 to 500 μg/mL) in a total volume of 100 μL in 10 mM ammonium acetate buffer (pH 6). Control reactions contained buffer in place of the purified enzyme. Enzymatic reactions were carried out in LC/MS vials at 50 ◦C for 16 h at which point the enzyme was inactivated by heating at 65 ◦C for 30 min [8]. In the experiment where increasing concentrations of GH-emn were used, the reactions mixture were incubated at 50 ◦C for 2 h instead of 16 h.

#### *3.3. Assays Using Purified Mycobacterial PIMs and Lipoglycans*

Purified *M. tuberculosis* H37Rv phosphatidyl-*myo*-inositol hexamannosides (PIM6) and *M. smegmatis* LAM were received from BEI resources. LM was extracted and purified from *M. smegmatis* whole cells as described previously [14]. Purified PIM6 (20 μg) was deacylated using monomethylamine following an established procedure [15] and further digested with GH-emn (0.25 mg/mL) in 10 mM ammonium acetate buffer (pH 6) for 24 h at 50 ◦C. Native LM from *M. smegmatis* (50 μg) was deacylated with 0.2 N NaOH (200 μL) at 37 ◦C and then neutralized with 10% aqueous glacial acetic acid. Deacylated LM (d-LM) was separated from sodium salts using an Amicon ultra-0.5 mL centrifugal filter (3 kDa MWCO) and resuspended in 10 mM ammonium acetate buffer (pH 6) prior to digestion with GH-emn as described for d-PIM6. D-LM and d-PIM6 before and after enzymatic digestion were analyzed by LC/MS (see next section).

Native LAM (40 μg) and LM (40 μg) from *M. smegmatis* digested with GH-emn (50 μg) in 100 mM citrate phosphate buffer pH 6.6 for 16 h at 50 ◦C were also was analyzed by SDS-PAGE followed by silver staining alongside 10 μg of undigested starting material.

#### *3.4. Analysis of Substrates and Reaction Products by Liquid Chromatography—Mass Spectrometry*

Separation of the GH-emn-digested products was performed using a reverse-phase X-bridge C18 column (50 mm × 2.1 mm; 1.7 μM) on a Waters ACQUITY UPLC system. The mobile phases used were water (solvent A), acetonitrile (solvent B) and 0.5 M ammonium acetate (solvent C) under the following gradient conditions: 0–0.3 min (10% B), 0.3–3 min (70% B), 3–4.8 min (98% B), and 4.8–6.8 min (10% B) at a flow rate of 400 μL/min. A constant 2% solvent C was maintained throughout the LC run.

Mass spectrometry (MS) was performed using a Bruker maXis plus II high-resolution quadrupole time-of-flight (Q-TOF). The electrospray ionization (ESI) source settings were as follows: end plate offset voltage 500 V, capillary voltage 3500 V, nebulizer gas pressure 3.0 bar, dry gas flow rate 10 L/min. Two different tune parameters were used for analyzing native and GH-emn-digested products. For detecting low molecular compounds, the tune parameters were: funnel RF 300 Vpp, multipole RF 300 Vpp, ion energy 3.0 eV, low mass range for ion transmission *m*/*z* 100, collision energy 8.0 eV, collision RF 450 Vpp, pre-pulse storage 5.0 μs, ion cooler RF 800 Vpp and transfer time 80.0 μs. For detecting high molecular weight, d-LM tune parameters were: funnel RF 400 Vpp, multipole RF 400 Vpp, ion energy 3.0 eV, low mass range for ion transmission *m*/*z* 600, collision energy 8.0 eV, collision RF 2500 Vpp, pre-pulse storage 5.0 μs, ion cooler RF 800 Vpp and transfer time 140 μs. A data analysis was performed using Bruker compass data analysis 4.4 SR1.
