Figure 2 | Scientific Reports

Figure 2

From: Identification of Two Mannosyltransferases Contributing to Biosynthesis of the Fungal-type Galactomannan α-Core-Mannan Structure in Aspergillus fumigatus

Figure 2

In vitro assay of CmsA mannosyltransferase activity. (A) Chromatograms of in vitro CmsA mannosyltransferase activity assays using p-nitrophenyl α-d-mannopyranoside as the substrate. A reaction mixture (20 µl) containing 25 mM HEPES-NaOH (pH 7.0), 50 mM NaCl, 15 mM KCl, 2.5% glycerol, 1.5 mM MnCl2, 1.5 mM α-Man-pNP (acceptor), 1.5 mM GDP-Man (donor), and 3.7 µg of purified CmsA was incubated at 37 °C for 24 h. Chromatograms at left show typical results of the assay without CmsA (−CmsA, upper panel) and with CmsA (+CmsA, lower panel). Assays without CmsA yielded only peaks from the substrate (α-Man-pNP) but no other peaks, while fractions with CmsA contained a new reaction product (termed product-cmsA) at 18.0 min. (B) Determination of product-cmsA structure using substrate-specific mannosidases. Upper panels show chromatographs of the purified product-cmsA. Purified product-cmsA was digested by α-1,2-mannosidase (middle panels) and α-1,6-mannosidase (lower panels). Product-cmsA could be digested only by α-1,2-mannosidase and converted to α-Man-pNP. (C) Chromatograms of in vitro CmsA mannosyltransferase activity assays using α-(1 → 2)-mannobiose (a) or α-(1 → 6)-mannobiose (b) as substrates. A reaction mixture (20 µl) containing 25 mM HEPES-NaOH (pH 7.0), 50 mM NaCl, 15 mM KCl, 2.5% glycerol, 1.5 mM MnCl2, 3.7 µg of purified CmsA, 100 mM GDP-Man (donor), and 25 mM α-(1 → 2)-mannobiose (α-Man-(1 → 2)-α-Man) or α-(1 → 6)-mannobiose (α-Man-(1 → 6)-α-Man) (acceptors) was incubated at 30 °C for 60 min. The reaction products were analyzed using HPLC after being labeled with 2-aminopyridine (PA). Chromatograms show typical results from the assay without CmsA (−CmsA, left panel) and with CmsA (+CmsA, right panel). Assays without CmsA yielded only peaks of the substrates (α-Man-(1 → 2)-α-Man-PA or α-Man-(1 → 6)-α-Man-PA) at 25.3 min, whereas those with CmsA exhibited a new reaction product (pyridylaminated product-a-(1 → 2)-M3 or pyridylaminated product-a-(1 → 6)-M3) to which mannose has been transferred and which eluted at 28.9 min.

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