Figure 6.

Hydropathy analysis of ranked glycopeptides and UDP–glucose glycoprotein-glucosyltransferase monoglucosylation of two purified tryptic glycopeptide substrates. (A) Hydropathy plots for monoglucosylated glycopeptides from acid phosphatase using the Kyte–Doolittle algorithm (see Methods). The x axis shows the position of each amino acid with respect to the N-linked glycan (position 0), where position 1 represents the amino acid immediately carboxy-terminal to the asparagine residue. The top panel shows the average Kyte–Doolittle value at each position for the group 1 glycopeptides from Fig. 4. The hydrophobic amino acids give positive values, and in a three-amino-acid window, a hydrophobic patch is defined as a positive average value for three consecutive amino acids. Note that the main difference between AcP_101–118 and AcP_101–114 is the hydrophobic patch downstream from residues 12–14. (B) Monoglucosylation of purified glycopeptides. AcP (280 μg) was digested with trypsin. Purification of glycopeptides T_248–270 and T_386–395 of AcP was followed by micro-liquid-chromatography–electrospray-massspectrometry as described for Fig. 2B. Each purified glycopeptide was then monoglucosylated with recombinant UDP–glucose glycoprotein:glucosyltransferase (25 nM) in separate reactions containing 14, 28 or 56 μM each purified glycopeptide and 1 mM UDP–glucose at pH 8 for 1.5 h at 37 °C. The molar incorporation of glucose (peak ratio) for each reaction was calculated as for Fig. 2B.