Background Information

The results of digestion of a hypothetical protein with two N-linked carbohydrate chains as it moves through the ER and Golgi complex with various enzymes are shown in Figure 12.4.8. At 0 min, both N-linked chains are high-mannose type. They have lost their Glc residues and one Man residue in the ER. Both are sensitive to endo H and PNGase F digestion, yielding a protein with only two remaining GlcNAc residues in the case of endo H diges tion, and no carbohydrate at all in the case of PNGase F digestion. These sugar chains are resistant to the other enzyme digestions.

At 45 min, the protein is in the medial Golgi complex and both sugar chains have been processed by Golgi a-mannosidase I. However, one of the chains (left) has been partially processed by Golgi a-mannosidase III to an endo H-re-sistant/endo D-sensitive chain. The other chain (right) has received a GlcNAc residue from GlcNAc transferase I, but has not encountered

Figure 12.4.8 Schematic diagram showing results that could be obtained for a hypothetical protein with two N-linked glycosylation sites as it moves through the Golgi complex. Assume that the protein has been biosynthetically labeled with an amino acid precursor (such as [35S]Met) for 10 min and chased in the absence of label for 45 min and 120 min. The protein is then precipitated with a specific antibody. At each time point, equal amounts of the sample are analyzed by flourography after one-dimensional SDS-PAGE, either without any digestion (control; C) or following digestion with endo H (H), endo D (D), endo F2 (F2), PNGase F (PNG), or sialidase (Sia). Oligosaccharide structures consistent with the banding patterns are shown below the schematic gel pattern.

Figure 12.4.8 Schematic diagram showing results that could be obtained for a hypothetical protein with two N-linked glycosylation sites as it moves through the Golgi complex. Assume that the protein has been biosynthetically labeled with an amino acid precursor (such as [35S]Met) for 10 min and chased in the absence of label for 45 min and 120 min. The protein is then precipitated with a specific antibody. At each time point, equal amounts of the sample are analyzed by flourography after one-dimensional SDS-PAGE, either without any digestion (control; C) or following digestion with endo H (H), endo D (D), endo F2 (F2), PNGase F (PNG), or sialidase (Sia). Oligosaccharide structures consistent with the banding patterns are shown below the schematic gel pattern.

Post-Translational

Modification:

Glycosylation

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