Passage
Dystroglycan (Dg) is a transmembrane protein that mediates interactions between the cytoskeleton and the extracellular matrix (ECM) . It binds the ECM protein laminin in an interaction that is required for intercellular communication and muscle membrane structural stability. The Dg-laminin interaction is facilitated by a long carbohydrate chain called an O-mannosyl glycan, which is covalently linked to Dg (Figure 1) .
Figure 1 Schematic of interaction between Dg and laminin, mediated by the Dg-linked O-mannosyl glycanThe O-mannosyl glycan is added to Dg by a series of enzymes called glycosyltransferases. The first enzyme in this series, protein O-mannosyltransferase (POMT) , attaches D-mannose to the hydroxyl groups of serine and threonine residues in an α-linkage through the anomeric carbon. The next sugar in the chain then forms a β-1,4 linkage to the mannose residue, followed by several more steps to produce the full glycan. Genetic inactivation of any of the enzymes in the pathway results in an incomplete glycan and failure to bind laminin, and is the underlying cause of several severe forms of muscular dystrophy known as dystroglycanopathies.Researchers investigating dystroglycanopathies recently discovered the functions of two enzymes in the pathway: protein O-mannose kinase (POMK) and fukutin. POMK phosphorylates Dg-linked mannose at carbon 6, and fukutin adds the sugar ribitol 5-phosphate to the glycan chain. To assess the roles of POMK and fukutin relative to each other, scientists conducted experiments in which lysates from muscle cells deficient in either POMK or fukutin were incubated with ³²P-labeled ATP or ³H-labeled activated ribitol 5-phosphate. Combined lysates containing both fukutin and POMK were also tested. Dg was then purified and detected by autoradiography and western blot using an antibody against Dg, as shown in Figure 2.
Figure 2 Autoradiograms and western blots of Dg purified from lysates incubated with ³²P-labeled ATP or ³H-labeled ribitol 5-phosphate
Adapted from Kanagawa M, Kobayashi K, Tajiri M, et al. Identification of a Post-translational Modification with Ribitol-Phosphate and Its Defect in Muscular Dystrophy. Cell Rep. 2016;14(9) :2209-23.
-Which conclusion about the functions of POMK and fukutin is supported by the autoradiogram in Figure 2?

Question

Passage
Dystroglycan (Dg) is a transmembrane protein that mediates interactions between the cytoskeleton and the extracellular matrix (ECM) . It binds the ECM protein laminin in an interaction that is required for intercellular communication and muscle membrane structural stability. The Dg-laminin interaction is facilitated by a long carbohydrate chain called an O-mannosyl glycan, which is covalently linked to Dg (Figure 1) .

Figure 1 Schematic of interaction between Dg and laminin, mediated by the Dg-linked O-mannosyl glycanThe O-mannosyl glycan is added to Dg by a series of enzymes called glycosyltransferases. The first enzyme in this series, protein O-mannosyltransferase (POMT) , attaches D-mannose to the hydroxyl groups of serine and threonine residues in an α-linkage through the anomeric carbon. The next sugar in the chain then forms a β-1,4 linkage to the mannose residue, followed by several more steps to produce the full glycan. Genetic inactivation of any of the enzymes in the pathway results in an incomplete glycan and failure to bind laminin, and is the underlying cause of several severe forms of muscular dystrophy known as dystroglycanopathies.Researchers investigating dystroglycanopathies recently discovered the functions of two enzymes in the pathway: protein O-mannose kinase (POMK) and fukutin. POMK phosphorylates Dg-linked mannose at carbon 6, and fukutin adds the sugar ribitol 5-phosphate to the glycan chain. To assess the roles of POMK and fukutin relative to each other, scientists conducted experiments in which lysates from muscle cells deficient in either POMK or fukutin were incubated with ³²P-labeled ATP or ³H-labeled activated ribitol 5-phosphate. Combined lysates containing both fukutin and POMK were also tested. Dg was then purified and detected by autoradiography and western blot using an antibody against Dg, as shown in Figure 2.

Figure 2 Autoradiograms and western blots of Dg purified from lysates incubated with ³²P-labeled ATP or ³H-labeled ribitol 5-phosphate
Adapted from Kanagawa M, Kobayashi K, Tajiri M, et al. Identification of a Post-translational Modification with Ribitol-Phosphate and Its Defect in Muscular Dystrophy. Cell Rep. 2016;14(9) :2209-23.
-Which conclusion about the functions of POMK and fukutin is supported by the autoradiogram in Figure 2?

Quizplus · Accepted Answer

11ecba2d_0e12_2f75_a3bf_3b524d9b586d_MD0008_00 In autoradiography, potentially radiolabeled biomolecules, including proteins, nucleic acids, and carbohydrates, are separated in gels. Radioactivity is then detected by exposure to radiation-sensitive film, and molecules that contain radioactive atoms such as ³²P or ³H show up as bands. Molecules without radioactive atoms do not produce visible bands. Figure 2 shows that ³²P from labeled ATP is incorporated into Dg in all cases tested except when POMK is absent. POMK is a kinase, meaning that it transfers a phosphate from ATP to a target molecule, so the lack of ³²P-incorporation in the absence of POMK is expected. Similarly, ³H from ribitol 5-phosphate is not incorporated when fukutin is absent, which is expected because fukutin attaches ribitol 5-phosphate to the growing glycan chain. However, the figure also shows that in the presence of fukutin, ribitol 5-phosphate is incorporated into Dg when POMK is present but not when POMK is absent. Therefore, POMK activity must occur before fukutin can catalyze the addition of ribitol 5-phosphate to the glycan, and must occur first. (Choice A) If fukutin activity needed to occur before POMK activity could, no ³²P-labeled band would appear in the lane where fukutin is absent. (Choice C) If fukutin and POMK activities could occur in any order, fukutin would be active in the second lane of the ³H autoradiogram, and a band would be visible. (Choice D) If POMK and fukutin activities needed to occur simultaneously, neither would show activity in the absence of the other and only the lanes with both enzymes would have visible bands in the autoradiogram. Educational objective: Autoradiography can detect the incorporation of radioactive atoms into biomolecules. It can help determine the conditions under which an enzyme can act on its substrate.

Passage Dystroglycan (Dg) Is a Transmembrane Protein That Mediates Interactions Between