A child appears to be suffering from I-cell disease, but when a sample of his proteins (lane 3* below), isolated from skin fibroblasts, is compared to protein samples from fibroblasts of his healthy parents (lanes 1 and 2) and siblings (lanes 4€“6) using Western blot analysis and antibodies against N-acetylglucosamine phosphotransferase (~145 kDa) and actin (loading control, ~ 43 kDa), the following is seen:

In a second set of experiments, N-acetylglucosamine phosphotransferase was isolated from cells from the afflicted child and from his healthy parents, and used in an assay with 12P to measure enzyme activity and the production of mannose 6-phosphate. The assay yielded the following results:

a. Using fibroblasts cultured from the child, design an experiment using the N-acctylglucosamine phosphotransferase antibody and fluorescence microscopy and draw the results that could explain why the child presents symptoms similar to I-cell disease.

b. Given the results from these three different experiments, how would you explain the I-cell symptoms seen in the child and what experiment would you propose to test your hypothesis?

c. A laser scanning confocal micrograph of MDCK cells labeled with an antibody against the mannose 6-phosphate receptor shows the following:

How do you explain the labeling on the apical and basolateral surfaces, for a receptor whose function is to target enzymes from the trans-Golgi network (TGN) to the lysosome? Likewise, what explains the labeling seen at the RER?

Transcribed Image Text:

Molecular weight ladder 3* 4 5 6 150 kDa 102 kDa 86 kDa 49 kDa 31 kDa 2. 150 125 100 O Father 75 X Child O Mother 50 25 5 15 30 60 120 240 Time (min) at 37 °C Mannose 6-phosphate jw/Br