You are studying a neurodegenerative disease whose genetic cause maps to a mutat

Question

You are studying a neurodegenerative disease whose genetic cause maps to a mutation in the p175 gene, which from BLAST searches may belong to the family of 7-transmembrane cell surface receptors. [See scheme of the protein below with each predicted transmembrane segment shown with its most basic flanking region marked.] You quickly generate a polyclonal antibody to human p175, because you want to use Western blotting to show that the disease results from a lack of expression, or perhaps truncation, of the receptor. Unexpectedly though, the Westerns show that patient ("P") and normal ("N") nervous tissue express this protein at comparable levels and that the protein is apparently the same size whether the disease is present or not (see below, left 2 lanes). As a side experiment, you treated the intact, live cells with trypsin prior to Western analysis (below, right 2 lanes). This provided the only clue so far that anything was different about p175 in disease--p175 in normal cells was cleaved into several smaller fragments, whereas p175 in patient cells was trypsin insensitive. (1) Why does the protein product get digested into four discreet products though there are seven membrane anchors? If you inserted an epitope tag at the amino terminus and expressed the protein in cultured cells and did the same experiment, would the labeled species get smaller after trypsin treatment, not change, or disappear altogether? (2) Explain what this experiment tells you about the protein in the diseased nerve cells. Assuming that the protein in fact encodes some kind of hormone receptor normally present on the cell surface, what kind of mutation might cause the disease and how would it affect the proteins' function as a receptor? (3) Now suppose you took patient and normal cell extracts from the minus trypsin samples and digested them with and without endoglycosidase H prior to the Western analysis. What would you expect to see and how would that be consistent with your hypothesis about the disease in A? Is there a precedent for diseases like this? Schematic of p175 with predicted membrane anchors Western on extracts of patient and normal cell samples

Explanation / Answer

Q.No 1

Protein product gel digested into 4 products due to higher molecular weight bands and the target protein is posttranslational modified. Molecular weight of a protein increased by any one of the following process such as acetylation, methylation, myristoylation, phosphorylation and glycosylation. If the protein is treated with epitope that the antibody must be recognized between the proteins and multiple bands will be observed.

Q.No 2

Protein in the diseased nerve cells is sensitive to trypsin and this occurs due to mutation. UV light causes mutation in the cell surface. Proteins encoded by P175 gene can be accumulated in the nucleus of cells continuous exposure to UV. This causes delays in the cell cycle and it allowing time for DNA repair of UV photoproducts. An inverse relationship is observed between over expression of the protein product and actual mutations in P175 gene.

Q.No 3

Patient cells may show the positive bands with trypsin in Western Blot and normal cells may or may not produce discrete bands. This may be due to sensitive to the trypsin. This clearly indicates the person have neurogenetic disease.

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