You are studying the binding of proteins to the cytoplasmic

You are studying the binding of proteins to the cytoplasmic face of a cultured neuroblastoma cells and have found a method to create small liposomes containing lipids and proteins from the plasma membrane. Your preparations always contain both inside-out and right-side-out vesicles. To separate these, a friend suggests that you pass your vesicles over an affinity column made of lectin coupled to solid beads. The diagrams below show (a) the orientation of a membrane protein in the original plasma membrane, and (b) how the membrane protein would be oriented in an inside-out vs. a right- side-out vesicle. You can assume the membrane lipids are similarly oriented.

(a) lipid baayer extracellular space Plasma membrane A. What is the function of lectins? (b) Inside-out Right-side-out B. Do you predict a lectin-based column would be able to bind to inside-out and/or right-side- out vesicles? How does this achieve the goal of separating the inside-out and right-side- out vesicles? Explain your reasoning C. Another friend suggests you stain the vesicle mixture with fluorescently-labeled Anne V, which is a protein that binds to phosphatidylserine lipids. Which set of vesicles, inside- out or right-side-out, would you expect to show greater fluorescence intensity after this staining? Explain your reasoning

Solution

A.Lectins are proteins that bind to specific carbohydrate structure and are found in plants, animals and microorganisms.

The main function of lectin is to bring about cell to cell contact and bring about cell to cell interaction and communication.

Lectins like selectin play an important role in immune systems.

B.Lectins recognize and bind sugars on the glycosylated lipids and membrane proteins . when lectins are passed through column chromatography the right side out vesicles are bound to the carbohydrates on the cell surfaces and thus are weeded out and only the inside out vesicles are allowed to pass. Or in other words the glycosylation phase will face the inside out vesicle and the outward on the right side outward vesicles and hence lectins bind only to the right side out vesicles allowing the inside out vesicles to pass through them.