The overall goal of this proposal is to determine the role of N-linked glycans in the maturation and quality control of proteins that traverse the secretory pathway. The majority of proteins that travel through the secretory pathway receive multiple N-linked glycosylations. We and others have shown that these carbohydrates are used as maturation and quality control signals to convey information about the fitness of maturing nascent chains and help to determine their fate. Information about the conformation of a protein is encoded within the composition of the N-linked glycan by a series of glycosidases and transferases that reside in the endoplasmic reticulum (ER). These encoder proteins are able to modulate the glycan composition based on the structure of the protein. The UDP-glucose: glycoprotein glucosyltransferase (GT1) is currently the only known protein that can modify the glycan structure dependent upon the properties of the modified protein, yet little is known about its activity in live cells. The glycan composition is then deciphered by carbohydrate-binding proteins that recognize distinct glycan structures and act as molecular chaperones or sorting receptors to assist in maturation and quality control processes. The glucose arm of the carbohydrate supports binding by the lectin chaperones calnexin and calreticulin, which direct its co- and post-translational folding processes. In contrast, the mannose branches appear to serve important roles for directing the exit of proteins from the ER. Native proteins are sent to the Golgi, whereas aberrant proteins are sorted for destruction by the cytosolic proteasome through the ER-associated protein degradation (ERAD) pathway. Therefore, mannosidases appear to be involved in generating the quality control code that is recognized by carbohydrate-binding sorting receptors that control the trafficking of proteins from the ER. This ER network of enzymes that modify and recognize glycans based on the conformation of the modified protein plays a crucial role in assisting the maturation process and directing quality control traffic of glycoproteins in the secretory pathway.
Our specific aims : (1) to understand the role of glycans and the vectorial nature of the cellular folding reaction in the efficient maturation of prosaposin, a sequential domain containing protein;(2) to elucidate the function of GT1 in the maturation and quality control of prosaposin, an obligate GT1 substrate;and (3) to determine the role of the mannosidase-like protein, EDEM1, in the sorting and delivery of non-native proteins to the ER-associated protein degradation pathway.
Relevance: Protein misfolding and subsequent degradation is the basis for a number of human disease states including albinism, liver cirrhosis, emphysema, cystic fibrosis and some forms of heart disease to name a few. Knowledge of the maturation, quality control and degradation of secretory cargo will help us to understand these diseases more thoroughly. It will also assist in the development of methodologies for the efficient expression of a large variety of recombinant proteins that are currently being used as therapeutic agents.
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