The asparagine-linked (N-linked) oligosaccharides present on glycoproteins are known to carry out many important functions including targeting, recognition, and stability. Inability to synthesize the correct N-linked oligosaccharides is known to be the basis of certain inherited diseases and alterations in cellular oligosaccharides are often a characteristic of transformed cells. Furthermore, synthesis of the correct sugar structure has become an important consideration for recombinant proteins made for therapeutic uses. Thus, knowledge about the functions and synthesis of N-linked oligosaccharides is crucial for our understanding and alleviating human diseases. The factors that control the processing of N-linked oligosaccharides and determine the type of structure present on a given glycoprotein are just beginning to be elucidated. The goal of my research is to gain greater understanding of these factors by studying one of the early-acting processing enzymes - an endoplasmic reticulum (ER) alpha-mannosidase. This enzyme displays an anomalous characteristic in that it exists as two forms, soluble and membrane-associated, in lysed cells extracts. The relationship between these two forms of the enzyme has been unclear. I have isolated a full-length cDNA encoding a rat liver alpha-mannosidase which displays the properties of the ER alpha-mannosidase. Surprisingly, the deduced amino acid sequence of the enzyme lacks the known signals for ER membrane translocation and retention. Using a combination of molecular and biochemical techniques, I hope to elucidate the ER transport mechanism for this enzyme and to investigate the possibility that the transport may be a regulated process, given the apparent dual localization of the enzyme in cell lysates. In addition, I plan to examine the effect of over-expression of alpha-mannosidase activity on the processing of N-linked oligosaccharides. This experiment will address the role of enzyme activity level on the rate and pathway for trimming high mannose-type oligosaccharides and provide insights about the factors that control the early processing reactions. These studies will be fundamental to understanding both protein translocation across the ER membrane and control of oligosaccharide processing.
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