The long-term objective of these studies is to determine the role L-fucose (fucose) plays in affecting protein function, stability and turnover; and what biochemical and physiological ramifications result when fucose metabolism is altered in disease states.  Our hypothesis is that changing the fucosylation index of a protein will affect its turnover, but that its function will be unchanged.  Two multifaceted specific aims are proposed to address this problem.  A knowledge of how fucose affects protein turnover will allow greater control over in vivo efficacy and half-life of pharmacological proteins such as tissue plasminogen activator (tPA).  It will also help us understand how altered glycoprotein carbohydrate structure affects metastasis.

Research Questions
1.  How many secreted fucoproteins are produced by H4IIE rat hepatoma cells under basal conditions and what are their identities?  Does the fucosylation index of proteins change as the mass of substrates change or do mechanisms of regulation exist?
     a. What is the baseline oligosaccharide structure of transferrin secreted from H4IIE cells?
     b. How is this baseline structure altered as the exogenous fucose concentration increases?
     c. What happens to the oligosaccharide structure of transferrin if the mass of protein produced in H4IIE cells increases?
     d. Is O-fucosylation affected in the same way that fucosylation of N-linked oligosaccharides is?
2.  How does the absence or presence of fucose affect protein turnover?  How is transferrin receptor-mediated endocytosis (RME) and degradation affected by altered fucose content?
     a.  Does fucosylated transferrin bind to H4IIE cells with different affinity than non-fucosylated transferrin?
     b.   Does the in vitro rate of degradation change when transferrin is more or less fucosylated?
     c.   Does the in vivo rate of degradation change when transferrin is more or less fucosylated?
     d.   What are the half-lives of other fucoproteins synthesized by H4IIE cells, and how do they change under altered culture conditions?

     In these studies, equilibrium labeling of human hepatoma (Hep G2) cells is conducted using varying concentrations of fucose and tracer amounts of tritiated fucose.  Medium is then collected, proteins concentrated, and the number and identity of the proteins produced is determined by immunoprecipitation, electrophoresis and blotting.
     To assess the effect of increased protein production on oligosaccharide structure, the gene for transferrin, under control of the inducible metallothionein promoter, will be transfected into H4IIE cells.  Following selection of transfected cells, protein production is increased by the addition of ZnSO₄ to the cell culture medium.
     To determine the influence of fucose on protein turnover and degradation, isolated protein is separated into  fucosylated and nonfucosylated species, radiolabeled, and degradation of protein determined in Hep G2 culture medium and in the bloodstream of rats.
    Other projects which are being carried out to more completely understand the metabolism of fucose are things such as charachterizing enzymes involved in fucose metabolism:  alpha-L-fucosidase, fucosyltransferase, and fucokinase.

     Glycosylation of proteins represents the most complex post-translational modification of proteins known to occur.  It is becoming increasingly accepted that the oligosaccharide moieties of glycoproteins have an important role in protein stability, function and turnover, and one of the moieties shown to play these roles is fucose.  A large number of studies have reported that glycoprotein metabolism in general is altered in cancer and other diseases.  Data has been published indicating an increase in fucosylation of haptoglobin in several disease states.  Understanding fucose metabolism in greater depth may allow manipulation of the fucosylation state of proteins, thereby manipulating protein half-lives.  These studies will enable us to predict with greater clarity what biological effects will occur if fucose metabolism changes in disease.