The broad long term objectives of my research are to understand the molecular basis of Lactoferrin(Lf)-cell interactions, and the biological effects elicited by Lf on it's target cells.
These aims will be achieved by selective modification of Lf cDNA to obtain variant forms of Lf (truncations, point mutations) with altered Lf protein interactions (with receptor, and with AGE-modified protein). Binding and endocytosis of advanced glycation end-products (AGEs) adducts on protein have been implicated over time in aging and at an accelerated rate in diseases with symptoms of premature aging, e.g. diabetes, to result in vascular (arterio- and atherosclerosis) and renal disease (renal nephropathy). Lf along with RAGE (receptor for AGE), directly binds of AGE adducts on plasma protein and mediates their endocytosis. Plasma Lf and it's bound iron are cleared from mammalian circulation by the liver via clathrin-dependent receptor-mediated endocytosis, which constitutes a significant hepatic homeostatic function. This proposal's two specific aims outline studies that examine Lf's molecular interactions with it's receptor (isolated rat hepatocytes), and with AGE-modified protein (125I-AGE-BSA). BLf cDNA, has been successfully expressed in prokaryotes as indicated in preliminary results. Even though it is deglycosylated, and lacks the first seventy N-terminal amino acids, it binds hepatocytes with physiologically relevant affinity. The next, step therefore will be to test the recombinant deletion mutant for it's ability to complete with 125I-Lf-BSA (AGE-modified protein for binding. If it competes efficiently, we would have proved that (i) the first seventy amino acids are not important and that (ii) glycosylation is not important for AGE-BSA binding. If it does not complete efficiently, further analysis of recombinant forms of Lf will be done, to determine whether glycosylation is important for Lf's interactions with AGE- modified proteins. In the event that glycosylation is important, native and mutant forms of recombinant protein will be expressed using an eukaryotic expression system, followed by functional assays. More precise mapping of the -cell binding (receptor binding), and -AGE- modified-protein (125I-AGE-BSA) binding, domain will be done by, generating mutant forms of recombinant Lf (truncations, point mutations, etc.), expressing them in prokaryotes, and testing them in functional assays for their ability to complete with 125I-Lf, or with 125I-AGE-BSA, for binding.
| Sitaram, M P; Moloney, B; McAbee, D D (1998) Prokaryotic expression of bovine lactoferrin deletion mutants that bind to the Ca2+-dependent lactoferrin receptor on isolated rat hepatocytes. Protein Expr Purif 14:229-36 |
| Sitaram, M P; McAbee, D D (1997) Isolated rat hepatocytes differentially bind and internalize bovine lactoferrin N- and C-lobes. Biochem J 323 ( Pt 3):815-22 |
