The lysosome plays a critical role in the degradative metabolism of the cell. Its importance to the organism is evidenced by the existence of more than 40 human lysosomal storage diseases. The transport of newly synthesized soluble lysosomal enzymes to lysosomes in virtually all mammalian cells is dependent upon the mannose 6-phosphate receptors (MPRs). Two distinct, but homologous, MPRs have been identified. The 270-kDa insulin-like growth factor II/cation-independent (IGF-II/CI) MPR is a multifunctional glycoprotein that binds both mannose 6-phosphate (Man-6-P) on lysosomal enzymes and IGF-II, a nonglycosylated polypeptide hormone that is essential for normal fetal growth. The second receptor, the cation-dependent (CD) MPR, is a 46-kDa glycoprotein that does not bind IGF-II.
The specific aims of this proposal are to determine: 1) the expression and function of the MPRs during vertebrate evolution, 2) the factors which influence the conformation, ligand binding affinity, and stability of the MPRs, and 3) the mechanism by which a single protein (IGF-II/CI-MPR) can bind two very different ligands, a carbohydrate moiety (Man-6-P) and a protein determinant (IGF-II), with high affinity. The long range goal of this proposal is to define the three-dimensional structure of the two MPRs. To achieve these objectives, pentamannosyl phosphate-agarose affinity chromatography will be used to purify the MPRs from different classes of non-mammalian vertebrate species and the IGF-II/CI-MPR will be assayed for its affinity to bind IGF-II. The influence of N-linked oligosaccharides, ligand binding, and pH on the conformation, ligand binding affinity, and stability will be evaluated by circular dichroism spectroscopy and reactivity to monoclonal antibodies. In addition, soluble fragments of the CD-MPR's and IGF-II/CI-MPR's ligand binding sites will be produced in milligram amounts in a baculovirus expression system for collaborative studies to determine their three-dimensional structure by both x-ray crystallographic and NMR spectroscopy analyses. Taken together, these studies will lead to a better understanding of the structural elements involved in the functional expression of these essential receptors.
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