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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Physiological Chemistry Study Section (PC)
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Haft, Carol R
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Medical College of Wisconsin
Schools of Medicine
United States
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Olson, Linda J; Dahms, Nancy M (2018) Cloning, Expression, and Purification of the Glycosylated Transmembrane Protein, Cation-Dependent Mannose 6-Phosphate Receptor, from Sf9 Cells Using the Baculovirus System. Methods Mol Biol 1722:105-116
Baldwin, Aaron C; Naatz, Aaron; Bohnsack, Richard N et al. (2018) Cation-Independent Mannose 6-Phosphate Receptor Deficiency Enhances ?-Cell Susceptibility to Palmitate. Mol Cell Biol 38:
Miller, James J; Aoki, Kazuhiro; Moehring, Francie et al. (2018) Neuropathic pain in a Fabry disease rat model. JCI Insight 3:
Olson, Linda J; Orsi, Ramiro; Peterson, Francis C et al. (2015) Crystal Structure and Functional Analyses of the Lectin Domain of Glucosidase II: Insights into Oligomannose Recognition. Biochemistry 54:4097-111
Olson, Linda J; Castonguay, Alicia C; Lasanajak, Yi et al. (2015) Identification of a fourth mannose 6-phosphate binding site in the cation-independent mannose 6-phosphate receptor. Glycobiology 25:591-606
Olson, Linda J; Jensen, Davin R; Volkman, Brian F et al. (2015) Bacterial expression of the phosphodiester-binding site of the cation-independent mannose 6-phosphate receptor for crystallographic and NMR studies. Protein Expr Purif 111:91-7
D'Alessio, Cecilia; Dahms, Nancy M (2015) Glucosidase II and MRH-domain containing proteins in the secretory pathway. Curr Protein Pept Sci 16:31-48
Bohnsack, Richard N; Warejcka, Debra J; Wang, Lingyan et al. (2014) Expression of insulin-like growth factor 2 receptor in corneal keratocytes during differentiation and in response to wound healing. Invest Ophthalmol Vis Sci 55:7697-708
Olson, Linda J; Orsi, Ramiro; Alculumbre, Solana G et al. (2013) Structure of the lectin mannose 6-phosphate receptor homology (MRH) domain of glucosidase II, an enzyme that regulates glycoprotein folding quality control in the endoplasmic reticulum. J Biol Chem 288:16460-75
Maga, John A; Zhou, Jianghong; Kambampati, Ravi et al. (2013) Glycosylation-independent lysosomal targeting of acid ?-glucosidase enhances muscle glycogen clearance in pompe mice. J Biol Chem 288:1428-38

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