Lysosomes carry out degradative metabolism critical to many endocytic, phagocytic, and autophagic processes. Mannose 6-phosphate receptors (CI-MPR and CD-MPR) play a vital role in the biogenesis of lysosomes by delivering ~60 different newly synthesized hydrolytic enzymes with mannose 6- phosphate on their N-glycans to lysosomes. CI-MPR is the primary receptor responsible for this trafficking and the ability of CI-MPR to internalize these enzymes is capitalized upon in enzyme replacement therapy for several lysosomal storage diseases. Unlike CD-MPR, CI-MPR binds a diverse set of extracellular ligands that mediate CI-MPR's ability to function as a tumor suppressor and to regulate cell growth and differentiation. CI-MPR interacts with three components of the plasminogen activation system, plasminogen, urokinase-type plasminogen activator receptor (uPAR), and tissue-type PA (tPA). Previous studies showed differentiation of fibroblasts to myofibroblasts, a key process during wound healing that becomes aberrant in fibrosis, requires the cleavage of uPAR and decreased levels of surface-bound uPAR. Recently we showed that CI-MPR is required for the differentiation of human corneal fibroblasts to myofibroblasts. However, limited information is available concerning the molecular basis for CI-MPR's interaction with plasminogen and uPAR and how CI- MPR impacts cellular differentiation. In this proposal, we expand upon our preliminary data that reveal the first structural view of the entire extracellular region of CI-MPR by high-resolution electron microscopy (EM), and our identification of a new ligand (tPA) for CI-MPR that enhances plasminogen activation upon binding CI-MPR. The overall structure of CI-MPR's 2300-residue extracellular region comprised of 15 domains will be determined using an integrated approach combining single particle EM, X-ray crystallography and NMR spectroscopy (Aim 1). The first structure of a complex between CI-MPR and a lysosomal enzyme will be elucidated using EM and crystallographic approaches (Aim 1). The mechanism of acidic pH-dependent ligand dissociation, which is essential to the functioning of the MPRs and other endocytic receptors, will be probed using mutagenesis studies and NMR techniques (Aim 1). The role of tPA and uPA in cellular differentiation will be investigated (Aim 2). The structure of a complex between CI-MPR and uPAR, and CI-MPR and plasminogen will be solved by crystallographic and NMR methods (Aim 2). Cell-based assays and quantitative binding studies will be done to evaluate: 1) how the different ligands of CI-MPR impact CI-MPR's function in ligand binding and internalization (Aim 1), and 2) whether CI-MPR regulates uPAR's half-life and function (Aim 2). These studies will provide insight for the design of improved therapeutics for the treatment of lysosomal storage diseases, and novel inhibitors of the plasminogen activation system.

Public Health Relevance

Lysosomes are critical to many physiological processes, including the turnover of normal cellular proteins, disposal of abnormal proteins, down-regulation of signaling pathways, mobilization of intracellular stores for energy, antigen processing, and inactivation of pathogenic organisms. Receptors play a key role in the formation of functional lysosomes by delivering degradative enzymes to the lysosome. Understanding how these receptors deliver their cargo will aid in identifying new strategies for the treatment of lysosomal storage disorders and other human diseases dependent upon lysosomal function.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK042667-25
Application #
9605279
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Sechi, Salvatore
Project Start
1992-03-01
Project End
2019-11-30
Budget Start
2018-12-01
Budget End
2019-11-30
Support Year
25
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Biochemistry
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
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:
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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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