The 300kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46kDa cation- dependent MPR (CD-MPR) play a key role in lysosome biogenesis by delivering ~60 different newly synthesized acid hydrolases to the lysosome by binding to mannose 6-phosphate (M6P) residues on their N-glycans. Disruption of this essential targeting pathway results in the most severe of the human lysosomal storage disorders, mucolipidosis II. In addition to lysosomal enzymes, the repertoire of identified extracellular ligands of the CI-MPR includes a diverse spectrum of M6P-containing proteins, such as growth factors (e.g., transforming growth factor-2) and pathogenic viruses (e.g., herpes simplex, varicella-zoster), and their interaction with the MPRs can result in the growth factor's activation or degradation, and facilitate viral entry into mammalian cells. The multifunctional CI-MPR also binds the non-M6P-containing ligands plasminogen, urokinase-type plasminogen activator receptor (uPAR), insulin-like growth factor-II (IGF-II), and retinoic acid. Together, these unique binding properties of the CI-MPR mediate its ability to regulate cell growth and motility, and to function as a tumor suppressor. However, the molecular basis governing the intracellular trafficking and ligand binding properties of these receptors has not been fully defined. Recent studies from our laboratory have provided the first, and to date the only, structural views of the CD-MPR's extracellular domain, and two out of the three carbohydrate binding sites of the CI-MPR. In the current proposal, we will use crystallographic and NMR approaches to determine the structure of the MPRs'carbohydrate (Aims 1 &3) and plasminogen (Aim 2) binding sites under conditions which are physiologically relevant, including acidic conditions which are key for the ability of the receptors to release their cargo to undergo multiple rounds of lysosomal enzyme delivery. Phosphodiester-containing proteins will be identified using domain 5 of the CI-MPR as a novel affinity probe (Aim 1). Crystallographic and solution structures of the MPRs'cytoplasmic domain in the absence and presence of adaptor proteins will be determined (Aim 4). These studies will be complemented by EPR spectroscopic analyses of MPRs containing site-specific spin labels (Aim 4). The long term goal is to understand the molecular mechanisms by which these essential receptors carry out their diverse biological functions. These studies will also provide the structural basis for the design of improved therapeutics for the treatment of lysosomal storage disorders, and novel inhibitors of viral infection and growth factor activation.

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, release of internalized nutrients, 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 #
5R01DK042667-21
Application #
8661159
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Sechi, Salvatore
Project Start
1992-03-01
Project End
2015-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
21
Fiscal Year
2014
Total Cost
$455,674
Indirect Cost
$157,848
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; 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
Castonguay, Alicia C; Olson, Linda J; Dahms, Nancy M (2011) Mannose 6-phosphate receptor homology (MRH) domain-containing lectins in the secretory pathway. Biochim Biophys Acta 1810:815-26
Olson, Linda J; Sun, Guangjie; Bohnsack, Richard N et al. (2010) Intermonomer interactions are essential for lysosomal enzyme binding by the cation-dependent mannose 6-phosphate receptor. Biochemistry 49:236-46
Olson, Linda J; Peterson, Francis C; Castonguay, Alicia et al. (2010) Structural basis for recognition of phosphodiester-containing lysosomal enzymes by the cation-independent mannose 6-phosphate receptor. Proc Natl Acad Sci U S A 107:12493-8
Bohnsack, Richard N; Patel, Manish; Olson, Linda J et al. (2010) Residues essential for plasminogen binding by the cation-independent mannose 6-phosphate receptor. Biochemistry 49:635-44
Kim, Jung-Ja P; Olson, Linda J; Dahms, Nancy M (2009) Carbohydrate recognition by the mannose-6-phosphate receptors. Curr Opin Struct Biol 19:534-42
Bohnsack, Richard N; Song, Xuezheng; Olson, Linda J et al. (2009) Cation-independent mannose 6-phosphate receptor: a composite of distinct phosphomannosyl binding sites. J Biol Chem 284:35215-26
Song, Xuezheng; Lasanajak, Yi; Olson, Linda J et al. (2009) Glycan microarray analysis of P-type lectins reveals distinct phosphomannose glycan recognition. J Biol Chem 284:35201-14
Dahms, Nancy M; Olson, Linda J; Kim, Jung-Ja P (2008) Strategies for carbohydrate recognition by the mannose 6-phosphate receptors. Glycobiology 18:664-78

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