Lysosomes perform degradative metabolism critical to many endocytic, phagocytic, and autophagic processes. Cation-independent mannose 6-phosphate receptor (CI-MPR) plays a vital role in the biogenesis of lysosomes by delivering ~60 different newly synthesized hydrolytic enzymes with mannose 6-phosphate (M6P) on their N-glycans to lysosomes. Lysosomal storage diseases (LSDs) are caused by mutations in lysosomal proteins, mainly enzymes, that result in defective catabolism and substrate accumulation. Characteristic of the family of ~70 LSDs is their progressive and debilitating nature due to their impact on multiple organ systems. Treatment is symptomatic for most LSDs, with only 11 having FDA-approved therapies. CI-MPR's ability to internalize recombinant M6P- containing enzymes delivered to patients by bi-weekly intravenous infusion forms the basis of enzyme replacement therapy (ERT) for 9 of these therapies. However, structural knowledge of the interaction between CI-MPR and its cargo of ~60 different lysosomal enzymes is lacking. CI-MPR also binds a diverse set of extracellular non-M6P-containing ligands that mediate CI-MPR's tumor suppressor role and regulation of cell growth and differentiation. CI-MPR regulates plasma insulin-like growth factor 2 (IGF2) levels, and binds three components of the plasminogen activation system, plasminogen, urokinase-type plasminogen activator receptor (uPAR), and tissue-type PA (tPA). However, limited information is available concerning the molecular basis for CI-MPR's interaction with plasminogen and uPAR. Here we will expand upon our preliminary data that: 1) revealed the crystal structure of CI- MPR's N-terminal region that houses several ligand binding sites, 2) showed the first structural view of CI-MPR's entire extracellular region by high-resolution electron microscopy (EM), and 3) identified conformational changes elicited by pH and ligand binding. The overall structure of CI-MPR's 2300- residue extracellular region comprised of 15 domains will be determined alone and bound to ligands using an integrated approach combining single particle EM, mass spectrometry, X-ray crystallography and NMR spectroscopy. The first structure of a complex between CI-MPR and a lysosomal enzyme (Aim 1), plasminogen (Aim 2), and uPAR (Aim 2) will be elucidated. Kinetic analyses and cell-based assays will evaluate allosteric effects of each of CI-MPR's ligands (Aim 2). The mechanism of acidic pH-dependent ligand dissociation, which is essential to the functioning of CI-MPR and other endocytic receptors, will be probed using mutagenesis studies and NMR techniques (Aim 3). Our new rat model of the LSD Fabry disease will be used to test novel lysosomal enzyme-IGF2 fusion constructs for their ability to reduce substrate accumulation (Aim 4). These studies will provide insight for the design of improved therapeutics for the treatment of LSDs, and novel inhibitors of plasminogen activation.
Lysosomes are critical to many physiological processes, including the disposal of abnormal proteins, supplying substrates for energy production and cell survival, 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.
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