Jonathan H. LeBowitz

The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells

We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct (MPKCCD14) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-β-cyclodextrin added apically to MPKCCD14 cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 ± 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of MPKCCD14 cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.

Glycosylation-independent Lysosomal Targeting of Acid α-Glucosidase Enhances Muscle Glycogen Clearance in Pompe Mice

Background: Acid α-glucosidase, an enzyme replacement therapy for Pompe disease, is poorly targeted to lysosomes when relying on phosphomannose residues. Results: Fusing IGF-II to acid α-glucosidase resulted in more efficient uptake and glycogen clearance from muscle of Pompe mice. Conclusion: Enhanced binding to the cation-independent mannose 6-phosphate receptor (CI-MPR) enabled improved glycogen clearance in Pompe mice. Significance: BMN 701 is now being tested for Pompe disease in human clinical studies. We have used a peptide-based targeting system to improve lysosomal delivery of acid α-glucosidase (GAA), the enzyme deficient in patients with Pompe disease. Human GAA was fused to the glycosylation-independent lysosomal targeting (GILT) tag, which contains a portion of insulin-like growth factor II, to create an active, chimeric enzyme with high affinity for the cation-independent mannose 6-phosphate receptor. GILT-tagged GAA was taken up by L6 myoblasts about 25-fold more efficiently than was recombinant human GAA (rhGAA). Once delivered to the lysosome, the mature form of GILT-tagged GAA was indistinguishable from rhGAA and persisted with a half-life indistinguishable from rhGAA. GILT-tagged GAA was significantly more effective than rhGAA in clearing glycogen from numerous skeletal muscle tissues in the Pompe mouse model. The GILT-tagged GAA enzyme may provide an improved enzyme replacement therapy for Pompe disease patients.

Functional Regulation of the Epithelial Na+ Channel by IκB Kinase-β Occurs via Phosphorylation of the Ubiquitin Ligase Nedd4-2

We have previously shown that IκB kinase-β (IKKβ) interacts with the epithelial Na+ channel (ENaC) β-subunit and enhances ENaC activity by increasing its surface expression in Xenopus oocytes. Here, we show that the IKKβ-ENaC interaction is physiologically relevant in mouse polarized kidney cortical collecting duct (mpkCCDc14) cells, as RNA interference-mediated knockdown of endogenous IKKβ in these cells by ∼50% resulted in a similar reduction in transepithelial ENaC-dependent equivalent short circuit current. Although IKKβ binds to ENaC, there was no detectable phosphorylation of ENaC subunits by IKKβ in vitro. Because IKKβ stimulation of ENaC activity occurs through enhanced channel surface expression and the ubiquitin-protein ligase Nedd4-2 has emerged as a central locus for ENaC regulation at the plasma membrane, we tested the role of Nedd4-2 in this regulation. IKKβ-dependent phosphorylation of Xenopus Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for regulation of Nedd4-2 and thus ENaC activity. 32P labeling studies utilizing wild-type or mutant forms of Xenopus Nedd4-2 demonstrated that Ser-444, a key SGK1 and protein kinase A-phosphorylated residue, is also an important IKKβ phosphorylation target. ENaC stimulation by IKKβ was preserved in oocytes expressing wild-type Nedd4-2 but blocked in oocytes expressing either a dominant-negative (C938S) or phospho-deficient (S444A) Nedd4-2 mutant, suggesting that Nedd4-2 function and phosphorylation by IKKβ are required for IKKβ regulation of ENaC. In summary, these results suggest a novel mode of ENaC regulation that occurs through IKKβ-dependent Nedd4-2 phosphorylation at a recognized SGK1 and protein kinase A target site.

Utilizing ExAC to assess the hidden contribution of variants of unknown significance to Sanfilippo Type B incidence

Given the large and expanding quantity of publicly available sequencing data, it should be possible to extract incidence information for monogenic diseases from allele frequencies, provided one knows which mutations are causal. We tested this idea on a rare, monogenic, lysosomal storage disorder, Sanfilippo Type B (Mucopolysaccharidosis type IIIB). Sanfilippo Type B is caused by mutations in the gene encoding α-N-acetylglucosaminidase (NAGLU). There were 189 NAGLU missense variants found in the ExAC dataset that comprises roughly 60,000 individual exomes. Only 24 of the 189 missense variants were known to be pathogenic; the remaining 165 variants were of unknown significance (VUS), and their potential contribution to disease is unknown. To address this problem, we measured enzymatic activities of 164 NAGLU missense VUS in the ExAC dataset and developed a statistical framework for estimating disease incidence with associated confidence intervals. We found that 25% of VUS decreased the activity of NAGLU to levels consistent with Sanfilippo Type B pathogenic alleles. We found that a substantial fraction of Sanfilippo Type B incidence (67%) could be accounted for by novel mutations not previously identified in patients, illustrating the utility of combining functional activity data for VUS with population-wide allele frequency data in estimating disease incidence.

BMN 701-mediated receptor redistribution is responsible for increased uptake

BMN 701 is a novel chimeric fusion protein of Insulin-like Growth Factor 2 (IGF-2) and acid alpha-glucosidase (GAA) that is now being tested in the clinic for the treatment of Pompe disease. BMN 701 was designed to improve delivery of GAA to the lysosome of muscle cells by fusing a high affinity ligand for the cation independent mannose-6phosphate receptor (CI-MPR) to GAA. In uptake experiments with rat L6 myoblasts, the Kuptake improvedmarkedly compared to untagged rhGAA, a predicted consequence of higher affinity binding to the CI-MPR. Additionally, the total amount of BMN 701 taken up by cells (system capacity) was doubled compared to rhGAA. This surprising observation could be explained by a phenomenon reported previously, namely that IGF-1 and IGF-2 can induce a signaling cascade resulting in redistribution of the CI-MPR so as to increase the quantity of the CI-MPR on the cell surface. We have investigated the ability of BMN 701 to do the same. In vitro, we find: i) BMN 701 can bind to the IGF-1 receptor; ii) BMN 701 can transduce a signaling cascade from the IGF-1 receptor; iii) the elevated system capacity of BMN701 is dependent on this signaling from the IGF-1 receptor; and iv) system capacity for uptake of untagged rhGAA can be increased by IGF-1. These results are consistent with the higher system capacity resulting from tag-dependent signaling. This novel attribute of BMN 701 may contribute to its enhanced ability to reverse glycogen storage in Pompe mice.