Fabry disease Schwann cells release p11 to induce sensory neuron hyperactivity.

Patients with Fabry disease suffer from chronic debilitating pain and peripheral sensory neuropathy with minimal treatment options, but the cellular drivers of this pain are unknown. Here, we propose a mechanism we believe to be novel in which altered signaling between Schwann cells and sensory neurons underlies the peripheral sensory nerve dysfunction we observed in a genetic rat model of Fabry disease. Using in vivo and in vitro electrophysiological recordings, we demonstrated that Fabry rat sensory neurons exhibited pronounced hyperexcitability. Schwann cells probably contributed to this finding because application of mediators released from cultured Fabry Schwann cells induced spontaneous activity and hyperexcitability in naive sensory neurons. We examined putative algogenic mediators using proteomic analysis and found that Fabry Schwann cells released elevated levels of the protein p11 (S100A10), which induced sensory neuron hyperexcitability. Removal of p11 from Fabry Schwann cell media caused hyperpolarization of neuronal resting membrane potentials, indicating that p11 may contribute to the excessive neuronal excitability caused by Fabry Schwann cells. These findings demonstrate that sensory neurons from rats with Fabry disease exhibit hyperactivity caused in part by Schwann cell release of the protein p11.

Schwann cell release of p11 induces sensory neuron hyperactivity in Fabry disease.

Patients with Fabry disease suffer from chronic debilitating pain and peripheral sensory neuropathy with minimal treatment options, but the cellular drivers of this pain are unknown. Here, we propose a novel mechanism by which altered signaling between Schwann cells and sensory neurons underlies the peripheral sensory nerve dysfunction we observe in a genetic rat model of Fabry disease. Using in vivo and in vitro electrophysiological recordings, we demonstrate that Fabry rat sensory neurons exhibit pronounced hyperexcitability. Schwann cells likely contribute to this finding as application of mediators released from cultured Fabry Schwann cells induces spontaneous activity and hyperexcitability in naïve sensory neurons. We examined putative algogenic mediators using proteomic analysis and found that Fabry Schwann cells release elevated levels of the protein p11 (S100-A10) which induces sensory neuron hyperexcitability. Removal of p11 from Fabry Schwann cell media causes hyperpolarization of neuronal resting membrane potential, indicating that p11 contributes to the excessive neuronal excitability caused by Fabry Schwann cells. These findings demonstrate that rats with Fabry disease exhibit sensory neuron hyperexcitability caused in part by Schwann cell release of the protein p11.

Multiple Domains of Staphylococcal Superantigen-like Protein 11 (SSL11) Contribute to Neutrophil Inhibition.

Staphylococcus aureus is an opportunistic pathogen producing many immune evasion molecules targeting various components of the host immune defense, including the Staphylococcal superantigen-like protein (SSL 1-14) family. Despite sharing similar structures with the powerful superantigens (SAgs), which cause massive T cell activation, SSLs interfere with a wide range of innate immune defenses. SSLs are divided into two subgroups, SSLs that contain a conserved carbohydrate Sialyl Lewis X [Neu5Acα2-3Galß1-4(Fucα1-3) GlcNAcß, SLeX] binding site and SSLs that lack the SLeX binding site. SSL2-6 and SSL11 possess the SLeX binding site. Our previous studies showed that SSL11 arrests cell motility by inducing cell adhesion in differentiated HL60 (dHL60) cells, while SSL7 did not bind dHL60 cells. SSL7-based chimeras were engineered by exchanging the SSL7 sequence with the corresponding SSL11 sequence and assaying for a gain of SSL11 function, namely, the induction of cell spreading and motility arrest. In addition to the SLeX-binding site, we observed that three beta-strands ß6, ß7, and ß9 and the N-terminal residues, Y16 and Y17, transitioned SSL7 to gain SSL11 activities. These studies define the structure-function properties of SSL11 that may allow SSL11 to inhibit S. aureus clearance by the host innate immune system, allowing S. aureus to maintain a carrier state in humans, an understudied aspect of S. aureus pathogenesis.

Platelet and myeloid cell phenotypes in a rat model of Fabry disease.

Fabry disease results from a deficiency of the lysosomal enzyme ⍺-Galactosidase-A (⍺-Gal A) and is estimated to occur in approximately 1:4100 live births. Characteristic of the disease is the accumulation of α-Gal-A substrates, primarily the glycosphingolipids (GSLs) globotriaosylceramide and globotriaosylsphingosine. Thrombotic events are a significant concern for Fabry patients, with strokes contributing to a significant decrease in overall lifespan. Currently, the mechanisms underlying the increased risk of thrombotic events experienced by Fabry patients are incompletely defined. Using a rat model of Fabry disease, we provide an improved understanding of the mechanisms linking GSL accumulation to thrombotic risk. We found that ⍺-Gal A-deficient rats accumulate myeloid-derived leukocytes at sites of GSL accumulation, including in the bone marrow and circulation, and that myeloid-derived leukocyte and megakaryocyte populations were prominent among cell types that accumulated GSLs. In the circulation, ⍺-Gal A-deficient rats had increases in cytokine-producing cell types and a corresponding elevation of pro-inflammatory cytokines. Lastly, circulating platelets from ⍺-Gal A-deficient rats accumulated a similar set of ⍺-Galactosidase-A substrates as was observed in megakaryocytes in the bone marrow, and exhibited increased platelet binding to fibrinogen in microfluidic and flow cytometric assays.

Tissue plasminogen activator is a ligand of cation-independent mannose 6-phosphate receptor and consists of glycoforms that contain mannose 6-phosphate.

Plasmin is the key enzyme in fibrinolysis. Upon interaction with plasminogen activators, the zymogen plasminogen is converted to active plasmin. Some studies indicate plasminogen activation is regulated by cation-independent mannose 6-phosphate receptor (CI-MPR), a protein that facilitates lysosomal enzyme trafficking and insulin-like growth factor 2 downregulation. Plasminogen regulation may be accomplished by CI-MPR binding to plasminogen or urokinase plasminogen activator receptor. We asked whether other members of the plasminogen activation system, such as tissue plasminogen activator (tPA), also interact with CI-MPR. Because tPA is a glycoprotein with three N-linked glycosylation sites, we hypothesized that tPA contains mannose 6-phosphate (M6P) and binds CI-MPR in a M6P-dependent manner. Using surface plasmon resonance, we found that two sources of tPA bound the extracellular region of human and bovine CI-MPR with low-mid nanomolar affinities. Binding was partially inhibited with phosphatase treatment or M6P. Subsequent studies revealed that the five N-terminal domains of CI-MPR were sufficient for tPA binding, and this interaction was also partially mediated by M6P. The three glycosylation sites of tPA were analyzed by mass spectrometry, and glycoforms containing M6P and M6P-N-acetylglucosamine were identified at position N448 of tPA. In summary, we found that tPA contains M6P and is a CI-MPR ligand.

Auditory brainstem responses in aging dark agouti rats.

The present study examined auditory function across age in the dark agouti (DA) rat strain. Auditory brainstem responses (ABRs) were measured for frequencies 8, 16, and 32 kHz in male and female DA rats from 3 to 18 months of age. Hearing thresholds and absolute and interpeak latencies (IPLs) were analyzed. Male hearing thresholds remained stable for the first year of life and then significantly increased at 18 months across all frequencies; female hearing remained stable at all tested ages out to 18 months. At 12 months, male DA rats showed significantly longer absolute latencies by age (i.e., compared with 3-month-old males) and sex (compared with 12-month-old females), with no differences in IPLs. At 18 months, female DA rats showed significantly longer absolute latencies with age (compared with 3-month-old females) and sex (compared with 18-month-old males), particularly for the later waves. Female IPLs were also significantly longer with age and by sex for the later waves. This report supports the feasibility of using male DA rats in studies to investigate age-related hearing loss (ARHL; presbycusis).

Allosteric regulation of lysosomal enzyme recognition by the cation-independent mannose 6-phosphate receptor.

The cation-independent mannose 6-phosphate receptor (CI-MPR, IGF2 receptor or CD222), is a multifunctional glycoprotein required for normal development. Through the receptor's ability to bind unrelated extracellular and intracellular ligands, it participates in numerous functions including protein trafficking, lysosomal biogenesis, and regulation of cell growth. Clinically, endogenous CI-MPR delivers infused recombinant enzymes to lysosomes in the treatment of lysosomal storage diseases. Although four of the 15 domains comprising CI-MPR's extracellular region bind phosphorylated glycans on lysosomal enzymes, knowledge of how CI-MPR interacts with ~60 different lysosomal enzymes is limited. Here, we show by electron microscopy and hydroxyl radical protein footprinting that the N-terminal region of CI-MPR undergoes dynamic conformational changes as a consequence of ligand binding and different pH conditions. These data, coupled with X-ray crystallography, surface plasmon resonance and molecular modeling, allow us to propose a model explaining how high-affinity carbohydrate binding is achieved through allosteric domain cooperativity.

Progress in the understanding and treatment of Fabry disease.

BACKGROUND: Fabry disease is caused by α-galactosidase A deficiency. Substrates of this lysosomal enzyme accumulate, resulting in cellular dysfunction. Patients experience neuropathic pain, kidney failure, heart disease, and strokes. SCOPE OF REVIEW: The clinical picture and molecular features of Fabry disease are described, along with updates on disease mechanisms, animal models, and therapies. MAJOR CONCLUSIONS: How the accumulation of α-galactosidase A substrates, mainly glycosphingolipids, leads to organ damage is incompletely understood. Enzyme replacement and chaperone therapies are clinically available to patients, while substrate reduction, mRNA-based, and gene therapies are on the horizon. Animal models exist to optimize these therapies and elucidate disease mechanisms for novel treatments. GENERAL SIGNIFICANCE: Recent newborn screening studies demonstrate that Fabry disease is the most common lysosomal storage disease. As many countries now include Fabry disease in their screening panels, the number of identified patients is expected to increase significantly. Better knowledge of disease pathogenesis is needed to improve treatment options.

Rats deficient in α-galactosidase A develop ocular manifestations of Fabry disease.

Fabry disease is an X-linked lysosomal storage disease caused by deficiency of α-galactosidase A. Ocular findings, such as cornea verticillata, cataracts, and retinal vascular tortuosity, serve as important diagnostic markers. We aimed to evaluate ocular phenotypes in α-galactosidase A-deficient (Fabry) rats and hypothesized that these rats would manifest ocular signs similar to those observed in patients. Slit lamp biomicroscopy was used to evaluate the cornea and lens, and retinal vasculature was examined by fluorescein angiography in WT and Fabry rats. Mass spectrometry was used to characterize and quantify ocular glycosphingolipids, and histology and electron microscopy revealed the location of the glycosphingolipid storage. We found that Fabry rats developed corneal and lenticular opacities to a statistically greater degree than WT rats. Retinal vascular morphology did not appear grossly different, but there was vascular leakage in at least one Fabry rat. Fabry rat eyes accumulated substrates of α-galactosidase A, and these α-galactosyl glycoconjugates were found in corneal keratocytes, lens fibers, and retinal vascular endothelial cells. Electron-dense lamellar inclusions were observed in keratocytes. Because Fabry rats recapitulate many ocular phenotypes observed in patients, they can be used to study disease pathogenesis and determine whether ocular findings serve as noninvasive indicators of therapeutic efficacy.

α-Galactosidase A-deficient rats accumulate glycosphingolipids and develop cardiorenal phenotypes of Fabry disease.

Fabry disease is an X-linked lysosomal storage disease caused by α-galactosidase A (α-Gal A) deficiency. Kidney and heart failure are frequent complications in adulthood and greatly contribute to patient morbidity and mortality. Because α-Gal A-deficient mouse models do not recapitulate cardiorenal findings observed in patients, a nonmouse model may be beneficial to our understanding of disease pathogenesis. In this study, we evaluated disease processes in a recently generated Fabry rat model. We found that male Fabry rats weighed significantly less than wild-type (WT) males, whereas female Fabry rats weighed significantly more than WT females. Whereas no difference in female survival was detected, we observed that male Fabry rats had a decreased lifespan. Skin histology revealed that inflammation and lipoatrophy may be chief disease mediators in patients. With respect to the kidney and heart, we found that both organs accumulate α-Gal A substrates, including the established biomarkers, globotriaosylceramide and globotriaosylsphingosine. Longitudinal serum and urine chemistry panels demonstrated pronounced renal tubule dysfunction, which was confirmed histologically. Mitral valve thickening was observed in Fabry rats using echocardiography. We conclude that Fabry rats recapitulate important kidney and heart phenotypes experienced by patients and can be further used to study disease mechanisms and test therapies.-Miller, J. J., Aoki, K., Mascari, C. A., Beltrame, A. K., Sokumbi, O., North, P. E., Tiemeyer, M., Kriegel, A. J., Dahms, N. M., α-Galactosidase A-deficient rats accumulate glycosphingolipids and develop cardiorenal phenotypes of Fabry disease.

Neuropathic pain in a Fabry disease rat model.

Fabry disease, the most common lysosomal storage disease, affects multiple organs and results in a shortened life span. This disease is caused by a deficiency of the lysosomal enzyme α-galactosidase A, which leads to glycosphingolipid accumulation in many cell types. Neuropathic pain is an early and severely debilitating symptom in patients with Fabry disease, but the cellular and molecular mechanisms that cause the pain are unknown. We generated a rat model of Fabry disease, the first nonmouse model to our knowledge. Fabry rats had substantial serum and tissue accumulation of α-galactosyl glycosphingolipids and had pronounced mechanical pain behavior. Additionally, Fabry rat dorsal root ganglia displayed global N-glycan alterations, sensory neurons were laden with inclusions, and sensory neuron somata exhibited prominent sensitization to mechanical force. We found that the cation channel transient receptor potential ankyrin 1 (TRPA1) is sensitized in Fabry rat sensory neurons and that TRPA1 antagonism reversed the behavioral mechanical sensitization. This study points toward TRPA1 as a potentially novel target to treat the pain experienced by patients with Fabry disease.

Cation-Independent Mannose 6-Phosphate Receptor Deficiency Enhances ß-Cell Susceptibility to Palmitate.

Palmitate attenuates insulin secretion and reduces the viability of insulin-producing cells. Previous studies identified the aberrant palmitoylation or mispalmitoylation of proteins as one mechanism by which palmitate causes ß-cell damage. In this report, we identify a role for lysosomal protein degradation as a mechanism by which ß cells defend themselves against excess palmitate. The cation-independent mannose 6-phosphate receptor (CI-MPR) is responsible for the trafficking of mannose 6-phosphate-tagged proteins to lysosomes via Golgi sorting and from extracellular locations through endocytosis. RINm5F cells, which are highly sensitive to palmitate, lack CI-MPR. The reconstitution of CI-MPR expression attenuates the induction of endoplasmic reticulum (ER) stress and the toxic effects of palmitate on RINm5F cell viability. INS832/13 cells express CI-MPR and are resistant to the palmitate-mediated loss of cell viability. The reduction of CI-MPR expression increases the sensitivity of INS832/13 cells to the toxic effects of palmitate treatment. The inhibition of lysosomal acid hydrolase activity by weak base treatment of islets under glucolipotoxic conditions causes islet degeneration that is prevented by the inhibition of protein palmitoylation. These findings indicate that defects in lysosomal function lead to the enhanced sensitivity of insulin-producing cells to palmitate and support a role for normal lysosomal function in the protection of ß cells from excess palmitate.

Cloning, Expression, and Purification of the Glycosylated Transmembrane Protein, Cation-Dependent Mannose 6-Phosphate Receptor, from Sf9 Cells Using the Baculovirus System.

The cation-dependent mannose 6-phosphate receptor (CD-MPR) is a single-pass type I membrane protein. This protein functions to transport lysosomal enzymes displaying phosphomannosyl residues from the Golgi complex and the cell surface to the lysosome. This glycosylated protein contains three disulfide bridges in its 159-residue extracytoplasmic domain. One of the problems with studying eukaryotic membrane proteins is isolating sufficient quantities. Structural studies typically require several hundred milligrams of highly purified protein. Here we present a method to isolate milligram quantities of CD-MPR/Asn81 suitable for structural studies.

Crystal Structure and Functional Analyses of the Lectin Domain of Glucosidase II: Insights into Oligomannose Recognition.

N-Glycans are modified as part of a quality control mechanism during glycoprotein folding in the endoplasmic reticulum (ER). Glucosidase II (GII) plays a critical role by generating monoglucosylated glycans that are recognized by lectin chaperones, calnexin and calreticulin. To understand how the hydrolytic activity of GIIα is enhanced by the mannose 6-phosphate receptor (MPR) homology domain (MRH domain) of its ß subunit, we now report a 1.6 Å resolution crystal structure of the MRH domain of GIIß bound to mannose. A comparison of ligand-bound and unbound structures reveals no major difference in their overall fold, but rather a repositioning of side chains throughout the binding pocket, including Y372. Mutation of Y372 inhibits GII activity, demonstrating an important role for Y372 in regulating GII activity. Comparison of the MRH domains of GIIß, MPRs, and the ER lectin OS-9 identified conserved residues that are critical for the structural integrity and architecture of the carbohydrate binding pocket. As shown by nuclear magnetic resonance spectroscopy, mutations of the primary binding pocket residues and adjacent W409, all of which inhibit the activity of GII both in vitro and in vivo, do not cause a significant change in the overall fold of the GIIß MRH domain but impact locally the stability of the binding pocket. W409 does not directly contact mannose; rather, its indole ring is stabilized by binding into a hydrophobic pocket of an adjacent crystallographic neighbor. This suggests that W409 interacts with a hydrophobic region of the GIIß or GIIα subunit to modulate its effect on GII activity.

Bacterial expression of the phosphodiester-binding site of the cation-independent mannose 6-phosphate receptor for crystallographic and NMR studies.

The cation-independent mannose 6-phosphate receptor (CI-MPR) is a multifunctional protein that interacts with diverse ligands and plays central roles in autophagy, development, and tumor suppression. By delivering newly synthesized phosphomannosyl-containing acid hydrolases from the Golgi to endosomal compartments, CI-MPR is an essential component in the generation of lysosomes that are critical for the maintenance of cellular homeostasis. The ability of CI-MPR to interact with ∼60 different acid hydrolases is facilitated by its large extracellular region, with four out of its 15 domains binding phosphomannosyl residues. Although the glycan specificity of CI-MPR has been elucidated, the molecular basis of carbohydrate binding has not been determined for two out of these four carbohydrate recognition domains (CRD). Here we report expression of CI-MPR's CRD located in domain 5 that preferentially binds phosphodiester-containing glycans. Domain 5 of CI-MPR was expressed in Escherichia coli BL21 (DE3) cells as a fusion protein containing an N-terminal histidine tag and the small ubiquitin-like modifier (SUMO) protein. The His6-SUMO-CRD construct was recovered from inclusion bodies, refolded in buffer to facilitate disulfide bond formation, and subjected to Ni-NTA affinity chromatography and size exclusion chromatography. Surface plasmon resonance analyses demonstrated that the purified protein was active and bound phosphorylated glycans. Characterization by NMR spectroscopy revealed high quality (1)H-(15)N HSQC spectra. Additionally, crystallization conditions were identified and a crystallographic data set of the CRD was collected to 1.8Šresolution. Together, these studies demonstrate the feasibility of producing CI-MPR's CRD suitable for three-dimensional structure determination by NMR spectroscopic and X-ray crystallographic approaches.

Glucosidase II and MRH-domain containing proteins in the secretory pathway.

N-glycosylation in the endoplasmic reticulum (ER) consists of the transfer of a preassembled glycan conserved among species (Glc3Man9GlcNAc2) from a lipid donor to a consensus sequence within a nascent protein that is entering the ER. The protein-linked glycans are then processed by glycosidases and glycosyltransferases in the ER producing specific structures that serve as signalling molecules for the fate of the folding glycoprotein: to stay in the ER during the folding process, to be retrotranslocated to the cytosol for proteasomal degradation if irreversibly misfolded, or to pursue transit through the secretory pathway as a mature glycoprotein. In the ER, each glycan signalling structure is recognized by a specific lectin. A domain similar to that of the mannose 6-phosphate receptors (MPRs) has been identified in several proteins of the secretory pathway. These include the beta subunit of glucosidase II (GII), a key enzyme in the early processing of the transferred glycan that removes middle and innermost glucoses and is involved in quality control of glycoprotein folding in the ER (QC), the lectins OS-9 and XTP3-B, proteins involved in the delivery of ER misfolded proteins to degradation (ERAD), the gamma subunit of the Golgi GlcNAc-1-phosphotransferase, an enzyme involved in generating the mannose 6-phosphate (M6P) signal for sorting acidic hydrolases to lysosomes, and finally the MPRs that deliver those hydrolytic enzymes to the lysosome. Each of the MRH-containing proteins recognizes a different signalling N-glycan structure. Three-dimensional structures of some of the MRH domains have been solved, providing the basis to understand recognition mechanisms.

Identification of a fourth mannose 6-phosphate binding site in the cation-independent mannose 6-phosphate receptor.

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays an essential role in lysosome biogenesis by targeting ∼ 60 different phosphomannosyl-containing acid hydrolases to the lysosome. This type I membrane glycoprotein has a large extracellular region comprised of 15 homologous domains. Two mannose 6-phosphate (M6P) binding sites have been mapped to domains 3 and 9, whereas domain 5 binds preferentially to the phosphodiester, M6P-N-acetylglucosamine (GlcNAc). A structure-based sequence alignment predicts that the C-terminal domain 15 contains three out of the four conserved residues identified as essential for carbohydrate recognition by domains 3, 5 and 9 of the CI-MPR, but lacks two cysteine residues that are predicted to form a disulfide bond. To determine whether domain 15 of the CI-MPR has lectin activity and to probe its carbohydrate-binding specificity, truncated forms of the CI-MPR were tested for binding to acid hydrolases with defined N-glycans in surface plasmon resonance analyses, and used to interrogate a phosphorylated glycan microarray. The results show that a construct encoding domains 14-15 binds both M6P and M6P-GlcNAc with similar affinity (Kd = 13 and 17 µM, respectively). Site-directed mutagenesis studies demonstrate the essential role of the conserved Tyr residue in domain 15 for phosphomannosyl binding. A structural model of domain 15 was generated that predicted an Arg residue to be in the binding pocket and mutagenesis studies confirmed its important role in carbohydrate binding. Together, these results show that the CI-MPR contains a fourth carbohydrate-recognition site capable of binding both phosphomonoesters and phosphodiesters.

Expression of insulin-like growth factor 2 receptor in corneal keratocytes during differentiation and in response to wound healing.

PURPOSE: Insulin-like growth factor 2 receptor (IGF2R) associates with ligands that influence wound healing outcomes. However, the expression pattern of IGF2R and its role in the cornea is unknown. METHODS: Human keratocytes were isolated from donor corneas. Fibroblasts (fibroblast growth factor 2 [FGF2]-treated) or myofibroblasts (TGF-ß1-treated) were analyzed for IGF2R and α-smooth muscle actin (α-SMA) expression by Western blotting and immunolocalization. Mouse corneas were wounded in vivo and porcine corneas ex vivo. The IGF2R and α-SMA protein expression were visualized and quantified by immunohistochemistry. The IGF2R gene expression in human corneal fibroblasts was knocked-down with targeted lentiviral shRNA. RESULTS: The IGF2R is expressed in epithelial and stromal cells of normal human, mouse, and porcine corneas. The IGF2R increases (11.2 ± 0.4-fold) in the epithelial and (11.7 ± 0.9-fold) stromal layers of in vivo wounded mouse corneas. Double-staining with α-SMA- and IGF2R-specific antibodies reveals that IGF2R protein expression is increased in stromal myofibroblasts in the wounded cornea relative to keratocytes in the normal cornea (11.2 ± 0.8-fold). Human primary stromal keratocytes incubated with FGF2 or TGF-ß1 in vitro demonstrate increased expression (2.0 ± 0.4-fold) of IGF2R in myofibroblasts relative to fibroblasts. Conversion of IGF2R shRNA-lentiviral particle transduced corneal fibroblasts to myofibroblasts reveals a dependence on IGF2R expression, as only 40% ± 10% of cells transduced converted to myofibroblasts compared to 86% ± 3% in control cells. CONCLUSIONS: The IGF2R protein expression is increased during corneal wound healing and IGF2R regulates human corneal fibroblast to myofibroblast differentiation.

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.

Here we report for the first time the three-dimensional structure of a mannose 6-phosphate receptor homology (MRH) domain present in a protein with enzymatic activity, glucosidase II (GII). GII is involved in glycoprotein folding in the endoplasmic reticulum. GII removes the two innermost glucose residues from the Glc3Man9GlcNAc2 transferred to nascent proteins and the glucose added by UDP-Glc:glycoprotein glucosyltransferase. GII is composed of a catalytic GIIα subunit and a regulatory GIIß subunit. GIIß participates in the endoplasmic reticulum localization of GIIα and mediates in vivo enhancement of N-glycan trimming by GII through its C-terminal MRH domain. We determined the structure of a functional GIIß MRH domain by NMR spectroscopy. It adopts a ß-barrel fold similar to that of other MRH domains, but its binding pocket is the most shallow known to date as it accommodates a single mannose residue. In addition, we identified a conserved residue outside the binding pocket (Trp-409) present in GIIß but not in other MRHs that influences GII glucose trimming activity.

Glycosylation-independent lysosomal targeting of acid α-glucosidase enhances muscle glycogen clearance in pompe mice.

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.