Juvenile mucopolysaccharidosis plus disease caused by a missense mutation in VPS33A.

A rare and fatal disease resembling mucopolysaccharidosis in infants, is caused by impaired intracellular endocytic trafficking due to deficiency of core components of the intracellular membrane-tethering protein complexes, HOPS, and CORVET. Whole exome sequencing identified a novel VPS33A mutation in a patient suffering from a variant form of mucopolysaccharidosis. Electron and confocal microscopy, immunoblotting, and glycosphingolipid trafficking experiments were undertaken to investigate the effects of the mutant VPS33A in patient-derived skin fibroblasts. We describe an attenuated juvenile form of VPS33A-related syndrome-mucopolysaccharidosis plus in a man who is homozygous for a hitherto unknown missense mutation (NM_022916.4: c.599 G>C; NP_075067.2:p. Arg200Pro) in a conserved region of the VPS33A gene. Urinary glycosaminoglycan (GAG) analysis revealed increased heparan, dermatan sulphates, and hyaluronic acid. We showed decreased abundance of VPS33A in patient derived fibroblasts and provided evidence that the p.Arg200Pro mutation leads to destablization of the protein and proteasomal degradation. As in the infantile form of mucopolysaccharidosis plus, the endocytic compartment in the fibroblasts also expanded-a phenomenon accompanied by increased endolysosomal acidification and impaired intracellular glycosphingolipid trafficking. Experimental treatment of the patient's cultured fibroblasts with the proteasome inhibitor, bortezomib, or exposure to an inhibitor of glucosylceramide synthesis, eliglustat, improved glycosphingolipid trafficking. To our knowledge this is the first report of an attenuated juvenile form of VPS33A insufficiency characterized by appreciable residual endosomal-lysosomal trafficking and a milder mucopolysaccharidosis plus than the disease in infants. Our findings expand the proof of concept of redeploying clinically approved drugs for therapeutic exploitation in patients with juvenile as well as infantile forms of mucopolysaccharidosis plus disease.

The lysosomal disease caused by mutant VPS33A.

A rare lysosomal disease resembling a mucopolysaccharidosis with unusual systemic features, including renal disease and platelet dysfunction, caused by the defect in a conserved region of the VPS33A gene on human chromosome 12q24.31, occurs in Yakuts-a nomadic Turkic ethnic group of Southern Siberia. VPS33A is a core component of the class C core vacuole/endosome tethering (CORVET) and the homotypic fusion and protein sorting (HOPS) complexes, which have essential functions in the endocytic pathway. Here we show that cultured fibroblasts from patients with this disorder have morphological changes: vacuolation with disordered endosomal/lysosomal compartments and-common to sphingolipid diseases-abnormal endocytic trafficking of lactosylceramide. Urine glycosaminoglycan studies revealed a pathological excess of sialylated conjugates as well as dermatan and heparan sulphate. Lipidomic screening showed elevated ß-D-galactosylsphingosine with unimpaired activity of cognate lysosomal hydrolases. The 3D crystal structure of human VPS33A predicts that replacement of arginine 498 by tryptophan will de-stabilize VPS33A folding. We observed that the missense mutation reduced the abundance of full-length VPS33A and other components of the HOPS and CORVET complexes. Treatment of HeLa cells stably expressing the mutant VPS33A with a proteasome inhibitor rescued the mutant protein from degradation. We propose that the disease is due to diminished intracellular abundance of intact VPS33A. Exposure of patient-derived fibroblasts to the clinically approved proteasome inhibitor, bortezomib, or inhibition of glucosylceramide synthesis with eliglustat, partially corrected the impaired lactosylceramide trafficking defect and immediately suggest therapeutic avenues to explore in this fatal orphan disease.

Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular ß-glucosidases.

The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading ß-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-ß-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and (13)C6-labeled GlcChol as internal standard. In cells, the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice, GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C disease, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through ß-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer.

Inhibition of UDP-glucosylceramide synthase in mice prevents Gaucher disease-associated B-cell malignancy.

Clonal B-cell proliferation is a frequent manifestation of Gaucher disease - a sphingolipidosis associated with a high risk of multiple myeloma and non-Hodgkin lymphoma. Gaucher disease is caused by genetic deficiency of acid ß-glucosidase, the natural substrates of which (ß-d-glucosylceramide and ß-d-glucosylsphingosine) accumulate, principally in macrophages. Mice with inducible deficiency of ß-glucosidase [Gba(tm1Karl/tm1Karl)Tg(MX1-cre)1Cgn/0] serve as an authentic model of human Gaucher disease; we have recently reported clonal B-cell proliferation accompanied by monoclonal serum paraproteins and cognate tumours in these animals. To explore the relationship between B-cell malignancy and the biochemical defect, we treated Gaucher mice with eliglustat tartrate (GENZ 112638), a potent and selective inhibitor of the first committed step in glycosphingolipid biosynthesis. Twenty-two Gaucher mice received 300 mg/kg of GENZ 112638 daily for 3-10 months from 6 weeks of age. Plasma concentrations of ß-d-glucosylceramide and the unacylated glycosphingolipid, ß-d-glucosylsphingosine, declined. After administration of GENZ 112638 to Gaucher mice for 3-10 months, serum paraproteins were not detected and there was a striking reduction in the malignant lymphoproliferation: neither lymphomas nor plasmacytomas were found in animals that had received the investigational agent. In contrast, 14 out of 60 Gaucher mice without GENZ 112638 treatment developed these tumours; monoclonal paraproteins were detected in plasma from 18 of the 44 age-matched mice with Gaucher disease that had not received GENZ 112638. Long-term inhibition of glycosphingolipid biosynthesis suppresses the development of spontaneous B-cell lymphoma and myeloma in Gaucher mice.

Biomarkers for osteonecrosis in Gaucher disease.

INTRODUCTION: The search for surrogate biomarkers of osteonecrosis, a disabling complication of Gaucher disease, has intensified in the last decade. Biomarkers that predict osteonecrosis and monitor the effectiveness of therapies would improve clinical practice and enrich the molecular exploration of this disorder. AREAS COVERED: Here we discuss advances in biomarker research with special reference to those biomarkers associated with Gaucher disease and investigated in the context of enzyme therapy. Much progress has been made in the diversification of treatment for the condition and several biomarker molecules, which may ultimately improve risk assessment for osteonecrosis, have been identified. EXPERT OPINION: The discovery of prospective biomarkers of osteonecrosis such as CCL18/PARC, CXCL8/IL-8, CCL5/RANTES, CCL3/MIP-1α, CCL4/MIP-1ß, particularly during recurrent episodes occurring despite enzyme treatment, has the potential radically to change practices in the management of Gaucher disease and should improve therapeutic monitoring and prognostic evaluation. Ultimately, exploration of this field will provide the basis for a refined mechanistic understanding of pathogenesis.

Potential biomarkers of osteonecrosis in Gaucher disease.

BACKGROUND: To investigate the relationship between chemokines and cytokines and osteonecrosis in Gaucher disease, we conducted multiplex assays in a cohort of 100 adult patients. METHODS: Mean age was 45 years (18-86); 92 Gaucher patients received imiglucerase (median duration 8 years (2-18)). Forty-three had experienced osteonecrosis (ON), and eight had ON despite enzyme therapy. Serum cytokines/chemokines were determined by fluorimetric bead arrays in samples from Gaucher patients and healthy volunteers (10 males and 10 females). Intra-assay and inter-assay coefficients of variation were 2%-9.8% and 5.6%-15%, respectively. RESULTS: VEGF and CCL5/RANTES did not differ between Gaucher and control samples. Concentrations of CCL3/MIP-1α, CCL4/MIP-1ß, CCL2/MCP-1, CXCL8/IL-8, IL-1ra and CCL18/PARC were elevated in Gaucher patients (p<0.05 for each). Median CCL4/MIP-1ß, CXCL8/IL-8, CCL5/RANTES and CCL18/PARC concentrations were greater in the 43 osteonecrosis patients (88.6 pg/mL, 30.5 pg/mL, 89.6 ng/mL and 434 ng/mL, respectively) compared with the 57 patients who had no evidence of osteonecrosis (medians of 59.4, 13.3, 62.7 and 283, respectively, p<0.05). Moreover, the eight patients with ON despite imiglucerase had median concentrations of CCL3/MIP-1α, CCL4/MIP-1ß, CXCL8/IL-8, CCL5/RANTES and CCL18/PARC (73.2, 120.9, 36.3 pg/mL, 105 and 767 ng/mL, respectively), which significantly exceeded the values in 84 patients now free of ON (52.3, 71.2, 16.5 pg/mL, 69.5 and 315 ng/mL, respectively, p<0.05). Treatment exposures were similar. CONCLUSION: Numerous serum cytokines are elevated in Gaucher disease. CCL18/PARC, CCL3/MIP-1α, CCL4/MIP-1ß, CCL5/RANTES and CXCL8/IL-8 are potential biomarkers of osteonecrosis and may allow prediction of this disabling complication.

Western blot analysis of human and rat serotonin transporter in platelets and brain using site-specific antibodies: evidence that transporter undergoes endoproteolytic cleavage.

BACKGROUND: Serotonin transporter (SERT) is important target molecule for many antidepressive drugs and substances of abuse and is implicated in psychiatric disorders. We performed immunoblotting analysis of human and rat SERT in platelets and brain using the panel of eight site-specific SERT monoclonal and polyclonal antibodies (mAbs and pAbs). METHODS: SDS-PAGE/Western blotting was conducted using peroxidase-labeled DEAE and affinity purified SERT antibodies under conditions preventing SERT post-extraction degradation. RESULTS: Immunoreactive polypeptides of 14, 22, 32, 35, 37, 56, 68, and approximately 150-200 kDa were revealed in human platelet extracts using N-terminal and C-terminal SERT antibodies. In rat brain, C-terminal mAbs detected 68, 56, and 37 kDa proteins, in postmortem human brain predominated 35-37 kDa proteins. The immunoreactivity was abolished after antibody preadsorption with antigens. N-terminal pAbs recognized the 68 kDa protein, affinity purified on C-terminal mAbs, confirming its identity as full-size human SERT (the predicted size approximately 70.5 kDa). CONCLUSIONS: The explanation of the results of immunoblotting most likely is a site-specific SERT endoproteolytic cleavage and a marked difference in glycosylation rather than nonspecific protein degradation, cross-reactivity with other epitopes or SERT alternative splicing.

Analysis of the binding of bispecific monoclonal antibodies with immobilized antigens (human IgG and horseradish peroxidase) using a resonant mirror biosensor.

The interaction between two monoclonal antibodies (mAbs) and their corresponding bispecific antibody (bAb) with immobilized antigens has been examined using a resonant mirror biosensor (IAsys). BAbs were produced by cell fusion. The analysed panel of affinity-purified antibodies included two parental mAbs, one specific to human IgG (hIgG), and another specific to horseradish peroxidase (HRP), and a bAb derived thereof (anti-hIgG/HRP). The real-time analysis showed the drastic differences in the avidity of bivalent anti-HRP mAbs and anti-HRP shoulder of bAbs. Thus, the observed equilibrium association constant (K(ass)) of anti-HRP mAbs was about 50 times higher that of anti-HRP shoulder of bAbs. The ratio of association rate constants (k(ass)) of mAbs and bAbs was about two, due to the statistical factor of two binding sites per bivalent antibody molecule. However, the dissociation rate constant (k(diss)) of anti-HRP shoulder of bAbs was 21 times higher k(diss) of anti-HRP mAbs. The comparison with the theoretical model shows that these observations are consistent only with a situation in which bivalent binding of mAbs with immobilized HRP predominates over monovalent binding. On the contrary, the second parental mAb (anti-hIgG) did not show the increase in avidity due to bivalent binding, compared to the anti-hIgG shoulder of bAbs, suggesting that this mAb was bound monovalently to immobilized hIgG. The K(ass) values determined by solid-phase radioimmunoassay (RIA) yielded figures almost overlapping with those obtained by IAsys. The results of the comparison of bAbs and mAbs are discussed from the viewpoint of the use of bAbs in heterogeneous systems. On the other hand, these data demonstrate that real-time analysis of antibody binding parameters in IAsys biosensor is valuable for the selection of mAbs and bAbs with desired features, for different fields of application.