Detection of mutant protein in complex biological samples: glucocerebrosidase mutations in Gaucher's disease

We report a sensitive method to detect point mutations in proteins from complex samples. The method is based on surface-enhanced laser desorption/ionization time-of-flight (SELDI-ToF) MS but can be extended to other MS platforms. The target protein in this study is the lysosomal enzyme glucocerebrosidase (GC), the key enzyme in Gaucher's disease. Deficiency of GC activity results in accumulation of glucosylceramide in macrophages. The relationship between GC genotypes and Gaucher's patient phenotypes is not strict. The possibility to measure protein levels of GC in clinical samples may provide deeper insight into the phenomenology of Gaucher's disease. For this purpose, GC was isolated in a single enrichment step through interaction with an immobilized monoclonal antibody, 8E4. After on-chip digestion of the antibody-antigen complex with trypsin, a total of 25 GC peptides were identified (sequence coverage approximately 60%), including several peptides containing mutated amino acid residues. The described methodology allows mutational analysis on the protein level, directly measured on complex biological samples without the necessity of elaborate purification procedure

CCL18: a urinary marker of Gaucher cell burden in Gaucher patients

Glucosylceramide-laden tissue macrophages in Gaucher patients secrete large quantities of chitotriosidase and CC chemokine ligand 18 (CCL18), resulting in markedly increased plasma levels. We have comparatively investigated the occurrence of both parameters in plasma and urine samples of Gaucher patients. Chitotriosidase was high in urine samples of some symptomatic patients, but elevations did not correlate with increased plasma concentrations. Urinary chitotriosidase was particularly high in a patient with severe kidney involvement and local storage cell infiltration. Urinary levels of CCL18 were also highly elevated in samples from Gaucher patients as compared to controls. The median value of the CCL18/creatinine ratio in urine samples of 31 Gaucher patients was 143.3 pg/micromol (range 32-551) and in those of 12 normal subjects was 4.1 pg/micromol (range 1.3-6.8). In sharp contrast to chitotriosidase, increases in the low-molecular-mass chemokine CCL18 in urine and plasma specimens of Gaucher patients correlated well. A correlation was also observed for reductions in urinary and plasma CCL18 following therapy. It is concluded that assessment of urinary CCL18 of Gaucher patients gives insight into the total body burden on Gaucher cells, whereas that of chitotriosidase does not. Urinary chitotriosidase appears rather to be a reflection of renal patholog

Nanomolar affinity, iminosugar-based chemical probes for specific labeling of lysosomal glucocerebrosidase

Three different photoprobes were synthesized to label beta-glucosidases; one probe was based on glucose, two probes on the iminosugar deoxynojirimycin. The affinity of the probes for three different beta-glucosidases was determined. Furthermore, their labeling efficiencies, binding specificities through competition with deoxynojirimycin, and binding specificities in the presence of cell lysate, were evaluated. Especially one showed very high affinity towards non-lysosomal glucoceramidase (IC(50)=20nM

Long-Term Effect of Antibodies against Infused Alpha-Galactosidase A in Fabry Disease on Plasma and Urinary (lyso)Gb3 Reduction and Treatment Outcome

Introduction: Enzyme replacement therapy (ERT) with alpha-Galactosidase A (aGal A) may cause antibody (AB) formation against aGal A in males with Fabry disease (FD). Anti agalsidase ABs negatively influence globotriaosylceramide (Gb3) reduction. We investigated the impact of agalsidase AB on Gb3 and lysoGb3 and clinical outcome in Fabry patients on ERT. Methods: Adult male and female patients on ERT for at least one year were included. Urinary Gb3 was measured by HPLC, plasma lysoGb3 by LC-ESI-MS/MS and AB with a neutralization assay. Results: Of the 59 patients evaluable patients, 0/30 females and 17/29 males developed anti-agalsidase antibodies (AB+). Only 3/17 males had transient (low) titers (tolerized). All AB+ patients developed antibodies during the first year of treatment. Change of agalsidase preparation (or dose) did not induce antibody formation. AB+ males had significant less decline in plasma lysoGb3 compared to AB- males (p = 0.04). Urinary Gb3 levels decreased markedly in AB- but remained comparable to baseline in AB+ males (p <0.01). (Lyso) Gb3 reduction in plasma and urine on ERT was correlated with LVmass reduction in females and development white matter lesions and stroke. Conclusion: In male patients antibodies against aGal A remained present up to 10 years of ERT. The presence of these antibodies is associated with a less robust decrease in plasma lysoGb3 and a profound negative effect on urinary Gb3 reduction, which may reflect worse treatment outcom

Synthesis and evaluation of dimeric lipophilic iminosugars as inhibitors of glucosylceramide metabolism

Four dimeric and four monomeric lipophilic iminosugars were synthesized and subsequently evaluated on their inhibitory potential towards mammalian glucosylceramide synthase, glucocerebrosidase, beta-glucosidase 2, sucrase and lysosomal alpha-glucosidase. Compared to their monomeric counterparts the dimeric inhibitors showed decreased inhibition of glucosylceramide synthase and generally a comparable inhibitory potency for the glycosidases. (C) 2009 Elsevier Ltd. All rights reserve

Perguntamos: qual é o diagnóstico? We ask: what is the diagnosis?

Centro de Medicina Diagnóstica FleuryUniversidade Federal de São Paulo (UNIFESP)University of British ColumbiaUNIFESPSciEL

Exploring functional cyclophellitol analogues as human retaining beta-glucosidase inhibitors

The natural product, cyclophellitol and its aziridine analogue are potent mechanism-based retaining beta-glucosidase inhibitors. In this paper we explore the inhibitory potency of a number of cyclophellitol analogues against the three human retaining beta-glucosidases, GBA, GBA2 and GBA3. We demonstrate that N-alkyl cyclophellitol aziridine is at least equally potent in inhibiting the enzymes evaluated as its N-acyl congener, whereas the N-sulfonyl analogue is a considerably weaker inhibitor. Our results complement the literature on the inhibitory potency of cyclophellitol analogues and hold promise for the future design of more effective activity-based retaining glycosidase probes with respect to probe stability in physiological media