Biochemical characterization of chitotriosidase enzyme: comparison between normal individuals and patients with Gaucher and with Niemann-Pick diseases

OBJECTIVES: The aim of the present study was to establish the range of chitotriosidase (CT) activity in normal individuals, patients with Gaucher disease (GD) and Niemann-Pick disease (NPD), types A or B. The kinetics of CT in these three groups was also investigated. DESIGN AND METHODS: CT activity, as well as Km, Vmax, optimum pH, and thermal stability of the enzyme were determined in the plasma of control, GD, and NPD subjects. RESULTS: CT activity in GD and NPD patients was, respectively, around 600-fold and 30-fold greater than in normal individuals. We observed significant differences in optimum pH, Vmax, and thermal stability between the various groups. Km was different in normal individuals relative to GD and NPD patients. However, there was no significant difference between Km values in patients with GD and with NPD. CONCLUSIONS: Based on the differences found in the biochemical parameters studied, our results may be important to help the identification of patients not only with GD but also with NPD.     

Comparison of the measurement of lysosomal hydrolase activity in mycoplasma-contaminated and non-contaminated human fibroblast cultures treated with mycoplasma removal agent

OBJECTIVES: To investigate the effect of both mycoplasma contamination and of its remover (MRA), through human fibroblasts culture over the activity of some lysosomal hydrolases. DESIGN AND METHODS: Activity was measured in contaminated fibroblasts before and after the addition of MRA. Results were compared with the enzymatic activity in control fibroblasts with and without MRA. RESULTS: Only beta-glucosidase showed no significant alteration in the presence of either mycoplasma or MRA. Total hexosaminidase and beta-galactosidase underwent significant interference in the presence of the mycoplasma and the MRA. The % of hexosaminidase A and arylsulphatase A altered their activity only in the presence of MRA. Beta-glucuronidase changed its activity only in the presence of mycoplasma. CONCLUSIONS: The fibroblast enzymes behaved differently in the presence of MRA and/or mycoplasma, demonstrating the sensitivity of these hydrolases. Our work suggests that mycoplasma and MRA alter the activity of some lysosomal hydrolases.     

Elevated lysosomal pH in Mucolipidosis type IV cells

The lysosomal pH in Mucolipidosis type IV (ML-IV) and several other storage disease fibroblasts (Niemann Pick, type A; Niemann Pick, type C; Hunter (MPS II); and Farber) and in normal human skin fibroblasts was determined in situ. Cells were pulse labeled with a fluorescein-conjugated dextran to label the lysosomes. Quantitative fluorescence microscopy was then carried out on living cells to measure the ratio of fluorescence at two different excitation wavelengths. An image processing routine was used to quantify fluorescence from individual lysosomes. Ratiometric data were converted to an absolute value of pH using an appropriate standard curve. Lysosomal pH varied between 4.3 and 4.5 for all the cell types examined except ML-IV cells which was almost one pH unit higher (pH 5.2). Qualitatively similar results were obtained using acridine orange, another fluorophore whose fluorescence emission is pH dependent, ruling out the possibility that the stored molecules in ML-IV cells might induce an artifact in the fluorescein-based pH measurements. We conclude that elevated lysosomal pH is unique to ML-IV cells. This property may be an important factor, if not the cause, for the accumulation of the broad spectrum of substances, including sphingolipids, phospholipids, and acid mucopolysaccharides, even though the lysosomal hydrolases participating in the catabolism of these molecules appear to be normal.     

Effects of antisera raised against native and denatured human alpha-glucosidase and beta-hexosaminidases on native enzyme activity

Antisera were raised in rabbits against native and sodium dodecylsulfate denatured forms of human acid alpha-glucosidase and beta-hexosaminidases A and B. Anti-native enzyme antisera were able to precipitate all or nearly all enzyme activity from cell extracts, and to eliminate all stainable activity on electrophoresis. Antisera prepared against denatured enzymes precipitated only a minor part of enzyme activity. Electrophoretic analysis showed that these antisera were able to bind to the enzyme molecule. The result was a slowing down of the anodic migration but not immobilization. The use of variants with hexosaminidase deficiencies helped to clarify the action of the antisera on the various hexosaminidase isozymes.     

Lysosomal storage disorders: Novel and frequent pathogenic variants in a large cohort of Indian patients of Pompe,Fabry, Gaucher and Hurler disease

ObjectivesDiagnosis of lysosomal storage disorders (LSDs) remains challenging due to wide clinical, biochemical and molecular heterogeneity. The study applies a combined biochemical and genetic approach to diagnose symptomatic Indian patients of Pompe, Fabry, Gaucher and Hurler disease to generate a comprehensive dataset of pathogenic variants for these disorders.Design & methodsSymptomatic patients were biochemically diagnosed by fluorometric methods and molecular confirmation was carried out by gene sequencing. Genetic variants were analyzed according to the ACMG/AMP 2015 variant interpretation guidelines.ResultsAmongst the 2181 suspected patients, 285 (13%) were biochemically diagnosed. Of these, 22.5% (64/285) diagnosed with Pompe disease harboured c.1933G>A, c.1A>G, c.1927G>A and c.2783G>C as common and 10 novel pathogenic variants while 7.4% (21/285) patients diagnosed with Fabry disease carried c.851T>C, c.902G>A, c.905A>C and c.1212_1234del as frequent disease-causing variants along with 7 novel pathogenic variants. As many as 48.4% (138/285) patients were diagnosed with Gaucher disease and had c.1448T>C as the most common pathogenic variant followed by c.1342G>C and c.754T>C with 7 previously unreported disease-causing variants and in the 21.7% (62/285) diagnosed cases of Hurler disease, c.1469T>C, c.754delC c.568_581del and c.1898C>T were identified as the most common causative variants along with 21 novel pathogenic variants.ConclusionThis comprehensive data set of disease-causing frequent and novel pathogenic variants reported for the first time in such a large patient cohort for each of these four LSDs from the Indian sub-continent, along with their biochemical and clinical spectrum will contribute towards providing definitive diagnosis and treatment, identifying carrier status, as well as in counselling prenatal cases to reduce the morbidity and mortality associated with these disorders.     

Lysosomal glycohydrolases in normal T and non-T peripheral lymphocytes

The optimal assay conditions and the levels of seven lysosomal glycohydrolases (alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucosidase, beta-D-glucuronidase, beta-N-acetyl-D-glucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase), alpha-D-mannosidase, alpha-L-fucosidase) were determined in human peripheral unseparated lymphocytes, T and non-T lymphocyte subpopulations. From fifteen adult volunteers the enzymes were assayed by fluorimetric procedures using the corresponding 4-methylumbelliferyl glycosides as substrates. The enzyme assay procedures displayed good precision and reproducibility. All the tested enzymes had higher activities in non-T than T lymphocytes. This difference was statistically highly significant, especially when the enzyme contents were expressed on a DNA, rather than mg protein, basis. Unseparated lymphocytes displayed levels of lysosomal enzymes which corresponded to the proportion of T and non-T lymphocytes in the unseparated preparation, indicating that the process of lymphocyte fractionation caused neither loss nor activation of lysosomal enzymes. It is concluded that the observed difference in lysosomal enzyme levels is an authentic imprint of the two lymphocyte subpopulations, implying a differential role played by the lysosomal apparatus in the same cells.     

Biochemical and ultra-structural reactions to parenteral nutrition with two different fat emulsions in rats

Objective: To compare the effects on fat metabolism and Kupffer cell morphology by total parenteral nutrition (TPN) with two different fat emulsions. Design: Thirty-two male Sprague-Dawley rats, divided into three groups, were investigated. Rats fed orally were used as a reference group, and a group of rats receiving TPN with fat emulsions containing pure long-chain triglycerides (LCT) was compared to a group of rats receiving fat emulsions containing both long-chain triglycerides and medium-chain triglycerides (MCT/LCT). The TPN regimens were equicaloric and administered continuously via a jugular catheter for 10 days. Interventions: After suffocation, blood of the rats was collected for the determination of serum lipids. Epididymal fat and heart were collected for the analysis of lipoprotein lipase (LPL) activities, and liver specimens were saved for analyses of hepatic triglyceride concentration, as well as activities of hepatic lipase (HL) and lysosomal enzymes. Light and electron microscopy were used for examination of the Kupffer cell reaction. Results: Directly after termination of parenteral feeding, the levels of serum triglycerides and high density lipoprotein (HDL) triglycerides were higher in the MCT/LCT group than in the LCT group, while no differences concerning cholesterol and phospholipid concentrations were found. No significant difference in liver steatosis was found between the two TPN groups. Comparison of the TPN groups showed that the MCT/LCT group had significantly decreased LPL activity in adipose tissue, while the LCT group had significantly increased LPL activity in the heart. The activity of HL was low in both groups, but significantly lower in the LCT group. Lipid accumulation and an increased number of lysosomes were found in all Kupffer cell when TPN with LCT emulsions was used. Moreover, TPN induced a pronounced increase in various liver lysosomal enzyme activities, but there was no notable difference between LCT and MCT/LCT effects. Conclusions: Compared to treatment with pure LCT emulsions, treatment with MCT/LCT emulsions evoked weaker biochemical reactions in terms of lower activity of lipoprotein lipase in fat and heart together with higher serum and HDL triglyceride levels. Morphological signs of increased Kupffer cell activity such as the appearance of multiple lysosomes and fat vacuoles in the cytoplasm followed treatment with pure LCT emulsions. However, both TPN groups showed a marked increase in activities of liver lysosomal enzymes. Received: 27 December 1996 Accepted: 24 April 1998     

Current molecular genetics strategies for the diagnosis of lysosomal storage disorders

Lysosomal storage disorders (LSDs) are a group of almost 50 monogenic diseases characterized by mutations causing deficiency of lysosomal enzymes or non-enzyme proteins involved in transport across the lysosomal membrane, protein maturation or lysosomal biogenesis. Usually, affected patients are normal at birth and have a progressive and severe disease with high morbidity and reduced life expectancy. The overall incidence of LSDs is usually estimated as 1:5000, but newborn screening studies are indicating that it could be much higher. Specific therapies were already developed for selected LSDs, making the timely and correct diagnosis very important for successful treatment and also for genetic counseling. In most LSD cases the biochemical techniques provide a reliable diagnosis. However, the identification of pathogenic mutations by genetic analysis is being increasingly recommended to provide additional information. In this paper we discuss the conventional methods for genetic analysis used in the LSDs [restriction fragment length polymorphism (RFLP), amplification-refractory mutation system (ARMS), single strand conformation polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC), real-time polymerase chain reaction, high resolution melting (HRM), multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing] and also the newer approaches [massive parallel sequencing, array comparative genomic hybridization (CGH)].     

α-甘露糖苷贮积症的分子遗传学及其在临床诊断与防治方面的意义

MAN281基因突变导致α-甘露糖苷酶缺乏或活性降低是引起α-甘露糖苷贮积症的根本内因。对MAN2BI基因、LAMAN酶的结构和功能的研究、基因型与表现型的相关性研究以及诊防治方面的研究近年来都取得了诸多新进展。本文重点围绕这几方面作一综述。     

Behaviour of several enzymes of lysosomal origin in human plasma during pregnancy

The following enzymes of lysosomal origin were fluorimetrically determined in maternal plasma from the second to the ninth month of pregnancy at 1-mth intervals: beta-D-N-acetylglucosaminidase (EC 3.2.1.30), beta-D-glucuronidase (EC 3.2.1.31), beta-D-glucosidase (EC 3.2.1.21), beta-D-galactosidase (EC 3.2.1.22), alpha-D-galactosidase (EC 3.2.1.23), alpha-L-fucosidase (EC 3.2.1.51) and alpha-D-mannosidase (EC 3.2.1.24) (pH 4.0). As reference microsomal alpha-D-mannosidase (pH 5.7) was also studied. Thirty-eight healthy women, aged 18-37 yr, who had a normal pregnancy followed by normal parturition, were studied. All enzymes, with the only exception of beta-D-galactosidase, showed a progressive and statistically significant increase of activity throughout pregnancy. At the end of pregnancy, the increase ranged from a maximum of 5.6-fold for beta-D-N-acetylglucosaminidase to a minimum of 0.55-fold for alpha-D-mannosidase, pH 5.7. In the case of beta-D-N-acetylglucosaminidase, the level at the fifth month of pregnancy was significantly higher than that at the third month, and from the sixth to the ninth month each level significantly differed from that of the month immediately preceding.     

Lysosomal enzyme activities: new potential markers for Sjögren's syndrome

OBJECTIVE: To evaluate the changes in lysosomal enzyme activities in leukocytes of patients with Sj?gren's syndrome. METHODS: Leukocytes were obtained from 38 patients with Sj?gren's syndrome and 36 healthy subjects. The activities of the following glycosidases were measured: alpha-glucosidase (AGU), beta-galactosidase (BGA), alpha-mannosidase (AMAN), beta-glucuronidase (GCU), beta-hexosaminidase (HEX), and the following proteases: cathepsin B (CATH B), dipeptidyl peptidase I (DPP I), cathepsin H (CATH H), dipeptidyl peptidase II (DPP II), tripeptidyl peptidase I (TPP I), and cathepsin D (CATH D) activity. RESULTS: Activity of the glycosidases beta-galactosidase, alpha-mannosidase, beta-glucuronidase and beta-hexosaminidase, as well as of the peptidases cathepsin B, cathepsin D, dipeptidyl peptidase I, and tripeptidyl peptidase I, was elevated during the first 5 years of SS, and it increased further between 5 and 10 years after diagnosis. CONCLUSIONS: The elevated activities of the lysosomal enzymes in Sj?gren's syndrome patients may play a role in tissue damage by accelerated breakdown of glycoproteins in lysosomes.     

Genetic analysis of mucopolysaccharidosis type VI in Taiwanese patients

BACKGROUND: Glutathione peroxidase 1 (GPX1), the key antioxidant enzyme in vascular endothelial cells, has been shown to exert a protective effect against the presence of coronary artery disease (CAD). The 198Pro/leu variant, located at codon 198 of GPX1 gene, has recently been linked to cardiovascular disease, but data were inconsistent. We investigated the association between the occurrence of CAD and the 198Pro/leu variant in a Chinese population. METHODS: A total of 265 unrelated CAD patients and 265 age- and sex-matched control subjects were recruited in this study. The GPX1 198Pro/leu genotype was determined using polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Compared to the 198Pro/Pro carriers, subjects with the variant genotypes (198Pro/leu and 198Leu/leu) had a significantly higher risk of CAD (adjusted OR=2.02, 95%CI=1.27-3.22). In stratified analyses, the variant genotypes were significantly associated with increased CAD risk in subjects <64 y (adjusted OR=2.41, 95%CI=1.16-4.98), males (adjusted OR=1.86, 95%CI=1.09-3.18) and non-smokers (adjusted OR=2.40, 95%CI=1.15-5.01). However, no significant association was observed between this variant and the severity of CAD. CONCLUSION: These data provide evidence that GPX1 198Pro/leu variant genotypes are significantly associated with CAD risk in this Chinese population.     

Effects of antisera raised against native and denatured human α-glucosidase and β-hexosaminidases on native enzyme activity

Antisera were raised in rabbits against native and sodium dodecylsulfate denatured forms of human acid α-glucosidase and β-hexosaminidases A and B.

1. (1) Anti-native enzyme antisera were able to precipitate all or nearly all enzyme activity from cell extracts, and to eliminate all stainable activity on electrophoresis.

2. (2) Antisera prepared against denatured enzymes precipitated only a minor part of enzyme activity. Electrophoretic analysis showed that these antisera were able to bind to the enzyme molecule. The result was a slowing down of the anodic migration but not immobilization. The use of variants with hexosaminidase deficiencies helped to clarify the action of the antisera on the various hexosaminidase isozymes.