Morphological and biochemical studies of human beta-mannosidosis: identification of a novel beta-mannosidase gene mutation.

BACKGROUND: There are seven well-known lysosomal storage diseases that produce angiokeratoma corporis diffusum clinically. beta-Mannosidosis (MANB1; OMIM248510), first reported in humans in 1986, is a rare hereditary lysosomal storage disease caused by a deficiency of the enzyme beta-mannosidase. Since then, 13 cases of beta-mannosidase deficiency in ten families have been described. A human beta-mannosidase mutation has been reported only by Alkhayat et al. in 1998. OBJECTIVES: To clarify its pathogenesis we did electron microscopic, biochemical and molecular biological investigations of a Japanese patient with beta-mannosidosis. METHODS: Ultrastructural analyses, enzyme assays, cell culture and mRNA and genomic DNA were sequenced to find mutations in the beta-mannosidase gene. RESULTS: Electron microscopy of skin biopsy specimens from the patient showed cytoplasmic vacuolation of lysosomes in blood and lymph vessels, endothelial cells, fibroblasts, secretory portions of eccrine sweat glands, neural cells and basal keratinocytes in the epidermis. This vacuolation was also observed in cultured keratinocytes and fibroblasts. Assays of seven enzyme activities in plasma and cultured skin fibroblasts showed a marked decrease of beta-mannosidase activity. Sequencing the beta-mannosidase cDNA revealed a four-base (ATAA) insertion between exons 7 and 8, resulting in a frameshift at codon 321 and termination at codon 325. Analysis of the patient's genomic DNA revealed a novel homozygous A(+1)-->G splice site mutation in intron 7. CONCLUSIONS: To our knowledge, this is the first case of beta-mannosidosis reported in Japan and the second report in which a gene mutation is identified. The biological importance of beta-mannose moieties in glycoproteins in basal keratinocytes is suggested.

beta-hexosaminidase, alpha-D-mannosidase, and beta-mannosidase expression in serum from patients with carbohydrate-deficient glycoprotein syndrome type I.

The activity of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside substrate, and of beta-D-mannosidase was significantly higher in the serum of patients with carbohydrate-deficient glycoprotein (CDG) syndrome type IA (phosphomannomutase deficiency) than in controls. No significant differences were observed in the activity of beta-hexosaminidase, determined using 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate as substrate, and the activity of alpha-D-mannosidase. Using DEAE-cellulose chromatography, a greater amount of hexosaminidase B than hexosaminidase A was detected in CDG serum. In CDG serum, hexosaminidase A was eluted in a more basic position in the salt gradient. An isoenzyme of alpha-D-mannosidase and beta-D-mannosidase was identified in control and CDG sera. alpha-D-Mannosidase isoenzyme was eluted in a slightly more basic position in CDG serum than in control serum, whereas beta-D-mannosidase isoenzyme was eluted in the same position.

[Effects of bioflavonoids on the toxicity of T-toxin in rats. A biochemical study]. / Vliianie bioflavonoidov na toksichnost' T-2-toksina dlia krys. Biokhimicheskie issledovaniia.

The enrichment of a diet of rats by flavonoids of milk thistle, Silybum marianum, reduced toxicity of T-2 toxin and was accompanied by reduction of a degree of change of total and nonsedimentable activity of lysosomal enzymes and microsomal xenobiotic metabolizing enzymes.

Effects of locoweed (Oxytropis sericea) on reproduction in cows with a history of locoweed consumption.

Locoweed (Oxytropis sericea) was fed to 4 open cycling cows that had repeatedly consumed locoweed in previous grazing trails. They received locoweed at 20% of their diet for 30 d (0.68-0.76 mg swainsonine/kg/d). Locoweed induced an immediate rise in serum swainsonine (the locoweed toxin) and a concomitant drop in serum alpha-mannosidase activity in all cows accompanied by abnormal estrus behavior, increased estrous cycle lengths and failure to conceive. Serum progesterone (P4) profiles demonstrated that estrous cycles lengthened from an average of 19 d before locoweed feeding to an average of 34 d (range 24-43 d) while on locoweed. After locoweed feeding ceased, normal estrous cycles returned within an average of 14 d (range 7-25 d). Two of the 4 cows conceived on their first post-locoweed estrus at 7 and 25 d. The third cow bred twice at 13 and 31 d after lowoweed feeding stopped, and the fourth cow bred 3 times at 11, 31 and 52 d before conception occurred. Pregnancies in all 4 cows progressed normally to 7 mo gestation when locoweed was again fed at 20% of the diet for 40 d (gestation days 213 and 253) to 2/4 cows, 1 of which aborted 10 d after lowoweed feeding stopped (263 days gestation). The other cow fed lowoweed calved normally as did the 2 pregnancy control cows. Serum P4 and estradiol (E2) profiles during pregnancy appeared normal before, during and after locoweed feeding except in the cow that aborted, whose P4 declined and E2 increased prematurely. The general trend of serum prolactin was similar in locoweed-fed and control cows.

Characterization of a soluble class I alpha-mannosidase in human serum.

Class I alpha-mannosidases are thought to exist exclusively as integral membrane proteins that play intracellulary an essential role in the N-glycan biosynthesis. Using [3H]Man9GlcNAc2 as a substrate, we were able to identify a soluble alpha-mannosidase in human serum that trims the substrate Man9GlcNAc2 to Man(5-8)GlcNAc2 with Man6GlcNAc2 being the major product. This serum mannosidase is Ca2+-dependent, sensitive to 1-deoxymannojirimycin but insensitive to the class II inhibitor swainsonine and, hence, belongs to class I mannosidases. The enzymatic properties of the serum class I mannosidase are similar to that of the membrane bound class I mannosidases Golgi-mannosidase IA and IB and Man9-mannosidase.

The pathogenesis and toxicokinetics of locoweed (Astragalus and Oxytropis spp.) poisoning in livestock.

Locoweed poisoning is a chronic disease that develops in livestock grazing for several weeks on certain Astragalus and Oxytropis spp. that contain the locoweed toxin, swainsonine. The purpose of this review is to present recent research on swainsonine toxicokinetics and locoweed-induced clinical and histologic lesions. Swainsonine inhibits cellular mannosidases resulting in lysosomal storage disease similar to genetic mannosidosis. Diagnosis of clinical poisoning is generally made by documenting exposure, identifying the neurologic signs, and analyzing serum for alpha-mannosidase activity and swainsonine. All tissues of poisoned animals contained swainsonine, and the clearance rates from most tissues was about 20 hours (T1/2 half life). The liver and kidney had longer rate of about 60 hours (T1/2). This suggests that poisoned animals should be allowed a 28-day withdrawal to insure complete swainsonine clearance. Poisoning results in vacuolation of most tissues that is most obvious in neurons and epithelial cells. Most of these histologic lesions resolved shortly after poisoning is discontinued; however, some neurologic changes are irreversible and permanent.

Mutant analysis reveals an alternative pathway for N-linked glycosylation in Drosophila melanogaster.

The mas-1 gene of Drosophila melanogaster encodes Golgi mannosidase I (MAS-1), and flies homozygous for small deletions of the gene are viable. They show but few abnormalities and those have a low penetrance [Kerscher, S., Albert, S., Wucherpfennig, D., Heisenberg, M. & Schneuwly, S. (1995) Dev. Biol. 168, 613-626]. Here we sequence the N-linked oligosaccharides associated with a reporter protein, and with membrane proteins prepared from wild type and MAS-1 null organisms. The results show that the null organisms synthesise the same range of oligosaccharides as wild type, albeit with different ratios. There is an accumulation of the Man8GlcNAc2 which is one of the substrates for the MAS-1 enzyme. This supports the suggestion of Kerscher et al. that the lack of severe phenotypic disturbances in the null organisms is due to the presence of an alternative pathway(s), but it also shows that this alternative pathway(s) does not entirely compensate for the normal pathway.

Plasma tumor necrosis factor-a (TNF-a) levels in Gaucher disease.

Tumor necrosis factor-a (TNF-a) levels were measured in the plasma of patients with different types of Gaucher disease (GD) and patients with other lysosomal storage diseases. The highest TNF-a levels were observed in the most severe neuronopathic type of GD, exceeding those found in healthy individuals as well as patients with other lysosomal disorders. Type I GD cases showed a wide range of TNF-a levels ranging from normal to 2.5 x the highest control value. TNF-a is a pleiotropic cytokine produced mainly by activated macrophages. Our data suggest that it may play a role in the pathophysiology of GD disease.

Lysosomal hydrolases in serum from human immunodeficiency virus-infected patients.

beta-Hexosaminidase isoenzymes were separated by DEAE-cellulose chromatography in the serum of 23 patients infected with human immunodeficiency virus at different stage of the disease. Forms corresponding to hexosaminidase B, I and A were present in pathological sera. There is an increase in the percentage of hexosaminidase I in pathological sera, that could be used as an additional marker to monitor the clinical stage of the disease. Furthermore, total activities of some lysosomal enzymes were determined in these sera. Activities of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside substrate, alpha-mannosidase and beta-mannosidase were significantly higher in the serum of patients at the C3 stage of disease than in controls. No significant differences were observed in the activity of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate substrate, beta-glucuronidase and beta-galactosidase.

Increased levels of several lysosomal enzymes in sera from women using oral contraceptives.

The activities of eight lysosomal enzymes were measured by spectrophotometric/spectrofluorimetric techniques in the blood sera of 19-24 apparently healthy women using an oral contraceptive (progestin and oestradiol synthetic derivative, desogestrel+ethinyloestradiol) in comparison with 15-16 non-pregnant women not using contraceptives (controls), in a randomised, double-blind, controlled study. beta-Glucuronidase and arylesterase showed statistically increased activities (P < or = 0.05) in the experimental group in comparison to the controls. No significant differences were found for the remaining enzymes assayed (beta-N-acetylhexosaminidase, alpha-L-fucosidase, alpha-mannosidase, beta-galactosidase, alpha-galactosidase and acid phosphatase). Similar results were obtained when the contraceptive formed by the combination of levonorgestrel and ethinyloestradiol was used by an experimental group of eight healthy women. These results suggest that the significant increases in the above-mentioned activities might be the physiological response of the organism (through catabolic processes catalysed by lysosomal enzymes) to the administration of exogenous synthetic compounds, such as the oral contraceptives used.

Lysosomal enzymuria in preeclampsia.

We hypothesize that the preeclamptic patient has proximal tubule epithelial injury, which leads to the release of lysosomal enzymes, and that the excretion of these enzymes might serve as a diagnostic or predictive marker in preeclamptic women. The study group consisted of 14 women with preeclampsia (10 severe and 4 mild, as defined by The American College of Obstetricians and Gynecologists criteria) and 28 normotensive controls with singleton pregnancies at 27 to 41 weeks. There were no significant differences between the two groups for gestational age, maternal age, or race. Maternal serum and urine specimens were prospectively obtained and analyzed for beta-glucuronidase, beta-hexosaminidase, alpha-galactosidase, beta-galactosidase, and alpha-mannosidase using fluorometric assays. Median serum and urine activities and fractional excretions of each of the five hydrolases were compared between the two study groups using the Mann-Whitney two-sample rank test. The serum enzyme activities of beta-hexosaminidase (P = 0.002), alpha-galactosidase (P = 0.0001), and alpha-mannosidase (P = 0.02) were significantly lower in preeclamptic patients than in controls. The urine enzyme activities of beta-glucuronidase (P = 0.001), alpha-galactosidase (P = 0.002), beta-galactosidase (P 0.0003), and alpha-mannosidase (P = 0.003) were significantly higher in the preeclamptic patients. The fractional enzyme excretions of all five lysosomal hydrolases were higher in preeclamptic patients than in controls with P < or = 0.0003 for each enzyme. Preeclampsia is associated with a significant decrease in serum activities of three of the five hydrolases studied, a significant increase in urine enzyme activities in four of the five hydrolases studied, and a significant increase in the fractional excretion of all five lysosomal hydrolases.

[Effects of stress on indices for assessing zinc nutrition status].

To evaluate the specificity of some functional indices in assessment of body zinc nutrition status, we used experimental rat model to observe the effects of some factors, such as forced swimming, starvation, trauma and alcohol intoxication on zinc the status. Plasma zinc levels of rats significantly decreased after trauma increased after starvation. Liver zinc content showed a rising tendency in trauma and starvation rats. Activities of superoxide dismutase in red blood cells and alkaline phosphatase, mannosidase, 5'-nucleotidase in plasma of rats with alcohol intoxication declined significantly. Starvation led to decreased activities of alkaline phosphatase and angiotensin-converting enzyme, but increased activities of mannosidase. Trauma and forced swimming could cause increase of angiotensin-converting enzyme activity and decrease of 5'-nucleotidase activity, respectively. These results indicate that physiological and pathological effects should be excluded from of the above indices as plasma zinc index, in the assessment of body zinc nutrition status.

Tissue and serum swainsonine concentrations in sheep ingesting Astragalus lentiginosus (locoweed).

Locoweed intoxication or locoism results when animals continuously graze certain plants of the general Astragalus or Oxytropis. The locoweed toxin, swainsonine, is water soluble and is rapidly absorbed and eliminated. The purpose of this study was to determine the distribution of swainsonine in tissues of sheep eating locoweed and to determine if the tissue swainsonine concentrations change with continued locoweed ingestion. Fifteen cross-breed whethers were divided into 3 groups of 5 each and fed alfalfa pellets (Group 1) or alfalfa pellets with 10% Astragalus lentiginosus for 13 d (Group 2) or for 21 d (Group 3). After the feeding periods, the animals were slaughtered and tissues were collected, frozen and later analyzed for swainsonine using an in vitro, alpha-mannosidase inhibition assay. Significant alpha-mannosidase inhibitory activity (interpreted as ng/ml of swainsonine) was detected in whole blood, skeletal muscle, brain, kidney, liver, thyroid and urine. The swainsonine concentrations in tissues were significantly correlated with daily swainsonine intake (r = 0.58 to 0.96). With the exception of kidney, longer exposure did not result in significant increases in the swainsonine concentrations in blood, muscle, brain, liver or thyroid. Liver had the highest swainsonine concentrations with 3049 +/- 1952 and 3947 +/- 457 ng/ml (mean +/- SD) in Groups 2 and 3 respectively. Swainsonine concentrations varied widely within the groups suggesting individual animal variability in swainsonine absorption, metabolism and excretion. These findings suggest that swainsonine is present in tissues of animals eating locoweed and that in most tissues the amount was directly correlated to the swainsonine dose ingested, but not to the length of exposure.

Elevated levels of activities of beta-hexosaminidase and alpha-mannosidase in human immunodeficiency virus-infected patients.

To determine their value as markers of the clinical stage of human immunodeficiency virus (HIV) disease, plasma activities of lysosomal glycosidases were determined in the plasma of 97 HIV-infected patients: molecular forms of cathepsin D were characterized by Western blot to examine the mode of enzyme release. In patients with Centers for Disease Control and Prevention stage II and III of HIV disease, plasma activity of beta-hexosaminidase was significantly increased. In patients with stage III infection, alpha-mannosidase activity was also significantly increased and cathepsin D was elevated and present only in its premature form. Thus, determination of plasma activities of beta-hexosaminidase and alpha-mannosidase in HIV-positive persons may be useful for distinguishing the clinical stage of disease. The elevation of precursors of cathepsin D in patients with stage III HIV disease indicates that secretion of lysosomal enzymes rather than leakage of enzymes from damaged cells is markedly elevated.

Platelet glycohydrolase activities: characterization and release.

Granules containing acid hydrolases have been detected in human platelets but have not been thoroughly characterized. We have studied the activity and characteristics of glycohydrolases present in normal human platelets, evaluated their release upon stimulation with thrombin, and assessed the contribution of platelet - released lysosomal contents to the glycohydrolase activity present in normal serum. Platelets contained a remarkable glycohydrolase activity with a prevalence of beta - N-acetylhexosaminidase. All glycohydrolases were released to some extent upon stimulation with thrombin and contributed to the glycohydrolase activity found in human serum. alpha-Mannosidase and alpha-galactosidase were partially inactivated after release by a mechanism as yet undefined. In addition, thrombin stimulation affects the intraplatelet isoenzyme pattern of beta-N-acetylhexosaminidase by producing the appearance of a new form.

Serum swainsonine concentration and alpha-mannosidase activity in cattle and sheep ingesting Oxytropis sericea and Astragalus lentiginosus (locoweeds).

Serum alpha-mannosidase activity and swainsonine concentration were determined in 4 cattle and 15 sheep (3 groups of 5 each) that were administered ground locoweed (Oxytropis sericea or Astragalus lentiginosus) containing swainsonine at dosages of approximately 0.8 mg/kg of body weight/d (cows, 30 days each) and 0, 1.0, and 1.5 mg/kg/d (sheep, 11 days each). The cattle developed mild clinical signs of locoism, including signs of depression, lethargy, and slight intention tremors. Clinical signs of toxicosis were not observed in the sheep. Within 24 hours of initial treatment, serum alpha-mannosidase activity in cows and sheep, measured by the release of 4-methylumbelliferone from an artificial substrate, was markedly decreased to 28 and 40 mumol of 4-methylumbelliferone/L, respectively. Mean serum alpha-mannosidase activity of control cows and sheep was 400 +/- 94 and 422 +/- 42 (mean +/- SD), respectively. In the treated animals, decreased serum alpha-mannosidase activities returned to normal or higher activities within 6 days after treatment was discontinued. Using a jack bean alpha-mannosidase assay, increased swainsonine activity (153, 209, and 381 ng/ml, respectively) was detected in the serum of cattle and of sheep in the high- and low-dose groups within 24 hours after treatment with locoweed. Swainsonine concentration remained high, with mean concentrations of 204, 432, and 395 ng/ml (cows and 2 sheep groups, respectively) during the treatment period. After treatment, swainsonine was rapidly cleared, with estimated serum half-life of 16.4, 17.6, and 20.3 hours (cows, and high- and low-dose sheep groups, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma lysosomal enzyme levels in patients with motor neuron disease.

beta-Hexosaminidase and acid-alpha-mannosidase were estimated in 17 adult patients with motor neuron disease. Normal plasma levels of beta-hexosaminidase ((A+B) and A) were found in all patients studied. Plasma acid alpha-mannosidase levels were normal in all but two patients with the spinal muscular atrophy type of the disorder. In addition, altered biochemical properties of acid alpha-mannosidase (i.e. Km, thermal stability) were found in the low-activity cases.

Serum alpha-mannosidase activity and the clinicopathologic alterations of locoweed (Astragalus mollissimus) intoxication in range cattle.

Subclinical intoxication of livestock with Astragalus and Oxytropis species (locoweeds) results in decreased animal feed conversion, reduced weight gains, and reproductive failure. Sensitive diagnostic methods to definitively diagnose and monitor intoxication are needed to minimize these losses and better manage locoweed-infested pastures and rangelands. Sera from cattle grazing locoweed were evaluated for alpha-mannosidase activity, serum biochemical values, electrolytes, and thyroid hormone concentrations. As the cows began to ingest locoweed, the mean serum alpha-mannosidase activities dropped significantly (400.0 microM to 72.5 microM). Changes in other serum chemistry values were less specific; however, individual animals (generally those ingesting more locoweed) had elevated levels of alkaline phosphatase (ALP), aspartate aminotransferase, and lactate dehydrogenase, with decreased serum total protein (5.8 +/- 0.8 g/dl) and albumin (2.3 +/- 0.3 g/dl). Mean serum thyroid concentrations (both T4 and T3) were lower in animals that were ingesting locoweed. The calculated swainsonine dose correlated statistically with serum alpha-mannosidase activity, ALP, albumin, Cl, CO2, and thyroid hormone T3. This correlation suggests that serum alpha-mannosidase activity along with potential changes in ALP, albumin, and thyroid hormone concentrations is a sensitive indicator of locoweed exposure and intoxication. These parameters may also be useful for monitoring intoxication and allowing subclinically affected cattle to be removed from infested areas before irreversible damage occurs.