Urinary lysosomal hydrolases in mucolipidosis II and mucolipidosis III.

Investigation of the binding characteristics of acid beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase, alpha-D-galactosidase and alpha-L-fucosidase from patients with mucolipidosis II and mucolipidosis III to concanavalin A--Sepharose 4B revealed a 2--10-fold decrease in the proportion of enzyme activities from patients with mucolipidoses II and III that adsorbed on the lectin. Neuraminidase treatment of the unadsorbed enzyme fraction did not significantly increased the proportion of enzyme activities that bound to the concanavalin A--Sepharose 4B. Characterization of acid beta-D-galactosidase from the adsorbed and unadsorbed enzyme fractions of mucolipidosis II and mucolipidosis III patients demonstrated identical apparent Km values of 0.22 mM with respect to 4-methylumbelliferyl beta-D-galactopyranoside, altered pH--activity profiles and heterogeneous isoelectric-focusing patterns. The results of this study support the suggestion of an alteration of a post-translational modification (possibly glycosylation) occurring in mucolipidosis II and mucolipidosis III common to the lysosomal hydrolases that affects the mannoserelated properties of these enzymes.     

Altered serum alpha-D-mannosidase activity in mucolipidosis II and mucolipidosis III.

Normal human serum contains at least three forms of α-D-mannosidase: an acidic form which has a pH optimum of 4.25, is inhibited by Co²⁺ and is thermostable; an intermediate form, which has a pH optimum of 5.6–5.7, is stimulated by Co²⁺ and is heat labile at 50°C; and a neutral form with a pH optimum of 6.0–6.5. In Mucolipidosis II and III sera, the acidic α-mannosidase activity persists while the intermediate activity is absent or altered. Heating the serum does not affect the pH activity curve, the electrofocusing profile or the response to Co²⁺ of α-mannosidase. During heat inactivation at 55°, 90–100% of the pH 5.6 α-mannosidase activity is lost in normal sera while less than 40% is lost from ML sera. The effect on sera from ML obligate heterozygotes is intermediate. The absent or altered intermediate mannosidase may be responsible for aberrant biochemical properties reported for other glycosidases in Mucolipidosis II and Mucolipidosis III.     

Rat hepatic uroporphyrinogen III co-synthase. Purification and evidence for a bound folate coenzyme participating in the biosynthesis of uroporphyrinogen III.

Rat hepatic uroporphyrinogen III co-synthase was isolated and purified 73-fold with a 13% yield by (NH4)2SO4 fractionation and sequential chromatography on DEAE-Sephacel, Sephadex G-100 (superfine grade) and folate-AH-Sepharose 4B. The purified co-synthase has an Mr of approx. 42 000, and is resolved into two bands, each possessing co-synthase activity, by polyacrylamide-gel electrophoresis. A factor was dissociated from the purified co-synthase. Results of both microbiological and competitive protein-binding assays suggest that it is a pteroylpolyglutamate. The isolated pteroylpolyglutamate factor was co-eluted with authentic N5-methyltetrahydropteroylheptaglutamate on DEAE-Sephacel. Uroporphyrinogen III is formed by cosynthase-free preparations of uroporphyrinogen I synthase in the presence of tetrahydropteroylglutamate. Tetrahydropeteroylheptaglutamate is also able to direct the formation of equivalent amounts of uroporphyrinogen III at a concentration approximately one-hundredth that of tetrahydropteroylmonoglutamate. These results suggest that a reduced pteroylpolyglutamate factor is associated with rat hepatic uroporphyrinogen III co-synthase, and that this may function as a coenzyme for the biosynthesis of uroporphyrinogen III.     

Mutation in the sphingolipid activator protein 2 in a patient with a variant of Gaucher disease

The lysosomal degradation of glucosylceramide requires the hydrolase, glucosylceramide-beta-glucosidase and a sphingolipid activator protein (Gaucher factor, SAP-2, saposin C). Genetic defects in either of these lysosomal proteins cause phenotypically similar disorders in man, the Gaucher disease. SAP-2 originates from a gene which generates a mRNA that codes for four homologous proteins. In a patient with an immunologically proven SAP-2 deficiency a G1154----T transversion (counted from A of the initiation codon ATG) was found in the mRNA of the SAP-2 precursor which results in the substitution of Phe for Cys385 in the mature SAP-2. The rest of the coding sequence remained entirely normal.     

N-terminal amino-acid sequence of a sphingolipid activator protein missing in a new human Gaucher disease variant

A naturally occurring non-enzymic sphingolipid activator protein (A1a activator) shown previously to be immunochemically not detectable in a new variant of human Gaucher disease (glucosylceramide-lipidosis) without glucosylceramidase deficiency was characterized by partial sequence analysis. The N-terminal amino-acid sequence of the A1a activator--a glycoprotein with high carbohydrate content--could be determined up to position 38. About 20% of the polypeptide chain are shorter by two amino-acid residues at the N-terminal end. Position 22 seems to be occupied by a carbohydrate-binding asparagine. The N-terminus of the A1a activator does not show any homology with the activator for the enzymic sulfatide degradation.     

Calcitonin biosynthesis: evidence for a precursor.

Calcitonin biosynthesis has been studied in chicken ultimobranchial glands incubated in vitro in the presence of radioactive amino acids. The results obtained suggest the existence of a biosynthetic precursor of higher molecular weight or procalcitonin. This precursor has been identified by pulse-chase experiments, molecular weight determinations, biological activity measurements and analysis of tryptic peptides. Its molecular weight is about 13000 (calcitonin, about 3500) as determined by polyacrylamide gel electrophoresis. Procalcitonin is present in small amounts in chicken ultimobranchial glands and it is biologically active in rats.     

Defective phosphorylation and processing of beta-hexosaminidase by intact cultured fibroblasts from patients with mucolipidosis III

Previous studies of the synthesis, phosphorylation, and processing of β-hexosaminidase in cultured fibroblasts from normal individuals and from patients with mucolipidosis II (I-cell disease) (A. Hasilik and E. F. Neufeld, 1980, J. Biol. Chem.225, 4937–4946) have been extended to fibroblasts derived from patients with a related genetic disorder, mucolipidosis III (pseudo-Hurler polydystrophy). The enzyme was biosynthetically labeled in pulse-chase experiments with [³H]leucine and ³³P_i, and isolated from cells and medium by immunoprecipitation. The constitutent α and β chains of the enzyme were separated by polyacrylamide gel electrophoresis under reducing and denaturing conditions, visualized by autoradiography and fluorography, extracted from the gel, and quantitated by liquid scintillation spectrometry. Enzyme produced by fibroblasts from mucolipidosis III patients had a very low but detectable phosphate content; a high proportion of newly made enzyme was secreted, though some remained within the cells and was processed to mature enzyme; the presence of NH₄Cl during the labeling and chase did not significantly increase the amount of enzyme secreted. The β-hexosaminidase produced by mucolipidosis III fibroblasts thus resembled more closely that produced by fibroblasts from patients with mucolipidosis II than the normal enzyme. β-Hexosaminidase made by fibroblasts from mucolipidosis II heterozygotes was similar to the normal enzyme with respect to phosphorylation, processing, and secretion. Mucolipidosis II and III fibroblasts could endocytose normal precursor β-hexosaminidase and process it to the mature form. The deficiency of mature enzyme in the patients' cells may therefore be attributed to failure of the unphosphorylated enzyme to be incorporated into lysosomes, where processing would normally occur.     

Gaucher disease. III. Substrate specificity of glucocerebrosidase and the use of nonlabeled natural substrates for the investigation of patients.

A reproducible and convenient method for assaying glucocerebrosidase activity using the natural substrates has been developed. From the insoluble pellet fraction of cultured skin fibroblast homogenates, released glucose was measured enzymically using hexokinase coupled with the glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP) system. Optimal enzyme assay conditions required both Triton X-100 and sodium taurocholate, pH 4.8. Glucocerebrosidase activities from three patients with type 1 Gaucher disease were 17.5%, 15.8%, and 11.2% of normal (normal = 198 +/- 14 nmol/hr per mg protein, n = 3). The first patient had normal beta-glucosidase activity with the artificial fluorogenic umbelliferone substrate. Interference with the accuracy of the glucose-dependent assay system by either glycolytic or gluconeogenic enzyme activites was not detected under these experimental conditions, and when substrates with long fatty-acid chain lengths (C = 22) were used, markedly decreased glucocerebrosidase activity occurred in both normal individuals and patients. The apparent Km's for the natural substrates were 0.56 +/- 0.05 mM for controls and 2.2-3.3 mM for Gaucher fibroblasts. These data further support the hypothesis that a structurally altered and catalytically deficient enzyme is synthesized in patients with type 1 Gaucher disease and illustrate the value of the natural substrate in investigating patients.     

The ovine Type II Gaucher disease model recapitulates aspects of human brain disease.

Acute neuronopathic (type II) Gaucher disease (GD) is a devastating, untreatable neurological disorder resulting from mutations in the glucocerebrosidase gene (GBA1), with subsequent accumulation of glucosylceramide and glucosylsphingosine. Patients experience progressive decline in neurological function, with onset typically within the first three-to-six months of life and premature death before two years. Mice and drosophila with GD have been described, however little is known about the brain pathology observed in the naturally occurring ovine model of GD. We have characterised pathological changes in GD lamb brain and compared the histological findings to those in GD patient post-mortem tissue, to determine the validity of the sheep as a model of this disease. Five GD and five age-matched unaffected lamb brains were examined. We observed significant expansion of the endo/lysosomal system in GD lamb cingulate gyrus however TPP1 and cathepsin D levels were unchanged or reduced. H&E staining revealed neurons with shrunken, hypereosinophilic cytoplasm and hyperchromatic or pyknotic nuclei (red neurons) that were also shrunken and deeply Nissl stain positive. Amoeboid microglia were noted throughout GD brain. Spheroidal inclusions reactive for TOMM20, ubiquitin and most strikingly, p-Tau were observed in many brain regions in GD lamb brain, potentially indicating disturbed axonal trafficking. Our findings suggest that the ovine model of GD exhibits similar pathological changes to human, mouse, and drosophila type II GD brain, and represents a model suitable for evaluating therapeutic intervention, particularly in utero-targeted approaches.     

Spectroscopic evidence for a porphobilinogen deaminase-tetrapyrrole complex that is an intermediate in the biosynthesis of uroporphyrinogen III

Incubation of porphobilinogen (PBG) with PBG deaminase from Rhodopseudomonas sphaeroides in carbonate buffer (pH 9.2) to total PBG consumption resulted in low yields of uroporphyrinogen I (uro'gen I). In the reaction mixture a pyrrylmethane accumulated, which at longer incubation periods was transformed into uro'gen I. The accumulated pyrrylmethane gave an Ehrlich reaction which was different from that of a 2-(aminomethyl)dipyrrylmethane or 2-(aminomethyl)tripyrrane. It resembled that of a bilane (tetrapyrrylmethane) but was different from that of a 2-(hydroxymethyl)bilane. The 13C NMR spectra of incubations carried out with [11-13C]PBG indicated that the pyrrylmethane was a tetrapyrrole with methylene resonances at 22.35-22.50 ppm. It was loosely bound to the deaminase, and when separated from the enzyme by gel filtration or gel electrophoresis, it immediately cyclized to uro'gen I. No enzyme-bound methylene could be detected by its chemical shift, suggesting that its line width must be very broad. When uro'gen III-cosynthase was added to the deaminase-tetrapyrrole complex, uro'gen III was formed at the expense of the latter in about 75% yield. The tetrapyrrole could only be partially displaced from the enzyme by ammonium ions, although a small amount of 2-(aminomethyl)bilane was always formed together with the tetrapyrrole intermediate. A protonated uro'gen I structure for this intermediate was ruled out by incubations using [2,11-13C]PBG. Uro'gen III formation from 2-(hydroxymethyl)bilane (HMB) and from the deaminase-tetrapyrrole intermediate was compared by using deaminase-cosynthase and cosynthase from several sources. It was found that while the HMB inhibited uro'gen III formation at higher concentrations and longer incubation times, uro'gen III formation from the complex did not decrease with time.     

Increased levels of sialic acid associated with a sialidase deficiency in I-cell disease (mucolipidosis II) fibroblasts.

Cultured fibroblasts from three unrelated patients with I-cell disease (mucolipidosis II) have a 3 to 4 fold increase in total sialic acid when compared to control fibroblasts. Sialic acid levels in a number of other lysosomal disorders, i.e., mucopolysaccharidosis I, II, III, VI, metachromatic leukodystrophy, G_M1 gangliosidosis, mannosidosis, Gaucher's and Sandhoff's disease are within the normal range suggesting that this is a finding specific for I-cells. Additionally, sonicates of cultured fibroblasts from controls were shown to have an acid sialidase capable of removing sialic acid from added fetuin at pH 4.2 in 0.05M acetate buffer. In contrast, I-cell fibroblasts, within the limits of the assay, lack this enzyme activity.     

Glucocerebrosidase "processing" and gene expression in various forms of Gaucher disease

Immunoblots were prepared using extracts of fibroblasts derived from five controls and from four unrelated patients with type I, three with type II, and two with type III Gaucher disease. Five monoclonal antisera and two rabbit sera, crude and affinity purified, were utilized to detect antigen transferred to nitrocellulose paper. Only a band of 63,000 molecular weight (Mr) was consistently detected. We found no 56-K band either in normal or in Gaucher disease fibroblast extracts. Thus, using a variety of antisera, we are unable to verify the claim that the types of Gaucher disease can be differentiated from one another by immunoblotting.     

Spatio-temporal parameters and the three-dimensionality of apparent motion: evidence for two types of processing

The minimum ISI required for perceiving apparent motion in depth was measured as a function of the 2D separation of stimuli and the physical separation of stimuli in depth. It was found that temporal thresholds increased as a function of the separation of stimuli in depth. This supports the results of previous research indicating that the perceived three-dimensionality of apparent motion in depth increases with ISI. In addition, the rate of threshold increase was significantly greater in displays with short 2D separations of stimuli than in displays with large 2D separations. This robust functional dissociation of thresholds indicates that the short-range system may be involved in the processing of apparent motion in depth in the former case.     

Analysis of the multiple forms of Gaucher spleen sphingolipid activator protein 2.

Gaucher spleen sphingolipid activator protein 2 was fractionated into concanavalin A binding- and non-binding fractions. These fractions each contained several bands on non-denaturing polyacrylamide gel electrophoresis (PAGE). The two fractions were further fractionated by electroblotting the proteins from preparative gels onto nitrocellulose, staining with Ponceau S to locate the bands of protein and then eluting the protein components from the nitrocellulose. A total of ten fractions, each containing only one or two major components, was collected. All of these subfractions activated beta-glucocerebrosidase and sphingomyelinase and most subfractions also activated beta-galactocerebrosidase. The structural relationship of the bands was investigated using endoglycosidase digestions. The results indicated that the two bands with the fastest mobility on non-denaturing PAGE did not contain any carbohydrate. The remaining bands showed only limited or partial digestion with endoglycosidase H and endoglycosidase D, but were readily hydrolysed with endoglycosidase F. The products of these digestions included bands with similar mobilities to the non-carbohydrate containing bands.     

Deficiency of steriod beta-glucosidase in Gaucher disease.

A deficiency in the activity of steroid: β-glucosidase has been observed in the particulate fraction of Gauchers tissues. There was no diminution of the “soluble” form of this enzyme in adult tissue samples. In contrast, there was a marked reduction in the soluble steroid β-glucoside hydrolytic activity in the brain and spleen, and not liver from the infantile form of the disease.     

Complete amino-acid sequence and carbohydrate content of the naturally occurring glucosylceramide activator protein (A1 activator) absent from a new human Gaucher disease variant

Two naturally occurring non-enzymic glucosylceramide activator proteins (A1a and A1b activator) shown previously to be immunochemically not detectable in a new variant of human Gaucher disease (glucosylceramide lipidosis) without glucosylceramidase deficiency, were characterized by amino-acid sequence and carbohydrate content. The complete amino-acid sequence of the A1a activator was determined. The protein consists of 80 amino-acid residues including three disulfide bridges lacking arginine and tryptophan. The molecular mass is 8.95 kDa. About 20% of the polypeptide chain are shorter by two amino-acid residues at the N-terminal end. The A1b activator was characterized by the amino-acid compositions of all tryptic peptides and of the entire protein; sequencing was performed of the regions 1-34 and 42-56. Identical results were obtained for the polypeptide chains of both A1 activators. This suggests that they do not differ in their primary structures which is in agreement with the immunochemical results. The difference between A1a and A1b activator is due to the carbohydrate part. The total amount of 49% carbohydrate in A1a and 76.7% in A1b consists mainly of hexoses. Both chains contain two moles of N-acetylglucosamine per mole protein bound to asparagine in position 22. A comparison of the primary structure of the A1 activator with the sulfatide activator sequence revealed an interesting similarity, especially of the cysteine residues and the carbohydrate-binding asparagine. Sequence homology was also found between a part of the A1 activator sequence and the hemagglutinin neuraminidase of influenza virus as well as to a hypothetical glycoprotein of the Epstein-Barr virus. The comparison with human lysosomal glucosylcerebrosidase showed no sequence similarity.     

Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. III. Genetic evidence for utilization of phosphatidylcholine and phosphatidylethanolamine as precursors

In the preceding paper, we reported that Chinese hamster ovary (CHO) cells contain two different serine-exchange enzymes (I and II) which catalyze the base-exchange reaction of phospholipid(s) with serine and that a phosphatidylserine-requiring mutant (strain PSA-3) of CHO cells is defective in serine-exchange enzyme I and lacks the ability to synthesize phosphatidylserine (Kuge, O., Nishijima, M., and Akamatsu, Y. (1986) J. Biol. Chem. 261, 5790-5794). In this study, we examined precursor phospholipids for phosphatidylserine biosynthesis in CHO cells. When mutant PSA-3 and parent (CHO-K1) cells were cultured with [32P]phosphatidylcholine, phosphatidylserine in the parent accumulated radioactivity while that in the mutant was not labeled significantly. On the contrary, when cultured with [32P]phosphatidylethanolamine, the mutant incorporated the label into phosphatidylserine more efficiently than the parent. Furthermore, we found that mutant PSA-3 grew normally in growth medium supplemented with 30 microM phosphatidylethanolamine as well as phosphatidylserine and that the biosynthesis of phosphatidylserine in the mutant was biosynthesis of phosphatidylserine in the mutant was normal when cells were cultured in the presence of exogenous phosphatidylethanolamine. The simplest interpretation of these findings is that phosphatidylserine in CHO cells is biosynthesized through the following sequential reactions: phosphatidylcholine----phosphatidylserine----phosphatidylethanolamine--- - phosphatidylserine. The three reactions are catalyzed by serine-exchange enzyme I, phosphatidylserine decarboxylase, and serine-exchange enzyme II, respectively.     

Preliminary evidence for a glucan acceptor in the yeast Candida albicans.

Two membrane preparation containing glucan synthase activity were obtained by lysis of regenerating sphaeroplasts (enzyme A) or mechanical breakage (enzyme B) of yeast (Candida albicans) cells. The reaction products of both enzymes (glucans A and B respectively) were characterized as linear beta-1,3-linked glucans on the basis of chemical and enzymic analysis. In addition, two pools of glucan could be distinguished in glucan A preparations on the basis of their susceptibility to an exoglucanase. In no case were the reaction products synthesized de novo; rather the radioactive chains were added to the non-reducing end of non-radioactive preformed glucan chains or to an acceptor of a different nature. At least some of the performed chains of glucan A, but not those of glucan B, showed a free reducing terminal. Glucan A preparations were endowed with endoglucanase activity, which, under appropriate conditions, released glucose, laminaribiose and laminaritriose. These sugars were also found in cell-wall autolysates. On the basis of the origin of both enzyme preparations it is suggested that glucan molecules are synthesized while they are bound to a non-glucan acceptor that is subsequently excised, presumably by cell-wall-associated glucanases.