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.     

Origin of α-mannosidase activity in CSF

The α-mannosidase activity in human frontal gyrus, cerebrospinal fluid and plasma has been analyzed by DEAE-cellulose chromatography to investigate the origin of the α-mannosidase activity in cerebrospinal fluid (CSF). The profile of α-mannosidase isoenzymes obtained in CSF was similar to that in the frontal gyrus but different from that in human plasma. In particular the two characteristic peaks of lysosomal α-mannosidase, A and B, which have a pH-optimum of 4.5 and are found in human tissues, were present in both the frontal gyrus and CSF. In contrast the majority of α-mannosidase activity in human plasma was due to the so called intermediate form, which has a pH-optimum of 5.5. The results suggest that the intermediate form of α-mannosidase in plasma does not cross the blood–brain barrier and that the α-mannosidase activity present in the cerebrospinal fluid is of lysosomal type and of brain origin. Thus the α-mannosidase activity in cerebrospinal fluid might mirror the brain pathological changes linked to neurodegenerative disorders such as Parkinson's disease.     

Biochemical Characterization of a Lysosomal α-Mannosidase from the Starfish <Emphasis Type="Italic">Asterias rubens</Emphasis>

Acidic α-mannosidase is an important enzyme and is reported from many different plants and animals. Lysosomal α-mannosidase helps in the catabolism of glycoproteins in the lysosomes thereby playing a major role in cellular homeostasis. In the present study lysosomal α-mannosidase from the gonads of echinoderm Asterias rubens was isolated and purified. The crude protein sample from ammonium sulfate precipitate contained two isoforms of mannosidase as tested by the MAN2B1 antibody, which were separated by anion exchange chromatography. Enzyme with 75 kDa molecular weight was purified and biochemically characterized. Optimum pH of the enzyme was found to be in the range of 4.5–5 and optimum temperature was 37 °C. The activity of the enzyme was inhibited completely by swainsonine but not by 1-deoxymannojirimycin. Ligand blot assays showed that the enzyme can interact with both the lysosomal enzyme sorting receptors indicating the presence of mannose 6-phosphate in the glycan surface of the enzyme. This is the first report of lysosomal α-mannosidase in an active monomeric form. Its interaction with the receptors suggest that the lysosomal enzyme targeting in echinoderms might follow a mannose 6-phosphate mediated pathway similar to that in the vertebrates.     

Alpha-mannosidase activity in stallion epididymal fluid and spermatozoa

The expression of α-D-mannosidase activity was fluorometrically and electrophoretically assessed in spermatozoa, epididymal fluid and homogenates of stallion epididymal tissue. Enzyme activity had regional differences; it was higher (P < 0.05) in samples from the cauda epididymal region than in samples from the proximal caput region (largely composed of efferent ducts). Based on enzyme activity, as a function of pH of the assay substrate, electrophoretic analysis in native and native/SDS-PAGE conditions, and the effect of inhibitors or activators, we inferred the presence of at least two catalytically active forms of α-D-mannosidase. The neutral form of the enzyme (α-mannosidase II) was activated by Co²⁺, whereas the acid form (optimum pH 3.5 to 4.0) was sensitive to swainsonine (an inhibitor of α-mannosidase I), stabilized or stimulated by Zn²⁺, and not activated by Co²⁺ (activator of the neutral form). The activity of the acid form of the enzyme was highest in the epididymal fluid, where it seemed to be mainly in a secretory form. This form of the enzyme may have a role in plasma membrane remodeling associated with sperm maturation. In contrast, the activity of α-mannosidase II was higher in mature spermatozoa. It has been postulated that α-mannosidase II may act as a receptor in the recognition and binding of the complementary carbohydrate moieties present on the zona pellucida. With non-denaturing electrophoresis, α-D-mannosidase had an electrophoretic mobility of 0.35 and 0.24. When resolved by 1D and 2D SDS-PAGE (under denaturing conditions) the enzyme had a major protein band of molecular weight 154 kDa in spermatozoa and epididymal samples. Based on its properties under native conditions, we inferred that this enzyme might interact with other proteins and form transitory aggregates.     

The effect of periodate oxidation and α-mannosidase treatment of Dolichos biflorus lectin

The effects of periodate and α-mannosidase treatment of the Dolichos biflorus lectin were determined. Destruction by periodate of 16% of the mannose residues of the lactin had no effect on its ability to agglutinate type A erythrocytes, precipitate blood group A + H substance or to be precipitated by concanavalin A. Removal of up to 40% of the mannose by either periodate or α-mannosidase rendered the lecton nonprecipitable by concanavalin A. The lectrin treated by α-mannosidase retained its ability to agglutinate erythrocytes and precipitate blood group A + H substance, but the lectin treated with periodate lost most of its activity.The results suggest that the complete integrity of the carbohydrate unit of the lectin is not necessary for its activity and that the periodate may be affecting the protein portion of the molecule as well as its carbohydrate residues. No conversion of form A to form B of the lectin was observed with either periodate oxidation or α-mannosidase treatment.     

The separation of bovine brain beta-N-acetyl-D-hexosaminidases. Abnormal gel-filtration behaviour of beta-N-acetyl-D-glucosaminidase C.

Bovine brain tissue was extracted and the 50 000g supernatant was separated by electrophoresis, DEAE-Sephadex chromatography and gel filtration on Sephadex G-200 and Bio-Gel P-200. The electrophoretic separation showed that the beta-N-acetyl-D-hexosaminidases (hexosaminidases) of bovine brain tissue were composed of four different fractions. Two fractions (A and B) exerted both glucosaminidase and galactosaminidase activity, a third fraction (C) showed only glucosaminidase activity, whereas a fourth form (D) with specificity towards the galactosaminide moiety was found to be present. DEAE-Sephadex chromatography at pH 7.0 showed that the B form was eluted with the void volume, whereas the A and D forms could be eluted in one peak by raising that salt concentration. The C form could not be detected in the eluate. Gel filtration on Sephadex G-200 showed that the B, A and D forms had almost equal molecular weights. In this case also the C form could not be detected in the column eluates. Gel filtration on Bio-Gel P-200 revealed that the C form was eluted with the void volume.     

GLYCOSIDE HYDROLASES OF SEA URCHIN SPERMATOZOA AND THEIR POSSIBLE INVOLVEMENT IN SPERM ISOAGGLUTINATION BY EGG WATER*

The whole sperm of sea urchins, Anthocidaris crassispin and Hemicentrotus pulcherrimus, were found to contain neuraminidase and 4-methylumbelliferyl α-mannosidase. The maximal activity was attained after a lag of a few minutes. The sperm hydrolyzed 8 kinds of 4-methylumbelliferyl glycosides including hexosides, hexosaminides and a fucoside. The enzymes were found to be membrane-bound and solubilized by KCl, sodium taurocholate or Triton X-100. When the enzymes were solubilized, the pH optima were at about 5.5, although the optima were at about 7.5 in the whole sperm. Divalent cation requirement was not detectable. On gel filtration, neuraminidase was eluted as a low molecular weight form, whereas α-mannosidase was eluted near the void volume as a high molecular weight form. The activity hydrolyzing other 4-methylumbelliferyl glycosides was found to be concentrated in the fraction showing α-mannosidase activity. Our results suggest that when the sperm are mixed with egg water, glycosidases on the sperm is fixed at sugar residues in the heterosaccharide chains of the jelly network and hydrolyze the glycosidic bonding after a lag period. The sequence of reactions is supposedly responsible for the reversible agglutination of the sperm by egg water.     

Purification and properties of a β-N-acetylaminoglucohydrolase from malted barley

β-N-Acetylaminoglucohydrolase (β-2-acetylamino-2-deoxy-D-glucoside acetylaminodeoxyglucohydrolase, EC 3.2.1.30) was extracted from malted barley and purified. The partially purified preparation was free from α-and β-glucosidase, α- and β-galactosidase, α-mannosidase and β-mannosidase. This preparation was free from α-mannosidase only after affinity chromatography with p-amino-N-acetyl-β-D-glucosaminidine coupled to Sepharose. The enzyme was active between pH 3 and 6.5 and had a pH optimum at pH 5. A MW of 92000 was obtained by sodium dodecyl sulfate-acrylamide gel electrophoresis and a sedimentation coefficient of 4.65 was obtained from sedimentation velocity experiments. β-N-Acetylaminoglucohydrolase had a K_m of 2.5 × 10^?4 M using the p-nitrophenyl N-acetyl β-D-glucosaminidine as the substrate.     

Partial purification of potato tuber invertase and its proteinaceous inhibitor

Improved purification of potato tuber invertase was achieved by utilizing a form of affinity chromatography between the enzyme and Concanavalin A (Con A) bound to Sepharose. Twenty-fold increases in specific activity were routinely obtained with this step and the enzyme was purified 190-fold over that found in the crude homogenate. The Con A-Sepharose chromatography step gave a greater purification than any other step in the invertase isolation procedure. There was up to 170% recovery of the activity loaded onto the column. α-Methyl-d-mannoside, sucrose, d-glucose and d-fructose eluted the enzyme from the Con A-Sepharose column with similar recoveries, although the volume of eluent required varied with the sugar. This unusually high recovery of invertase activity was obtained with some batches of tubers but not with others. There was evidence to suggest that the high recovery, or activation, may be due to the release of an inhibitor from the enzyme in the presence of Con A-Sepharose. Adsorption of invertase to Con A-Sepharose could be eliminated by incubation of the enzyme with α-mannosidase and β-glucosidase, indicating that potato tuber invertase is a glycoprotein. Proteinaceous inhibitor purification was improved by treatment of the tuber extract at low pH.     

Analysis of unsaturated fatty acids, and their hydroperoxy and hydroxy derivatives by high-performance liquid chromatography.

A simple procedure for the detection of endo-β-N-acetylglucosaminidase H activity is described. The method utilizes N-[¹⁴C]methylribonuclease B as substrate. This is prepared from ribonuclease B by reductive alkylation of free amine groups in the protein with [¹⁴C]formaldehyde. Because the carbohydrate moiety of ribonuclease B has α-mannosyl residues at nonreducing terminal positions, the radioactive molecule binds to Sepharose-concanavalin A. Endo-β-N-acetylglucosaminidase action releases this mannose-containing oligosaccharide by splitting the di-N-acetylchitobiosyl residue that links it with the peptide and thereby renders the radioactive portion of the molecule unreactive with Sepharose-concanavalin A. This forms the basis of a convenient assay for screening column fractions during the purification of the endoglycosidase. Although protease or α-mannosidase activity might also be detected by the procedure, no difficulties were presented by these enzymes when the assay was used for the preparation of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus.     

Mannosidosis: detection of the disease and of heterozygotes using serum and leucocytes

α-D-mannosidase activity in serum and leucocytes from normal individuals, patients with mannosidosis and their parents was measured at pH 4.4 and pH 6.0. When the results were expressed as total activity or specific activity at pH 4.4 in both tissues, or as a ratio of enzyme activity at the two pH conditions in serum, the disease could be diagnosed, but the heterozygotes could not be distinguished from the controls. However, all three groups could be recognised when acid α-mannosidase activity was related to total

activity in serum and leucocytes. The distribution of α-mannosidases, as separated by ion-exchange chromatography was different in each tissue. The serum profile was unique, all components having substantial “neutral” activities which are unaltered in mannosidosis and carriers.     

Mannosidosis in Angus cattle. The enzymic defect

Normal calf alpha-mannosidase activity exists in at least three forms separable by chromatography on DEAE-cellulose and by starch-gel electrophoresis. Two components, A and B, have optimum activity between pH3.75 and 4.75, but component C has an optimum of pH6.6. Components A and B are virtually absent from the tissues of a calf with mannosidosis and the residual activity is due to component C. The acidic and neutral forms of alpha-mannosidase differ in their molecular weights and sensitivity to EDTA, Zn(2+), Co(2+) and Mn(2+). An acidic alpha-mannosidase component (pH optimum 4.0) accounts for most of the activity in normal plasma but it is absent from the plasma of a calf with mannosidosis. Although the acidic alpha-mannosidase component is probably related to tissue components A and B, it can be distinguished from them by ion-exchange chromatography and gel filtration. The optimum pH of the low residual activity in the plasma from a calf with mannosidosis is pH5.5-5.75. The results support the hypothesis that Angus-cattle mannosidosis is a storage disease caused by a deficiency of lysosomal acidic alpha-mannosidase activity.     

Interconversion of the A and B forms of ceramide trihexosidase from human plasma

The human plasma α-galactosidases which specifically hydrolyze galactosyl-(α1→4)galactosyl(β1→ 4)glucosylceramide consist of an A group with optimal enzymatic activity at pH 5.4, and a B group, which is characterized by optimal activity at pH 7.2. The relationship between the A and B groups of these α-galactosidases (ceramide trihexosidases) has been investigated with regard to their sialic acid content. Partial neuraminidase treatment of the most acidic (A-1) form of ceramide trihexosidase yields a complex mixture of 14 enzymatically active proteins separable by isoelectric focusing. Exposure to neuraminidase for a longer time causes an almost complete conversion of the A-1 form to a protein which has the same electrophoretic properties as the least acidic (B-V) form. Conversely, a crude kidney sialyltransferase preparation can be used to incorporate either CMP[1-¹⁴C]sialic acid or UDP-N-acetyl[1-¹⁴C]glucosamine into the B-V form of the enzyme. Sialyltransferase treatment causes the formation of a complex mixture of enzymatically active proteins, one of which has the same electrophoretic characteristics as the A-1 and A-2 forms of ceramide trihexosidase. On the basis of these studies it is suggested that the multiple forms of plasma ceramide trihexosidase are glycoproteins which differ primarily in their sialic acid content.     

The separation and characterization of marmoset kidney beta-D-galactosidase and beta-D-glucosidase.

beta-D-Galactosidase and beta-D-glucosidase activities were determined in homogenates of marmoset kidney by using the appropriate 4-methylumbelliferyl glycoside, beta-D-Galactosidase activity was separated into two main components by ion-exchange chromatography on DEAE-cellulose, starch-gel electrophoresis, isoelectric focusing and gel filtration on Sephadex G-200. One form designated A had a pI of 5.1, was loosely bound to DEAE-cellulose at pH7.0, remained near the origin on starch-gel electrophoresis at pH 7.0 and had an apparent molecular weight of 160000. The second beta-D-galactosidase component, designated B, was associated with the total beta-D-glucosidase activity, had a pI of 4.3, was firmly bound to DEAE-cellulose, migrated rapidly towards the anode on starch-gel electrophoresis and had an apparent molecular weight of 50000. The optimum pH values of beta-D-galactosidase A and B were 4.5 and 6.0 respectively. beta-D-Galactosidase A was activated by 0.1 M-NaC1 but the activity of the B form was inhibited by 1 M-NaC1 at pH 4.5. beta-D-galactosidase had a bimodal distribution, the A form being recovered in the lysosomal fraction whereas the B form was present in the soluble fraction, as was the major portion of the beta-D-glucosidase activity. The lysosomal and soluble forms were further characterized by DEAE-cellulose chromatography.     

Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization.

Human N-acetylglucosamine-6-sulphate sulphatase was purified at least 50,000-fold to homogeneity in 78% yield from liver with a simple three-step four-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, chromatofocusing and Cu2+-chelating Sepharose chromatography. In all, four forms were isolated and partially characterized. Forms A and B, both with a pI greater than 9.5 and representing 30% and 60% respectively of the recovered enzyme activity, were separated by hydroxyapatite chromatography of the enzyme preparation obtained from the Cu2+-chelating Sepharose step. Both forms A and B had native molecular masses of 75 kDa. When analysed by SDS/polyacrylamide-gel electrophoresis, form A consists of a single polypeptide of molecular mass 78 kDa, whereas form B contained 48 kDa and 32 kDa polypeptide subunits. Neither form A nor form B was taken up from the culture medium into cultured human skin fibroblasts. The two other forms (C and D), with pI values of 5.8 and 5.4 respectively, represented approx. 7% and 3% of the total recovered enzyme activity. The native molecular masses of forms C and D were 94 kDa and approx. 75 kDa respectively. Form C contained three polypeptides with molecular masses of 48, 45 and 32 kDa. N-Acetylglucosamine-6-sulphate sulphatase activity was measured with a radiolabelled disaccharide substrate derived from heparin. The development of this substrate enabled the isolation and characterization of N-acetylglucosamine-6-sulphate sulphatase to proceed efficiently. Forms A, B and C had pH optima of 5.0, Km values of 11.7, 14.2 and 11.1 microM respectively and Vmax. values of 105, 60 and 53 nmol/min per mg of protein respectively. The molecular basis of the multiple forms of this sulphatase is not known. It is postulated that the differences in structure and properties of the four enzyme forms are due to differences in the state of processing of a large subunit.     

Purification of a β-mannanase enzyme from lucerne seed by substrate affinity chromatography

A technique which employs substrate affinity chromatography on glucomannan- or mannan-AH-Sepharose, has been developed for the purification of legume seed β-mannanases. Using this technique, lucerne seed β-mannanase B has been purified to near homogeneity with a final specific activity of 1080 nkat per mg protein. The preparation was completely devoid of α-galactosidase and β-mannosidase activities. β-Mannanases of microbial origin can also be purified using these materials.     

Biochemical characterization of a recombinant thermostable β-mannosidase from Thermotoga maritima with transglycosidase activity

A β-mannosidase gene (TM1624) from Thermotoga maritima MSB8, the hyperthermophilic bacterium was expressed as a soluble C-terminal His-tagged protein in E. coli. Heat treatment of cell lysate followed by metal affinity- and anion-exchange chromatographic techniques the recombinant β-mannosidase was purified to apparent homogeneity. The recovery of the purified protein from the crude lysate was 23%. Results of SDS-PAGE analysis (96.8 kDa) and gel permeation chromatography (93.2 kDa) indicated monomeric nature of the β-mannosidase protein. The enzyme displayed its maximal activity at pH 7.0 with pH stability over a range of pH 5.0–9.0. Similarly, the optimum temperature for maximal activity was found to be 95 °C and thermostability of up to 85 °C. The substrate specificity and kinetics of the enzyme was studied using different mannooligosaccharides and pNP-β-d-mannopyranoside. The K_m value of the purified enzyme for pNPM was 0.49 mM. Different mannooligosaccharides tested as enzyme substrates were hydrolysed in an exo-wise manner when checked by thin-layer chromatography (TLC). The enzyme also exhibited transglycosidase activity when the reaction was carried out with 10% (w/v) of mannobiose in the presence of alcohols or galactose. Because of extreme thermostability and transglyocosylation properties of β-mannosidase from T. maritima, the enzyme may be of industrial applications in future. This is the first report on the purification and characterization of a β-mannosidase from T. maritima.     

A specific concanavalin A-mediated binding of bovine serum α-mannosidase to cultured human skin fibroblasts

A specific elevation of cell-associated α-mannosidase was observed in human skin fibroblasts cultured with concanavalin A for 12–72 hours. There was a latency of several hours before the increase of the enzyme activity occurred. When the cells were washed with α-methylmannoside, α-mannosidase activity was not increased. Other lysosomal enzymes including β-mannosidase showed a slight decrease in activity. It was concluded that the elevation of this enzyme activity was the result of a specific binding to the cell surface mediated by concanavalin A.     

Purification and properties of a low-molecular-weight α-mannosidase from Cellulomonas sp.

Cellulomonas sp. isolated from soil produces a high level of α-mannosidase (α-mannanase) inductively in culture fluid. The enzyme had two different molecular weight forms, and the properties of the high-molecular-weight form were reported previously (Takegawa, K. et al.: Biochim. Biophys. Acta, 991, 431–437, 1989). The low-molecular-weight α-mannosidase was purified to homogeneity by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was over 150,000 by gel filtration. Unlike the high-molecular-weight form, the low-molecular-weight enzyme readily hydrolyzed α-1,2- and α-1,3-linked mannose chains.