The catabolism of mammalian glycoproteins. Comparison of the storage products in bovine, feline and human mannosidosis.

Analysis of the neutral urinary oligosaccharides in bovine, feline and human mannosidosis by thin-layer and gel-permeation chromatography has shown that the patterns of stored oligosaccharides in the three species are different. In bovine and feline mannosidosis the most abundant urinary oligosaccharide is also the most abundant in the tissues of each species. The predominant oligosaccharides were purified by a combination of gel-filtration, ion-exchange and thin-layer chromatography and shown to contain only mannose and N-acetylglucosamine by g.l.c. and g.l.c.--mass spectrometry. The probable composition and size of each oligosaccharide were predicted from its chromatographic properties, sugar composition and the known structure of asparagine-linked oligosaccharides. The bovine and feline oligosaccharides belonged to a homologous series of general composition Mann (GlcNAc)2, whereas the human oligosaccharides belong to a different series, MannGlcNAc. These structures suggest that lysosomal endohexosaminidase is not present in bovine and feline tissues. The predominant feline storage product, Man3(GlcNAc)2, was the expected storage product from the catabolism of complex asparagine-linked glycans. In contrast, the predominant bovine oligosaccharide, Man2(GlcNAc)2, probably lacks one of the alpha-linked mannose residues in the core region. A similar situation occurs in human mannosidosis. It is predicted that in these species either that the residual mutant alpha-D-mannosidase retains activity towards one of the core alpha-linked mannose residues or that another form of lysosomal alpha-D-mannosidase that is unaffected in these disorders occurs. It is concluded that the differences in storage products are due to differences in the catabolic pathways of glycoproteins among the species.     

Specificity studies on alpha-mannosidases using oligosaccharides from mannosidosis urine as substrates.

Oligosaccharides containing terminal non-reducing alpha(1 leads to 2)-, alpha(1 leads to 3)-, and alpha(1 leads to 6)-linked mannose residues, isolated from human and bovine mannosidosis urines were used as substrates to test the specificities of acidic alpha-mannosidases isolated from human and bovine liver. The enzymes released all the alpha-linked mannose residues from each oligosaccharide and were most effective on the smallest substrate. Enzyme A in each case was less active on the oligosaccharides than alpha-mannosidase B2, even though the apparent Km value for the substrates was the same with each enzyme. The human acidic alpha-mannosidases were also found to be more active on substrates isolated from human rather than bovine mannosidosis urine. Human alpha-mannosidase C, which has a neutral pH optimum when assayed with a synthetic substrate, did not hydrolyse any of the oligosaccharides at neutral pH, but was found to be active at an acidic pH.     

Characterization of oligosaccharides from the urine of loco-intoxicated sheep

Two major oligosaccharides were isolated by preparative HPLC from the urine of a locoweed-fed sheep. Analysis by gas-liquid chromatography and mass-spectrometry indicated compositions of (Man)4(GlcNAc)2 and (Man)5(GlcNAc)2, respectively. Structures were determined by digestion with alpha-D-mannosidase and endo-beta-N-acetylglucosaminidases D and H, and comparison of the products by HPLC with synthetic standards, and oligosaccharides isolated from human mannosidosis urine. Incubation with an exo-beta-N-acetylglucosaminidase was without effect.     

The storage products in genetic and swainsonine-induced human mannosidosis.

Swainsonine induces the accumulation of mannose-rich oligosaccharides in human fibroblasts. The composition of the storage products shows that swainsonine completely inhibits lysosomal alpha-D-mannosidase and alters processing of glycoproteins by inhibiting Golgi alpha-D-mannosidase II. Comparison of the storage products in genetic and swainsonine-induced mannosidosis suggests that human fibroblasts contain a lysosomal alpha-D-mannosidase that is unaffected in genetic mannosidosis.     

Characterization of human liver alpha-D-mannosidase purified by affinity chromatography.

Human liver acidic alpha-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral alpha-mannosidase did not bind to the concanavalin A-Sepharose so the two types of alpha-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was alpha-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that alpha-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of alpha-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human alpha-mannosidase. The purified enzyme completely removed the alpha-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic alpha-mannosidase.     

Oligosaccharides from placenta: early diagnosis of feline mannosidosis

High-pressure liquid chromatography analysis of oligosaccharides from placentas allowed the diagnosis of alpha-mannosidosis in three litters of kittens. The chromatography also afforded a detailed comparison of the oligosaccharide pattern and levels in placenta, liver, brain, urine and ocular fluid of the affected animals. In all cases, two series of compounds were observed, with one or two residues of N-acetylglucosamine at the reducing terminus, respectively, and between two and nine mannose residues. This pattern is unlike that of human mannosidosis, and resembles that of ruminants, except that the major oligosaccharide contains three mannose residues instead of two.     

Lymphocytes transfer only the lysosomal form of alpha-D-mannosidase during cell-to-cell contact

We have examined the changes in the activities of the different types of alpha-D-mannosidase when fibroblasts from patients deficient in the lysosomal form of the enzyme are cultured together with normal lymphocytes. Our results show that whereas the mannosidosis cells acquired high levels of this enzyme, the activities of both the Golgi and the endoplasmic reticulum forms of alpha-D-mannosidase remained the same as in the fibroblasts cultured alone in the absence of lymphocytes. The increase in the activity of the lysosomal enzyme in the cocultured fibroblasts was not affected by the presence of mannose 6-phosphate or alpha-methyl mannoside, inhibitors of receptor- and lectin-mediated uptake of lysosomal enzymes, respectively, but it did require cell-to-cell contact. Ion-exchange HPLC and electrophoresis in polyacrylamide gradient gels showed that the acquired enzyme had the same elution profile and molecular size as the lysosomal form of the enzyme present in the lymphocytes. Immunoprecipitation studies using antibody specific for the lymphocyte type of lysosomal alpha-D-mannosidase confirmed that the increased activity in the cocultured mannosidosis cells resulted from the acquisition of the lymphocyte enzyme. Cytochemical examination revealed, however, that the transferred lymphocyte enzyme was localized in cytoplasmic organelles in the peripheral regions of the recipient fibroblasts. These results show that lymphocytes transfer only the lysosomal form of alpha-D-mannosidase during cell-to-cell contact with mannosidosis cells.     

Direct enzyme transfer from lymphocytes corrects a lysosomal storage disease

Fibroblasts from patients with mannosidosis, the lysosomal storage disease resulting from an inherited deficiency of lysosomal alpha-D-mannosidase (EC 3.2.1.24), accumulate specific mannose-containing oligosaccharides which are characteristic of the disease (1,2). The present study shows that these substances were extensively degraded following transfer of the missing enzyme from normal lymphocytes to mannosidosis fibroblasts on direct contact in tissue culture. Moreover, prolonged correction of the metabolic abnormality of the recipient cells was sustained if contact with fresh donor lymphocytes was periodically renewed. These findings may be highly relevant to lymphocyte function in enzyme replacement therapy by transplantation procedures currently being attempted.     

Rat epididymal alpha-D-mannosidase: purification, carbohydrate composition, substrate specificity, and antibody production

Two alpha-D-mannosidases have previously been identified in rat epididymis. This communication reports the purification and characterization of the "acid" alpha-D-mannosidase. The enzyme was purified over 1000-fold to near homogeneity by acetone and (NH4)2SO4 precipitation followed by ion-exchange and hydroxylapatite chromatography. The molecular weight of the enzyme was estimated to be 220,000 by gel filtration. Polyacrylamide gel electrophoresis of the native enzyme under two conditions of buffer and pH showed a single band when stained for protein while electrophoresis under denaturing conditions resulted in bands of apparent Mr 60,000 and 31,000. The enzyme is a glycoprotein containing about 5.6% hexose. In addition to mannose (3.1%) and glucosamine (2.0%), the enzyme also contained small amounts of glucose, fucose, and galactose. Chemical analysis indicated the absence of sialic acid. The substrate specificity of the purified enzyme was investigated using linear and branched mannose-containing oligosaccharides. The enzyme cleaved linear oligosaccharides [Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc and Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc] very efficiently. However, little or no activity was observed toward high mannose oligosaccharides (Man9GlcNAc through Man5GlcNAc) or the branched trimannosyl derivative Man3GlcNAc. This specificity is very similar to that observed with rat kidney lysosomal alpha-D-mannosidase. Additional evidence that the epididymal enzyme is essentially a lysosomal alpha-D-mannosidase is the fact that polyclonal antibody prepared against the purified epididymal enzyme cross-reacted with lysosomal alpha-D-mannosidase from several rat tissues and with acidic alpha-D-mannosidase of a human cell line, results suggesting that the antibody will be useful in studying the biosynthesis and turnover of lysosomal alpha-D-mannosidases in at least two species.     

GM1 gangliosidosis (type 1) in a cat.

A kitten with clinical and morphological symptoms of a neurovisceral lysosomal-storage disease has been shown to have a marked deficiency of acidic beta-D-galactosidase in the brain, kidney and spleen. Chromatography on concanavalin A-Sepharose and inhibition studies with 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, a selective inhibitor of the neutral broad-specificity beta-D-galactosidase, have shown that the residual beta-D-galactosidase at pH 4.0 in the tissues of the affected cat is due to the neutral beta-D-galactosidase and that there is a complete deficiency of the acidic (lysosomal) beta-D-galactosidase. There is marked accumulation in all tissues and excretion in the urine of neutral oligosaccharides. Analysis of these oligosaccharides by fast-atom-bombardment mass spectrometry and g.l.c. suggests that they arise from the incomplete catabolism of N-glycans of glycoproteins. The ganglioside content of all the tissues is elevated, and it has been shown by t.l.c. that the concentration of a ganglioside fraction with a mobility similar to that of GM1 ganglioside is particularly increased. There is also some evidence of accumulation of glycosaminoglycans in the brain. The clinical symptoms, the complete deficiency of acidic beta-D-galactosidase and the storage products in visceral organs all suggest that this is a case of feline GM1-type gangliosidosis comparable with the severe infantile (Type 1) form of the disease in humans.     

Apparently normal extracellular acidic alpha-mannosidase in fibroblast cultures from patients with mannosidosis.

Fibroblasts from patients with mannosidosis, cultured in medium supplemented with fetal calf serum from which acidic alpha-mannosidase (alpha-D-mannoside mannohydrolase, E.C.3.2.1.24) has been removed, secreted a normal amount of apparently unaffected acidic alpha-mannosidase into fetal calf serum-free medium. Both the intracellular and extracellular acidic alpha-mannosidase activities were completely precipitated by antiserum to placenta alpha-mannosidase B. In contrast to the heat-lability of the intracellular acidic alpha-mannosidase and its low affinity for artificial mannoside substrate, the extracellular enzyme exhibited both normal thermostability and normal kinetics. Mixing experiments with the intercellular enzymes suggested that the decreased activity in the patients' fibroblasts is not the effect of an inhibitor or absence of an activator. However, incubation of the mannosidosis extracellular enzyme with either normal or patient cell lysate resulted in a partial loss of activity, whereas an additive value was observed with the normal extracellular enzyme. In contrast to normal culture medium, the medium from mannosidosis cell culture was unable to enhance the rate of reduction of intracellular radioactivity in mucolipidosis type II fibroblasts precultured in the presence of radiolabeled mannose. These findings suggest that the defect in mannosidosis is expressed only after the enzyme has been delivered to lysosomes and presumably undergone some form of processing there.     

Neutral alpha-D-mannosidase activity in human granulocytes

The presence of neutral soluble alpha-D-mannosidase activity was shown in human granulocytes. For detection of the enzyme different methods were used: addition of stabilizing agents; sorption of acid alpha-D-mannosidase on concanavalin A-sepharose; inhibition of acid alpha-D-mannosidase; determination of neutral alpha-D-mannosidase in granulocytes of patients with inherited defect of acid alpha-D-mannosidase (mannosidosis). The specific activity of neutral alpha-D-mannosidase in granulocytes of donors calculated in nmol/min/mg of protein was near to the activity in lymphocytes. However the activity in granulocytes calculated in nmol/min/10(8) of cells was approximately 3 times lower than that in lymphocytes. The activity of neutral alpha-D-mannosidase in immature myeloid cells of a patient with chronic myeloid leukaemia was 10 times higher than in natural granulocytes of the same patient. This high activity may be in connection with the process of cell differentiation or the result of malignant transformation.     

Two forms of acid alpha-D-mannosidase in monkey brain: evidence for the co-existence of high mannose and complex oligosaccharides in one form

Lysosomal alpha-D-mannosidase of monkey brain existed in two forms. One form of mannosidase was bound to the Ricinus communis agglutinin120 (RCA1)-Sepharose and could be specifically eluted with lactose. The other form did not bind to the RCA1-Sepharose. Both forms of mannosidase could bind to a similar extent to the immobilized brain lysosomal receptor protein. Both the forms were purified to apparent homogeneity. Neutral sugar analysis by GLC showed the presence of glucose, mannose and galactose in the RCA1-Sepharose bindable mannosidase and glucose and mannose in the non-bindable mannosidase. Several other brain lysosomal hydrolases did not bind to the RCA1-Sepharose. The results suggested the existence of only high mannose oligosaccharides in the RCA1 non-bindable mannosidase and both high mannose and complex oligosaccharides in the bindable mannosidase.     

Substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases and effects of swainsonine suggest a role of the cytosolic enzyme in glycoprotein catabolism

Swainsonine is a potent inhibitor of lysosomal alpha-D-mannosidase, causes the production of hybrid glycoproteins, and is reported to produce a phenocopy of hereditary alpha-mannosidosis. We now report that the effects of swainsonine administration in the rat are different in two respects from those found in other animals thus far studied. Swainsonine caused the accumulation of oligosaccharide in kidney and urine but not in liver or brain. The accumulated oligosaccharides were mainly Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, Man(alpha 1-3)[Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4) GlcNAc, and Man(alpha 1-3)[Man(alpha 1-6)]Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc. Analogous branched Man4 and Man5 structures are found in pig and sheep tissues, but they are N, N'-diacetylchitobiose derivatives. The substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases were investigated because in one type of hereditary alpha-mannosidosis, that occurring in man, the major storage products are linear rather than branched oligosaccharides. The lysosomal enzyme showed much greater activity toward linear oligosaccharides than toward the branched oligosaccharides induced in the kidney by swainsonine. On the other hand, cytosolic alpha-D-mannosidase preferred the branched oligosaccharides, a result suggesting that this mannosidase might be inhibitable by swainsonine and that the enzyme might play a normal role in glycoprotein catabolism. Swainsonine was indeed found to inhibit this enzyme at relatively high concentrations (I50 at 100 microM swainsonine), and concentrations of this magnitude were in fact found in the cytosol of kidney of swainsonine-fed rats. The kidney cytosolic alpha-D-mannosidase levels were reduced in these rats and, more important, the accumulated oligosaccharides were present mainly in the cytosol rather than in lysosomes. These results point to possible involvement of cytosolic alpha-D-mannosidase in glycoprotein degradation in the rat.     

Cell contact induces the synthesis of a lysosomal enzyme precursor in lymphocytes and its direct transfer to fibroblasts

The activity of a lysosomal enzyme, alpha-D-mannosidase (EC 3.2.1.24), increased markedly in normal lymphocytes when they were cultured together with fibroblasts from a patient with an inherited deficiency of this enzyme. Cell-to-cell contact was obligatory for this increase in activity, which also required new protein synthesis. The enzyme induced in the co-cultured lymphocytes was a high molecular weight form of alpha-D-mannosidase that was not detected in lymphocytes cultured alone, which had only the low molecular weight mature enzyme. It was this precursor form alone that was directly transferred to the mannosidosis fibroblasts, where it was present initially in organelles of low density. When the culture period was extended the lymphocyte precursor enzyme was transported to the heavy lysosomes in the recipient cells, and correctly processed to the functionally effective mature enzyme.     

Swainsonine affects the processing of glycoproteins in vivo

Rats, sheep and guinea pigs treated with swainsonine excrete 'high mannose' oligosaccharides in urine. The major rat and guinea pig oligosaccharide is (Man)5GlcNAc, whereas sheep excrete a mixture of oligosaccharides of composition (Man)2-5GlcNAc2 and (Man)3-5GlcNAc. The presence of these oligosaccharides suggests that Golgi alpha-D-mannosidase II as well as lysosomal alpha-D-mannosidase is inhibited by swainsonine resulting in storage of abnormally processed asparagine-linked glycans from glycoproteins. Altered glycoprotein processing appears to have little effect on the health of the intoxicated animal, but the accompanying lysosomal storage produces a disease state.     

Purification and properties of major alpha-D-mannosidase in the luminal fluid of porcine epididymis.

A lysosomal type alpha-D-mannosidase was successfully purified by DEAE-Sephacel, Red-Amicon and Superdex 200 column chromatographies from porcine cauda epididymal fluid. The purified enzyme consisted of 63 and 51 kDa subunits at equimolar amounts. It cleaved alpha1-2 linked mannosyl residues and less but significantly cleaved alpha1-3 and alpha1-6 linked mannosyl residues in the high-mannose oligosaccharides. The optimal pH to hydrolyze oligosaccharide was in the acidic pH range (pH 3.5 approximately 4.0).Total alpha-D-mannosidase activities in the porcine epididymal fluid increased from proximal to distal caput epididymis, which maintained to cauda epididymis. At least two kinds of alpha-D-mannosidase (lysosomal type enzyme and 135 kDa alpha-D-mannosidase (MAN2B2)) were contained in the porcine epididymal fluid. The activity of the lysosomal type enzyme is much higher than MAN2B2 at the physiological pH.These results suggest that the lysosomal type alpha-D-mannosidase is the predominantly active enzyme in the luminal fluid of porcine epididymis and that it participates in the glycoprotein modification on the sperm surface during epididymal transit.     

Mannosidosis: separation and characterization of two acid alpha-mannosidase forms in mutant fibroblasts

Two acidic forms of alpha-mannosidase activity, A and B, are separated and characterized in fibroblasts from controls and patients with mannosidosis. In normal cells, A and B differ in adsorption on DEAE-cellulose, electrophoretic mobility, stability at 70 degrees C and resistance to freezing at --20 degrees C. In 5 of 6 mutant cell lines, A and B both exhibit altered Km's for artificial substrates and decreased thermal stability. Zn2+ has no effect on A or B in mutant or normal cells. In contrast, Co2+ slightly inhibits B in normal cells but markedly enhances the activity of B in mutant cells. The mutation in mannosidosis clearly results in an alteration in the biochemical characteristics of both major acidic forms of alpha-mannosidase.     

The multiple forms of alpha-D-mannosidase in human plasma.

The acidic alpha-D-mannosidase in human plasma closely resembles liver acidic alpha-D-mannosidase in its affinity for concanavalin A-Sepharose, molecular weight and resolution into multiple components on DEAE-cellulose. A combination of chromatography on concanavalin A-Sepharose and gel filtration on Sephadex G-200 and Sepharose 6B suggests that four forms of intermediate alpha-D-mannosidase, which differ either in their molecular weight of affinity for concanavalin A, exist in human plasma. A practical classification and nomenclature for the multiple forms of intermediate alpha-D-mannosidase in plasma based on molecular weight and affinity for concanavalin A is proposed. Multiple forms of intermediate alpha-D-mannosidase were also observed by ion-exchange chromatography on DEAE-cellulose, but there was not a simple correlation between these forms and those obtained with the other separation procedures. The form of intermediate alpha-D-mannosidase least abundant in plasma, approx. 7% of the activity, has very similar properties to the neutral alpha-D-mannosidase in human liver. In contrast, the other three forms of intermediate alpha-D-mannosidase, which account for over 90% of the activity, do not appear to be present in liver, except perhaps in trace amounts.     

A urinary pentasaccharide in bovine mannosidosis.

Abnormally high amounts of low molecular weight mannose-rich carbohydrate material were found in the urine of an Angus calf with mannosidosis. At least five oligosaccharide fractions were detected by paper chromatography. The most abundant compound was purified by gel chromatography, zone electrophoresis, and two consecutive preparative paper chromatographic steps. The yield was 10 mg/liter of urine. From structural studies including nuclear magnetic resonance spectroscopy, optical rotation, sugar analysis, methylation analysis, and partial enzymatic degradation the following structure was deduced: alpha-D-Manp-(1 leads to 6)-beta-D-Manp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to 4)-D-GlcNAc. This oligosaccharide is distinct from all the oligosaccharides previously described which are excreted by patients with mannosidosis.