Lysosomal beta-mannosidase was purified 160,000-fold in 24% yield from bovine kidney by a four-step purification procedure, which included concanavalin A-Sepharose, immunoaffinity, TSK-butyl and h.p.l.c. cation-exchange chromatography. When analysed by SDS/PAGE and detected by Coomassie Blue or silver staining, the purified enzyme preparation consists of two prominent peptides (100 and 110 kDa) and a third minor peptide (84 kDa). These three peptides are immunologically related and are consistently associated with beta-mannosidase activity in all chromatographic steps. Removal of N-linked carbohydrate from the 84, 100 and 110 kDa peptides decreases their molecular sizes to 75, 86 and 91 kDa respectively. Bovine kidneys lacking beta-mannosidase, activity, acquired from calves affected with beta-mannosidosis, do not contain detectable quantities of the three beta-mannosidase peptides, as judged by monoclonal- and polyclonal-antibody reactivity.

beta-D-Mannosidase activity in selected normal adult, neonatal and foetal goat tissues and in tissues from animals affected with caprine beta-mannosidosis was examined with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. The enzyme in normal adult thyroid, kidney and brain exhibited a sharp unimodal pH optimum at pH 5.0, whereas the enzyme in both normal adult and mutant liver exhibited broad pH ranges of activity (pH 4.5-8.0). No residual enzyme was detectable in mutant kidney or brain; in contrast, residual activity in mutant liver was 52% of that in a neonatal control. Concanavalin A-Sepharose 4B (Con A-Sepharose) fractionation of normal adult liver beta-D-mannosidase resolved the enzyme into an unbound (non-lysosomal) from (52%) with a broad pH range of activity (pH 4.5-8.0) and a bound (lysosomal) form (48%) with a sharp pH optimum of 5.5. The enzyme in mutant liver consisted entirely of the unbound (non-lysosomal) form. Beta-D-Mannosidase activity in normal adult thyroid, kidney and brain was resolved by chromatofocusing into two major isoenzymes, with pI 5.5 and 5.9, and traces of a minor isoenzyme, with pI 5.0. In normal adult liver the enzyme was also resolved into three isoenzymes with similar pI values; however, that with pI 5.0 predominated. The predominant form of the enzyme in 60-day-foetal liver was bound by Con A, exhibited a unimodal pH optimum (5.0) and was resolved into two isoenzymes, with pI 5.4 and 5.8; only traces of an isoenzyme with pI 5.0 were detectable. Total hepatic beta-D-mannosidase activity increased progressively towards adult values during the last 90 days of gestation as a result of increasing non-lysosomal isoenzyme activity (pI 5.0). Lysosomal beta-D-mannosidase was shown to occur in all normal goat tissues studied as multiple isoenzymes, which are genetically and developmentally distinct from the non-lysosomal isoenzyme occurring predominantly, if not exclusively, in liver.

1. Goat kidney beta-mannosidase was purified 8500-fold to a specific activity of 65,000 nmol/h per mg of protein with a 6% yield by using multiple steps including cation-exchange and anion-exchange fast protein liquid chromatography. This is the first description of a highly purified preparation from goat tissue; however, it was not homogeneous, as judged by silver-stained SDS/polyacrylamide-gel electrophoresis. 2. The enzyme exhibited microheterogeneity when analysed by isoelectric focusing (pI 5.5-6.5). 3. Purified beta-mannosidase hydrolysed the terminal beta-(1----4)-linkage of oligosaccharides that accumulate in beta-mannosidosis.

Lysosomal storage diseases associated with early-onset pathological changes may require prenatal therapy to avert the profound effects of the metabolic error on organs, especially the central nervous system. The present investigation determined the extent of expression of beta-mannosidase deficiency in the caprine fetus at 62 days of gestation, near the end of the period of immunotolerance when donor cells can engraft in various organs without immune rejection and supply missing enzyme. Three pairs of obligate carrier goats from the beta-mannosidosis colony were mated. Out of six fetuses delivered at 62 days of gestation, one (V385) was identified by measurement of beta-mannosidase activity as the only fetus affected with beta-mannosidosis. Thin-layer chromatography and quantitation of oligosaccharides revealed the presence of tri- and disaccharides, typical of beta-mannosidosis, only in V385. Morphological analysis revealed cytoplasmic vacuolation typical of beta-mannosidosis in V385; in thyroid, spinal cord, and kidney, the pattern of vacuolation was similar to, but less severe than, that observed previously in newborn affected goats. On the basis of these results, it will be possible to determine the effects of prenatal cell transplantation therapeutic strategies performed during the period of immunotolerance by monitoring phenotypic characteristics after treatment.

An aberrant beta-mannosidosis phenotype in a 5-month-old triplet goat kid was characterized by a late postnatal onset of mild neurological symptoms. Necropsy examination revealed relatively normal myelination; however, the distribution of cytoplasmic vacuolation in the kidney and brain was similar to that observed in neonatal beta-mannosidosis. Variable engraftment of donor stem cells, resulting from transplacental transfusion of stem cells from a normal sibling during the immunotolerant period, may have modified the expected severe beta-mannosidosis phenotype. This investigation was designed to determine the effects of a possible chimeric state on organ-specific metabolic perturbations. Residual beta-mannosidase enzyme activity was found in plasma, kidney, liver and spleen but not in brain. Other lysosomal enzyme activities were comparable to normal values. Immunoreactive beta-mannosidase was estimated to be less than 10% of normal levels. Kidney, brain grey matter and brain white matter contained 33%, 12% and 4%, respectively, of the oligosaccharides expected in the organs of beta-mannosidosis animals. There were no detectable oligosaccharides or cytoplasmic vacuolation in the liver or spleen. Studies of this possible chimera provided preliminary evidence for the efficacy of prenatal treatment of early-onset neurodegenerative disorders.

Urinary oligosaccharides were studied in beta-mannosidosis, a newly identified, inherited glycoprotein catabolic disorder associated with severe neonatal neurological deficits, widespread lysosomal storage vacuoles and a deficiency of plasma and tissue beta-mannosidase. A preliminary analysis of the oligosaccharides was obtained by gel-permeation chromatography and mass chromatography. The major urinary oligosaccharides were then isolated by gel-permeation chromatography, DEAE-Sephadex column chromatography and preparative paper chromatography, and were analyzed by carbohydrate composition analysis, methylation studies, mass spectrometry and glycosidase digestion. As a result of these studies, beta-mannosyl-(1 leads to 4)-N-acetylglucosamine and beta-mannosyl-(1 leads to 4)-beta-N-acetylglucosaminyl-(1 leads to 4)-N-acetylglucosamine were identified as the major abnormal oligosaccharides. Galactosaminyl-(alpha 1 leads to 3)-[fucosyl-(alpha 1 leads to 2)]-galactose was also found in affected goat urine, while lactose was present in the urine of both control and affected goats.

The phenotype of bovine beta-mannosidosis (beta-mannosidase deficiency), recently identified in Salers cattle, is similar to the caprine form of the disease (Abbitt et al., 1991). This investigation was designed to characterize accumulated kidney oligosaccharides in bovine beta-mannosidosis. Oligosaccharides were extracted from the kidney of an affected Salers calf and purified by chromatographic techniques. The amount of accumulating oligosaccharides in 1 g of wet tissue was about 21 mumol. Structures of derivatized oligosaccharides were characterized by high-performance liquid chromatography, mass spectrometry, methylation analysis and sequential exoglycosidase digestions. The major accumulating oligosaccharides were Man beta 1-4GlcNAc and Man beta 1-4GlcNAc beta 1-4GlcNAc. Oligosaccharides accumulating in minor amounts were Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc, Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc and Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc beta 1-4GlcNAc. As in caprine beta-mannosidosis, oligosaccharides with terminal beta-mannose residues and cleaved as well as uncleaved chitobiose linkages were identified in bovine beta-mannosidosis kidney. The accumulating oligosaccharides in tissue were thus identical in bovine and caprine beta-mannosidosis; however, the source of the novel oligosaccharides remains to be determined.

Ultrasound-guided fetal fluid sampling was performed on 13 pregnant goats at Days 59 to 65 of gestation to establish safe techniques for accurate sampling and to determine the feasibility of prenatal diagnosis of beta-mannosidosis. Fluids were analyzed for electrolyte and creatinine content to assess accuracy of sampling. Values correlated well with previously reported concentrations for caprine and ovine fetal fluids at the same gestational stage. The single abortion which occurred following ultrasound-guided sampling was correlated with placentome penetration and aspiration of bloody fluids. Thin layer chromatography of amniotic and allantoic fluids was performed to detect oligosaccharides that accumulate in beta-mannosidosis. Abnormal accumulated oligosaccharides were identified in the allantoic but not amniotic fluid from a beta-mannosidase-deficient 62-d-old fetus. Thus, allantocentesis was shown to be an optimal, safe procedure for providing information at this gestational stage to diagnose caprine beta-mannosidosis.

A fatal case of malignant atrophic papulosis (Degos disease) with optic nerve and spinal cord involvement is described. Magnetic resonance imaging (MRI) of the optic nerve showed abnormal signal enhancement on fat suppressed T1 weighted images after intravenous meglumine gadopentetate infusion. On T2 weighted sagittal images, a sawtooth pattern was observed over seven vertebral segments of the spinal cord. On necropsy, a severe loss of myelinated nerve fibres in the left optic nerve was seen, with thrombotic obstruction of the central retinal artery. Spongy degeneration was observed in all levels of the spinal cord, with patchy and motheaten patterns caused by thromboses and endothelial proliferation in subarachnoid vessels. Findings on MRI were consistent with findings on pathological examination.

Dipeptidyl peptidase III (DPP III)(EC 3.4.14.4), which has a HELLGH-E (residues 450-455, 508) motif as the zinc binding site, is classified as a zinc metallopeptidase. The zinc dissociation constants of the wild type, Leu(453)-deleted, and E508D mutant of DPP III at pH 7.4 were 4.5 (+/-0.7) x 10(-13), 5.8 (+/-0.7) x 10(-12), and 3.2 (+/-0.9) x 10(-10) M, respectively. The recoveries of the enzyme activities by the addition of various metal ions to apo-DPP III were also measured, and Co(2+), Ni(2+), and Cu(2+) ions completely recovered the enzyme activities as did Zn(2+). The dissociation constants of Co(2+), Ni(2+), and Cu(2+) ions for apo-DPP III at pH 7.4 were 8.2 (+/-0.9) x 10(-13), 2.7 (+/-0.3) x 10(-12), and 1.1 (+/-0.1) x 10(-14) M, respectively. The shape of the absorption spectrum of Co(2+)-DPP III was very similar to that of Co(2+)-carboxypeptidase A or Co(2+)-thermolysin, in which the Co(2+) is bound to two histidyl nitrogens, a water molecule, and a glutamate residue. The absorption spectrum of Cu(2+)-DPP III is also very similar to that of Cu(2+)-thermolysin. The EPR spectrum and the EPR parameters of Cu(2+)-DPP III were very similar to those of Cu(2+)-thermolysin but slightly different from those of Cu(2+)-carboxypeptidase A. The five lines of the superfine structure in the perpendicular region of the EPR spectrum in Cu(2+)-DPP III suggest that nitrogen atoms should coordinate to the cupric ion in Cu(2+)-DPP III. All of these data suggest that the donor set and the coordination geometry of the metal ions in DPP III, which has the HExxxH motif as the metal binding site, are very similar to those of the metal ions in thermolysin, which has the HExxH motif.

Positional cloning approaches revealed that Tangier disease (TD), a genetic high density lipoprotein deficiency, is associated with mutations in the ATP-binding cassette transporter-1 (ABCA1) gene. However, the biological function of ABCA1 is still not fully investigated. Recently, we have reported that the cells from the patients with TD had abnormal actin cytoskeletons in association with decreased expression of Cdc42, a member of RhoGTPases family. In the present study, we have found that actin cytoskeletons were altered in HEK293 cells transfected with human ABCA1 (hABCA1) cDNA. Cells expressing hABCA1 were divided into the following two groups by the distinct morphology with altered actin cytoskeletons: one had increased formation of filopodia (designated as Type I) and the other had long protrusions (designated as Type II). Type I cells had morphology similar to that of cells transfected with dominant active form of Cdc42 (Cdc42-DA, V12Cdc42Hs-DA). Type II cells had morphology similar to that of cells transfected with neural Wiskott-Aldrich Syndrome Protein (N-WASP),one of the established downstream effector molecules of Cdc42. We have obtained the data showing a possible pathway of ABCA1/Cdc42/N-WASP by the following experiments. Introduction of mutant of Cdc42 (dominant negative form of Cdc42, N17Cdc42Hs-DN) and N-WASP (N-WASP lacking verprolin homology domain, N-WASPDeltaVPH), both of which are supposed to have potential to inhibit rearrangement of actin cytoskeletons, significantly inhibited the morphological changes induced by expression of hABCA1. Immunoprecipitation study with FLAG-tagged ABCA1 (hABCA1-FLAG) revealed that Cdc42 was coimmunoprecipitated with hABCA1-FLAG. In addition, we have demonstrated possible intracellular colocalization of these two molecules in the overexpressing cells by the confocal laser microscopy. These results may suggest that hABCA1 regulates actin organization through the possible interaction with Cdc42Hs.

Glycosynthases are precise molecular instruments for making specifically linked oligosaccharides. X-ray crystallography screening of ligands bound to the 1,3(4)-beta-d-glucanase nucleophile mutant E115S of Phanerochaete chrysosporium Laminarinase 16A (Lam16A) showed that laminariheptaose (L7) bound in an arch with the reducing and nonreducing ends occupying either side of the catalytic cleft of the enzyme. The X-ray structure of Lam16A E115S in complex with alpha-laminariheptaosyl fluoride (alphaL7F) revealed how alphaL7F could make a nucleophilic attack upon itself. Indeed, when Lam16A E115S was allowed to react with alphaL7F the major product was a cyclic beta-1,3-heptaglucan, as shown by mass spectrometry. NMR confirmed uniquely beta-1,3-linkages and no reducing end. Molecular dynamics simulations indicate that the cyclic laminariheptaose molecule is not completely planar and that torsion angles at the glycosidic linkages fluctuate between two energy minima. This is the first report of a glycosynthase that joins the reducing and nonreducing ends of a single oligosaccharide and the first reported synthesis of cyclic beta-glucan.

Four oligosaccharides containing a fructopyranosyl residue have been found from fermented beverage of plant extract and isolated from the beverage using carbon-Celite column chromatography and preparative high performance liquid chromatography. Structure confirmation of the saccharides was provided by methylation analysis, MALDI-TOF-MS and NMR measurements. These saccharides were identified as oligosaccharides of fructopyranoside series; beta-d-fructopyranosyl-(2-->6)-d-fructofuranose (1), beta-d-fructopyranosyl-(2-->1)-d-fructopyranose (2), beta-d-fructopyranosyl-(2-->1)-beta-d-fructofuranosyl-(2<-->1)-alpha-d-glucopyranoside (3), and beta-d-fructopyranosyl-(2-->6)-alpha-d-glucopyranosyl-(1<-->2)-beta-d-fructofuranoside (4). Saccharides 3 and 4 among novel saccharides 1, 3, and 4 were named 'pyrano-1-kestose (pyrano-isokestose)' and 'pyrano-neokestose', respectively.

alpha-mannosidosis is caused by the genetic defect of the lysosomal alpha-D-mannosidase (LAMAN) which is involved in the breakdown of free alpha-linked mannose-containing oligosaccharides originating from glycoproteins with N-linked glycans and thus manifests itself in an extensive storage of mannose-containing oligosaccharides. Here we demonstrate in a alpha-mannosidosis mouse model that native lysosomal proteins exhibit elongated N-linked oligosaccharides as shown by 2-D DIGE, deglycosylation assays and mass spectrometry. The analysis of cathepsin B derived oligosaccharides revealed a hypermannosylation of glycoproteins in alpha-mannosidosis mice as indicated by the predominance of extended Man3GlcNAc2 oligosaccharides. Treatment with recombinant human alpha-mannosidase partially corrected the hyperglycosylation of lysosomal proteins in vivo and in vitro. These data clearly demonstrate that LAMAN is not only involved in the lysosomal catabolism of free oligosaccharides but also in the trimming of asparagine-linked oligosaccharides on native lysosomal proteins.

Detailed structures of N-linked oligosaccharides of Defibrase, a highly active thrombin like enzyme (TLE) purified from the venom of Agkistrodon acutus, were successfully characterized using MALDI-TOF mass spectrometry in combination with sequential exoglycosidase digestion. Monosaccharide composition analysis was performed by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Galactose(Gal), mannose(Man), fucose(Fuc), N-acetylglucosamine (GlcNAc), and sialic acid (Neu5Ac) was detected and the total carbohydrate content was about 19.4%(w/w). The N-linked oligosaccharides were released by treatment with PNGase F, fluorescent labeled with 2-aminobenzamide, and fractionated by high performance liquid chromatography (HPLC). The main oligosaccharide fractions were collected and further digested with an array of exoglycosidase mixtures, and subsequent MALDI TOF MS analysis of the resulting products yielded information about structural features of the oligosaccharide. The combined data revealed the presence of five distinct oligosaccharide structures in Defibrase, which are mainly complex or hybrid type, with a small amount of oligomannosidic type. The complex type oligosaccharides are mostly tri-or bi-antennary and the hybrid oligosaccharides are all bi-antennary. Most oligosaccharides are also found to be fucosylated.

Poisoning by Sida carpinifolia belongs to a group of plant-induced phenotype which resembles lysosomal storage diseases. Saanen goats were fed aerial parts of green S. carpinifolia for up to 3 months. Concentrates complemented the nutritional requirements. Urine and blood samples were collected for oligosaccharide study (by thin layer chromatography-TLC) and hemogram analysis, respectively. Abnormal excretion of oligosaccharides was observed from the 2nd day of S. carpinifolia ingestion until one day after withdrawal of the plant from the diet. There were no changes in hemogram. Clinical signs were typical of poisoning caused by plants of this group and were seen from the 37th day on S. carpinifolia diet until seven days after withdrawal of the plant, when signs gradually became scarce and less evident. Results presented here suggest that detection of urinary oligosaccharides by TLC may be an useful method to assess swainsonine-containing plants exposure or an early diagnostic tool for poisoning by these plants.

Three ether-linked alkyl 5a-carba-glycopyranosides 1b,d, and 5b, and 5a'-carba-lactoside 7b were examined as potent primers in mouse B16 melanoma cells for their feasibility as building blocks for oligosaccharide biosynthesis. Uptake by B16 cells was first observed for all carba-glycoside primers, and, especially, the 5a-carba-sugar analogues of N-acetyl-beta-d-glucosaminide 1b and beta-d-glucoside 1d were shown to produce two-to-four-fold larger amounts of glycosylated products than the corresponding true sugar primers 1a and 1c. The carba glycoside uptake by cells resulted in beta-galactosylation and subsequent sialylation of the incorporated galactose residues, giving rise to glycosylated products 3b and 3d having similar glycan structures as the ganglioside GM3. According to efficient uptake in cells, in addition to stability of the ether-linked pseudo-reducing ends of the oligosaccharides that formed, the carba glycoside primers have been demonstrated to be versatile building blocks for these biocombinatorial syntheses of glycolipid oligosaccharide mimetics. On the other hand, uptake for 5a-carba-galactopyranoside residue was found to be decreased by one-third for dodecyl 5a-carba-beta-d-galactopyranoside 5b. Observation of similar levels for 5a'-carba-beta-lactoside 7b under both cellular and cell-free conditions suggested that enzymes are likely to recognize the pyranose oxygen atom.

The commercial enzyme preparation Pectinex Ultra SP-L containing fructosyltransferase activity was used to hydrolyze stachyose. During this reaction, besides the formation of mono-, di-, and trisaccharides (DP(3)), the presence of one pentasaccharide (DP(5)) and a new oligosaccharide (DP(6)) has been detected by gas chromatography. DP(5) and DP(6) oligosaccharides were isolated and fully characterized for the first time by an extensive nuclear magnetic resonance (NMR) study. Complete structure elucidation and full proton and carbon assignments were carried out using 1D ((1)H,(13)C) and 2D (gCOSY, multiplicity-edited gHSQC, gHSQC-TOCSY, and gHMBC) NMR experiments. The two oligosaccharides were shown to be stachyose-based structures; the pentasaccharide has a fructose unit linked to the C-1 of the fructose end of stachyose, and the hexasaccharide has a fructose unit linked to the C-1 of the fructose end of the pentasaccharide. The fructosyltransferase activity present in Pectinex Ultra SP-L allows new uses of this commercial enzyme preparation in the synthesis of oligosaccharides derived from alpha-galactosides.

Structural characterization of oligosaccharides from proteoglycans and other glycoproteins is greatly enhanced through the use of mass spectrometry and gel electrophoresis. Sample preparation for these sensitive techniques often requires enzymatic treatments to produce oligosaccharide sequences for subsequent analysis. This chapter describes several small-scale methods for in-gel, on-blot, and in-solution enzymatic digestions in preparation for graphitized carbon liquid chromatography-mass spectrometry (LC-MS) analysis, with specific applications indicated for glycosaminoglycans (GAGs) and N-linked oligosaccharides. In addition, accompanying procedures for oligosaccharide reduction by sodium borohydride, sample desalting via carbon microcolumn, desialylation by sialidase enzyme treatment, and small-scale oligosaccharide species fractionation are included. Fluorophore-assisted carbohydrate electrophoresis (FACE) is another useful method to isolate derivatized oligosaccharides. Overall, the modularity of these techniques provides ease and flexibility for use in conjunction with mass spectrometric and electrophoretic tools for glycomic research studies.

It is essential to establish a library of glycosaminoglycan oligosaccharides from the chondroitin and dermatan sulfates to investigate their biological functions and structure-activity relationships (SARs). There are several approaches to obtain oligosaccharides using chemical and enzymatic degradation procedures; however, purification of each resulting oligosaccharide is complicated because of the diversity of sulfonation patterns present in these oligosaccharides. We have developed a new method for the solvolytic degradation for chondroitin and dermatan sulfates to obtain an oligosaccharide mixture that can be easily purified into chondro/dermato oligosaccharides for characterization by both (1)H NMR and MALDI-TOFMS. These oligosaccharides have a methyl-esterified uronate residue and a methyl 2-acetamido-2-deoxy-d-galactofuranoside at the nonreducing and reducing ends, respectively. All other internal repeating disaccharide units were desulfonated, but maintained their core carbohydrate structures.

SUMMARY Endo-beta-mannosidase, which hydrolyzes the Manbeta1-4GlcNAc linkage of N-glycans in an endo-manner, was discovered in plants [Ishimizu, T. Sasaki, A., Okutani, S., Maeda, M., Yamagishi, M., Hase, S. J. Biol. Chem. 279, 38555-38562 (2004)]. During the course of the purification of the enzyme from lily flowers, we found a higher molecular mass form of the enzyme (designated as EBM II). EBM II was purified by column chromatgraphy to homogeneity and its molecular composition revealed EBM II to be comprised of endo-beta-mannosidase and an associated protein. The cDNA of this associated protein encodes a protein with slight homology to the fucosidase domain of bifidus AfcA. EBM II has alpha1,2-L-fucosidase activity and acts on a fucosylated xyloglucan nonasaccharide. The amino acid sequence of this associated protein has no similarity to known plant alpha-L-fucosidases. These results show that EBM II is a novel alpha1,2-L-fucosidase and a protein complex containing endo-beta-mannosidase.