A mouse model for fucosidosis recapitulates storage pathology and neurological features of the milder form of the human disease.

Fucosidosis is a rare lysosomal storage disorder caused by the inherited deficiency of the lysosomal hydrolase α-L-fucosidase, which leads to an impaired degradation of fucosylated glycoconjugates. Here, we report the generation of a fucosidosis mouse model, in which the gene for lysosomal α-L-fucosidase (Fuca1) was disrupted by gene targeting. Homozygous knockout mice completely lack α-L-fucosidase activity in all tested organs leading to highly elevated amounts of the core-fucosylated glycoasparagine Fuc(α1,6)-GlcNAc(ß1-N)-Asn and, to a lesser extent, other fucosylated glycoasparagines, which all were also partially excreted in urine. Lysosomal storage pathology was observed in many visceral organs, such as in the liver, kidney, spleen and bladder, as well as in the central nervous system (CNS). On the cellular level, storage was characterized by membrane-limited cytoplasmic vacuoles primarily containing water-soluble storage material. In the CNS, cellular alterations included enlargement of the lysosomal compartment in various cell types, accumulation of secondary storage material and neuroinflammation, as well as a progressive loss of Purkinje cells combined with astrogliosis leading to psychomotor and memory deficits. Our results demonstrate that this new fucosidosis mouse model resembles the human disease and thus will help to unravel underlying pathological processes. Moreover, this model could be utilized to establish diagnostic and therapeutic strategies for fucosidosis.

Lysosomal exoglycosidases in serum and urine of patients with pancreatic adenocarcinoma.

Lysosomal exoglycosidases: N-acetyl-ß-D-hexosaminidase (HEX), ß-D-galactosidase (GAL), α-L-fucosidase (FUC) and α-D-mannosidase (MAN) modify oligosaccharide chains of glycoconjugates in endoplasmatic reticulum and/or Golgi apparatus and degrade them in lysosomes. In acid environment of lysosome, exoglycosidases degrade oligosaccharide chains of glycoproteins, glycolipids and glycosaminoglycans by eliminating single sugars from the edges of oligosaccharide chains. Neoplasms change biochemical processes in tissues and may significantly change the activity of many enzymes including the activity of lysosomal exoglycosidasses in serum and urine of persons with neoplasmatic diseases. The aim of the present paper was evaluation the activity of HEX, GAL, FUC and MAN in serum and urine of patients with pancreatic adenocarcinoma. Serum and urine samples were collected from 15 patients with adenocarcinoma of the pancreas and 15 healthy persons. The activity of lysosomal exoglycosidases was determined by the method of Marciniak et al. adapted to serum and urine of patients with adenocarcinoma of the pancreas. Our results indicate significant decrease in activity of GAL (p=0.037) in serum of patients with pancreatic adenocarcinoma, significant increase in activity of HEX (p<0.001) and FUC (p=0.027) in serum, and HEX (p=0.003) in urine, as well as significant decrease of FUC (p=0.016) and MAN (p=0.029) in urine o patients with adenocarcinoma of the pancreas, in comparison to the control group. Increase in activity of some lysosomal enzymes in serum and urine of pancreatic adenocarcinoma patients, may indicate on destruction of pancreatic tissue by pancreatic adenocarcinoma. Determination of the HEX, GAL, FUC and MAN in serum and urine may be useful in diagnostics of pancreatic adenocarcinoma.

Identification of glycoconjugates in the urine of a patient with congenital disorder of glycosylation by high-resolution mass spectrometry.

More than 150 molecular species were detected in a single glycoconjugate fraction obtained from urine of a congenital disorders of glycosylation (CDG) patient by use of high-resolution FT-ICR MS. With respect to its high-mass accuracy and resolving power, FT-ICR MS represents an ideal tool for analysis of single components in complex glycoconjugate mixtures obtained from body fluids. The presence of overlapping nearly isobaric ionic species in glycoconjugate mixtures obtained from CDG patient's urine was postulated from fragmentation data of several precursor ions obtained by nanoESI Q-TOF CID. Their existence was confirmed by high-resolution/high-mass accuracy FT-ICR MS detection. High-resolution FT-ICR mass spectra can, therefore, be generally considered for glycoscreening of complex mixture samples in a single stage. From the accurate molecular ion mass determinations the composition of glycoconjugate species can be identified. Particular enhancement of identification is offered by computer-assisted calculations in combination with monosaccharide building block analysis, which can be extended by considerations of non-carbohydrate modifications, such as amino acids, phosphates and sulfates. Taking advantage of this strategy, the number of compositions assigned to mass peaks was significantly increased in a fraction obtained from urine by size exclusion and anion exchange chromatography.

Thin chip microsprayer system coupled to quadrupole time-of-flight mass spectrometer for glycoconjugate analysis.

A thin chip polymer-based microsprayer has been coupled to a hybrid quadrupole time-of-flight mass spectrometer (QTOF MS) and introduced in carbohydrate research. The feasibility of the approach is demonstrated for mapping, sequencing and structural elucidation of glycoconjugates originating from human body fluids and tissues such as a glycopeptide mixture from normal human urine and an isolated and purified GT1 ganglioside fraction from normal adult human brain. The optimization procedure required by each glycoconjugate category is described and the advantages of the system in terms of flexibility and adaptability to QTOF MS, stability of the ESI MS signal, carbohydrate ionization and sequencing, sensitivity, speed of analysis and sample consumption are discussed.

Fully automated chip-based mass spectrometry for complex carbohydrate system analysis.

Carbohydrates represent a major class of biopolymers, which occur in nature either as oligosaccharides or glycoconjugates, in which the sugar moiety is linked to proteins or lipids. The significance of mass spectrometry for highly sensitive analysis of complex carbohydrates increased after the introduction of the electrospray ionization and matrix assisted laser desorption/ionization methods and the possibility of tandem MS for sequencing of single molecular species in complex mixtures. Rapid and sensitive characterization of carbohydrates in biological systems by automated nanoscale liquid delivery and chip-based electrospray interface techniques have not been developed so far. In this contribution, the implementation and optimization of a fully automated chip-based nanoelectrospray assembly (NanoMate system), operating in the negative ion mode, in combination with QTOF-tandem MS for mapping/sequencing and computer-assisted structure assignment for carbohydrate components in complex mixtures is presented.

Tryptophan glycoconjugates in food and human urine.

Evaluating the formation of tryptophan glycoconjugates other than well-established Amadori rearrangement products, HPLC-tandem MS (MS/MS) analysis of human urine collected from several healthy individuals proved the presence of one distinct tryptophan C-glycosyl compound [Horiuchi, Yonekawa, Iwahara, Kanno, Kurihara and Fujise (1994) J. Biochem. (Tokyo) 115, 362-366]. After isolation, unambiguous identification of this novel tryptophan metabolite as 2-(alpha-mannopyranosyl)-l-tryptophan was achieved by tandem MS combined with NMR spectroscopy including homonuclear COSY, heteronuclear multiple-bond connectivity and (1)H-detected heteronuclear multiple-quantum coherence experiments. Remarkably, a thorough evaluation of vicinal proton-proton coupling constants in different solvents and nuclear Overhauser effect experiments demonstrate the predominant axial orientation of the hydroxymethyl group of the hexopyranosyl residue. Likewise this spatial arrangement indicates that the respective alpha-anomeric C-mannosylhexopyranose is preferentially adopting a (1)C(4) conformation in acidic methanol. Whereas only one distinct tryptophan mannoconjugate could be observed in human urine, HPLC-MS/MS analysis of food samples for the first time led to the identification of numerous N(1)-(beta-d-hexopyranosyl)-l-tryptophan, 2-(beta-d-hexopyranosyl)-l-tryptophan and 1-(1,2,3,4,5-pentahyd- roxypent-1-yl)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid derivatives derived from the condensation of tryptophan with aldohexoses. Taking into consideration the significant differences between profiles and configurations of tryptophan glycoconjugates originating from dietary sources and human urine, C-2 mannosylation of tryptophan residues [de Beer, Vliegenthart, Loeffler and Hofsteenge (1995) Biochemistry 34, 11785-11789] represents a novel enzymic pathway in tryptophan metabolism in humans.

Origin of non-dialysable urinary glucoconjugates.

A Gel exclusion chromatographic method was used to determine the molecular weight distribution, and therefore, the origin, of non-dialysable urinary glucoconjugates in normal men. The results showed a mixture of glucoconjugates with molecular weight ranges of 1605 to 141,000. It is suggested that the high molecular weight forms originally constitute the glucoconjugates, and that they are probably post-glomerular in origin. These may be degraded in vivo by glucosylhydrolases to produce the low molecular weight forms. The activities of the urinary enzymes may be reduced in male type 1 diabetic patients, and this may be responsible for the reported increase in their excretion of non-dialysable urinary glucoconjugates.

Structural analysis of the non-dialysable urinary glucoconjugates of normal men.

Enzymic methods were employed to analyse the structure of the non-dialysable urinary glucoconjugates of ten healthy males. The excretion of the urinary glucoconjugates was determined from the glucosyl:galactosyl ratio after acid hydrolysis, and a mean value of 0.27 was obtained. The results of the specific actions of alpha- and beta-glucosidases showed that the non-dialysable urinary glucoconjugates contain a branched alpha-glucan fraction with 1,4- and 1,6-glucosidic bonds, and a beta-glucan fraction containing 1,4-glucosidic bonds.

Metabolism in vivo of the tropane alkaloid, scopolamine, in several mammalian species.

1. In vivo metabolism of scopolamine was studied in rats, mice, guinea pigs and rabbits. The structures of eight urinary metabolites including unchanged drug were elucidated by mass and nuclear magnetic resonance spectrometry. Determination of these metabolites was achieved by a g.l.c. method using a semi-capillary column. 2. The major metabolites in rats were the three phenolic metabolites, p-hydroxy-, m-hydroxy- and p-hydroxy-m-methoxy-scopolamine. 3. Significant intra-species difference of the metabolism was observed in rabbits. Tropic acid was the major metabolite in two rabbits out of three, while the other rabbit excreted mainly unchanged scopolamine, accompanied by five metabolites. Tropic acid was also the major metabolite in guinea pigs, but was of minor importance in mice. 4. The dehydrated metabolites, aposcopolamine and aponorscopolamine, were abundantly excreted in guinea pigs, moderately in mice, and least in rabbits and rats. 5. Excretion of glucuronide conjugates of scopolamine and norscopolamine were high in mice compared with other species. On the other hand, phenolic metabolites in rat urine; and tropic acid in rabbit and guinea pig urine, were excreted as the free forms. 6. These results indicate that scopolamine metabolism is highly species-specific.

N(+)-glucuronidation of aliphatic tertiary amines, a general phenomenon in the metabolism of H1-antihistamines in humans.

1. Representative drugs of the various structural classes of H1 antihistamines were chosen for study. The drugs chosen (class name in parentheses) were chlorpheniramine maleate and pheniramine maleate (alkylamines), diphenhydramine hydrochloride and doxylamine succinate (ethanolamines), pyrilamine maleate and tripelennamine hydrochloride (ethylenediamines), promethazine hydrochloride (phenothiazine), cyclizine lactate (piperazine) and terfenadine (miscellaneous). In each case oral dose(s) were administered over no more than 6 h to two healthy volunteers and the total urine collected for 36 h. 2. Metabolites from urine were separated by h.p.l.c. and individually collected prior to mass spectrometric analysis in the fast atom bombardment mode. The structure of each metabolite identified as a quaternary ammonium-linked glucuronide metabolite was confirmed by direct comparison of its mass spectrum and chromatographic behaviour with that of a synthetic authentic compound. 3. For eight of the nine drugs studied, metabolism by the N(+)-glucuronidation pathway was observed in each of the volunteers. Terfenadine was the exception. 4. The amount of each N(+)-glucuronide in the urine was estimated by h.p.l.c. analysis. The mean proportion of dose excreted as the metabolite was 14.3%, 6.5% and 4.0% for cyclizine, tripelennamine and diphenhydramine, respectively. Promethazine was the only case where the N(+)-glucuronide accounted for less than 1.0% of the administered dose in both volunteers examined.

Novel lysosomal glycoaminoacid storage disease with angiokeratoma corporis diffusum.

A 46-year-old Japanese woman had disseminated angiokeratoma, confirmed by electron microscopy which showed numerous cytoplasmic vacuoles in cells of the kidney and skin. Enzyme activities against synthetic and natural substrates in leucocytes and fibroblasts were normal. Her urine contained a large amount of sialylglycoaminoacids, with predominant excretion of an O-glycoside-linked glycoaminoacid.

Glucuronoconjugates in chronic renal failure. Comparative determination with values in healthy adult.

We have isolated substances of molecular weight ranging between 350 and 2,000 daltons from ultrafiltrates of patients traited by maintenance haemodialysis for chronic renal failure (CRF). Such substances might have a role in the genesis of uremic toxicity. They were found to be glucuronoconjugates. We have studied 22 patients with CRF and 16 with end stage renal failure, 8 of them were traited by peritoneal dialysis, 8 by haemodialysis. We have evaluated the concentration of glucuronic acid in the serum, the urine and dialysis fluid. The patients with chronic renal failure have plasma levels of free and conjugate glucuronic acid higher than normal subjects. Urinary elimination is very lower in patient with CRF. Nevertheless the glucuronoconjugates are removed to a large extend by haemodialysis and peritoneal dialysis.