HILIC-UPLC-MS for high throughput and isomeric N-glycan separation and characterization in Congenital Disorders Glycosylation and human diseases.

N-glycan analyses may serve uncovering disease-associated biomarkers, as well as for profiling distinctive changes supporting diagnosis of genetic disorders of glycan biosynthesis named congenital disorders of glycosylation (CDG). Strategies based on liquid chromatography (LC) preferentially coupled to electrospray ionization (ESI) - mass spectrometry (MS) have emerged as powerful analytical methods for N-glycan identification and characterization. To enhance detection sensitivity, glycans are commonly labelled with a functional tag prior to LC-MS analysis. Since most derivatization techniques are notoriously time-consuming, some commercial analytical kits have been developed to speed up N-deglycosylation and N-glycan labelling of glycoproteins of pharmaceutical and biological interest such as monoclonal antibodies (mAbs). We exploited the analytical capabilities of RapiFluor-MS (RFMS) to perform, by a slightly modified protocol, a detailed N-glycan characterization of total serum and single serum glycoproteins from specific patients with CDG (MAN1B1-CDG, ALG12-CDG, MOGS-CDG, TMEM199-CDG). This strategy, accomplished by Hydrophilic Interaction Chromatography (HILIC)-UPLC-ESI-MS separation of the RFMS derivatized N-glycans, allowed us to uncover structural details of patients serum released N-glycans, thus extending the current knowledge on glycan profiles in these individual glycosylation diseases. The applied methodology enabled to differentiate in some cases either structural isomers and isomers differing in the linkage type. All the here reported applications demonstrated that RFMS method, coupled to HILIC-UPLC-ESI-MS, represents a sensitive high throughput approach for serum N-glycome analysis and a valuable option for glycan detection and separation particularly for isomeric species.

MAN1B1 deficiency: an unexpected CDG-II.

Congenital disorders of glycosylation (CDG) are a group of rare metabolic diseases, due to impaired protein and lipid glycosylation. In the present study, exome sequencing was used to identify MAN1B1 as the culprit gene in an unsolved CDG-II patient. Subsequently, 6 additional cases with MAN1B1-CDG were found. All individuals presented slight facial dysmorphism, psychomotor retardation and truncal obesity. Generally, MAN1B1 is believed to be an ER resident alpha-1,2-mannosidase acting as a key factor in glycoprotein quality control by targeting misfolded proteins for ER-associated degradation (ERAD). However, recent studies indicated a Golgi localization of the endogenous MAN1B1, suggesting a more complex role for MAN1B1 in quality control. We were able to confirm that MAN1B1 is indeed localized to the Golgi complex instead of the ER. Furthermore, we observed an altered Golgi morphology in all patients' cells, with marked dilatation and fragmentation. We hypothesize that part of the phenotype is associated to this Golgi disruption. In conclusion, we linked mutations in MAN1B1 to a Golgi glycosylation disorder. Additionally, our results support the recent findings on MAN1B1 localization. However, more work is needed to pinpoint the exact function of MAN1B1 in glycoprotein quality control, and to understand the pathophysiology of its deficiency.

Glycopeptide storage in skin fibroblasts cultured from a patient with alpha-mannosidase deficiency.

Patients with mannosidosis, an inherited deficiency of lysosomal alpha-mannosidase, accumulate large amounts of mannose-rich oligosaccharides (the "core" of the carbohydrate units of many glocoproteins) in brain and liver and excrete these partial degradation products in their urine. A profound alpha-mannosidase deficiency was demonstrated in fibroblasts cultured from a skin biopsy obtained from a child with mannosidosis. Further, abnormal glycopeptides rich in mannose and similar to oligosaccharides found in the patient's urine were isolated from fibroblast extracts by a variety of chromatographic procedures and by virtue of their binding to a concanavalin A-Sepharose 4B affinity column. This storage material contained mannose, N-acetylglucosamine, and asparagine in the ratio 3 : 1 : 1 together with a few toher amino acids and had a molecular weight of approximately 1,100. There was no evidence for excretion of storage material by mannosidosis fibroblasts or for any abnormality in cell surface glycoprotein composition. The glycopeptide nature of the storage material isolated from cultured skin fibroblasts may be attributed to the low level of N-aspartyl-beta-glucosamindase (EC 3.5.1.-) activity in these cells.

Structural analysis of the major caprine beta-mannosidosis urinary oligosaccharides.

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.

Amount and properties of uptake forms in preparations of alpha-mannosidase from pig kidney.

alpha-Mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) from pig kidney has been shown to exist in multiple forms differing in their capability to be endocytosed by alpha-mannosidase deficient cultured cells. A method is presented to evaluate the amount of "uptake" forms in different preparations of the enzyme. Preparations with different rates of uptake were shown to contain different amount of "uptake" forms and "non-uptake" forms. The content of "uptake" forms in a preparation was identical with that of enzyme molecules bearing a phosphorylated carbohydrate group necessary for the recognition by cell surface receptors.

The nature of mannose-containing material which accumulates in cultured fibroblasts from patients with mannosidosis.

Fibroblasts from a patient with mannosidosis were grown in a medium containing a radioactive monosaccharide (D-[U-14C]mannose or N-acetyl-D-[1-14C]-glucosamine). An accumulation of radioactive material was observed. It was possible to prevent the accumulation to a certain degree by the addition of human liver alpha-D-mannosidase to the fibroblast medium. After six days of fibroblast culture the majority of the accumulated material had a molecular weight in the oligosaccharide range and was stationary during high-voltage electrophoresis. Paper chromatography of the stationary material separated three radioactive compounds with the same chromatographic mobilities as the oligosaccharides alpha-D-Man-(1 leads to 3)-beta-D-Man-(1 leads to 4)-D-GlcNAc (I), alpha-D-Man-(1 leads to 2)-alpha-D-Man-(1 leads to 3)-beta-D-Man-(1 leads to 4)-GlcNAc (II), and alpha-D-Man-(1 leads to 2)-alpha-D-Man-(1 leads to 2)-alpha-D-Man-(1 leads to 3)-beta-D-Man-(1 leads to 4)-GlcNAc (III) previously isolated from the urine of patients with mannosidosis. Degradation of the three radioactive compounds with jack bean alpha-mannosidase gave D-mannose and a disaccharide (containing D-mannose and N-acetyl-D-glucosamine). Thus the three main compounts observed in the fibroblasts from patients with mannosidosis are most probably identical to the oligosaccharides I--III.

Golgi alpha-mannosidase II deficiency in vertebrate systems: implications for asparagine-linked oligosaccharide processing in mammals.

The maturation of N-glycans to complex type structures on cellular and secreted proteins is essential for the roles that these structures play in cell adhesion and recognition events in metazoan organisms. Critical steps in the biosynthetic pathway leading from high mannose to complex structures include the trimming of mannose residues by processing mannosidases in the endoplasmic reticulum (ER) and Golgi complex. These exo-mannosidases comprise two separate families of enzymes that are distinguished by enzymatic characteristics and sequence similarity. Members of the Class 2 mannosidase family (glycosylhydrolase family 38) include enzymes involved in trimming reactions in N-glycan maturation in the Golgi complex (Golgi mannosidase II) as well as catabolic enzymes in lysosomes and cytosol. Studies on the biological roles of complex type N-glycans have employed a variety of strategies including the treatment of cells with glycosidase inhibitors, characterization of human patients with enzymatic defects in processing enzymes, and generation of mouse models for the enzyme deficiency by selective gene disruption approaches. Corresponding studies on Golgi mannosidase II have employed swainsonine, an alkaloid natural plant product that causes "locoism", a phenocopy of the lysosomal storage disease, alpha-mannosidosis, as a result of the additional targeting of the broad-specificity lysosomal mannosidase by this compound. The human deficiency in Golgi mannosidase II is characterized by congenital dyserythropoietic anemia with splenomegaly and various additional abnormalities and complications. Mouse models for Golgi mannosidase II deficiency recapitulate many of the pathological features of the human disease and confirm that the unexpectedly mild effects of the enzyme deficiency result from a tissue-specific and glycoprotein substrate-specific alternate pathway for synthesis of complex N-glycans. In addition, the mutant mice develop symptoms of a systemic autoimmune disorder as a consequence of the altered glycosylation. This review will discuss the biochemical features of Golgi mannosidase II and the consequences of its deficiency in mammalian systems as a model for the effects of alterations in vertebrate N-glycan maturation during development.

Isolation and characterization of the gene encoding the mouse broad specificity lysosomal alpha-mannosidase1.

A genomic clone encoding the mouse lysosomal alpha-mannosidases was isolated and the gene was found to be encoded by 24 exons spanning approximately 14.5 kb of genomic DNA. The intron-exon boundaries were conserved between the mouse, human, and bovine lysosomal alpha-mannosidase genes as well as being partially conserved in several other species. In order to define the promoter of the mouse mannosidase gene, >1 kb of DNA sequence was obtained upstream from the respective initiation codon. The transcription start site was identified by a 5'-RACE procedure and putative promoter elements were identified by expression of promoter/reporter constructs. Fluorescence in situ hybridization analysis using the mouse and human mannosidase genomic clones as probes, localized the mouse gene to chromosome 8, at band position 8C2, and the human gene to chromosome 19p13.2, a region syntenic to the lysosomal mannosidase gene on mouse chromosome 8.

Evidence for two catabolic endoglycosidase activities in beta-mannosidase-deficient goat fibroblasts.

Cultured skin fibroblasts derived from Nubian goats deficient in lysosomal beta-mannosidase, which had previously been shown to accumulate storage oligosaccharides with the structures Man beta 4GlcNAc beta 4GlcNAc and Man beta 4GlcNAc (in the ratio of 2.7:1) were evaluated for their ability to catabolize exogenous [3H]GlcN-labelled glycoproteins isolated from the secretions of cultured goat or human fibroblasts. Regardless of the source of exogenous labelled glycoprotein, affected goat fibroblasts took up the labelled glycoprotein from the culture medium and subsequently accumulated the same major labelled oligosaccharide, identified as Man beta 4GlcNAc beta 4GlcNAc; no such oligosaccharide accumulated in normal goat fibroblasts under the same conditions. Tunicamycin-treated affected fibroblasts also took up labelled exogenous glycoprotein and accumulated labelled storage trisaccharide, further suggesting the direct accumulation of storage trisaccharide from impaired glycoprotein-associated oligosaccharide catabolism. Treatment of metabolically labelled affected fibroblasts with leupeptin, an inhibitor of lysosomal cathepsins, resulted in the 2- to 6-fold inhibition of trisaccharide accumulation, while having little effect on the uptake of [3H]GlcN or the accumulation of labelled disaccharide. The results are most consistent with the presence of two endoglycosidases, an endo-beta-N-acetylglucosaminidase and an endo-aspartylglucosaminidase, in goat fibroblasts. These two activities, rather than heterogeneous core oligosaccharide structures, are responsible for the ultimate accumulation of storage oligosaccharides with one and two GlcNAc residues at their reducing terminus.

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.

Estimation of genotype distributions and posterior genotype probabilities for beta-mannosidosis in Salers cattle.

beta-Mannosidosis is a lethal lysosomal storage disease inherited as an autosomal recessive in man, cattle and goats. Laboratory assay data of plasma beta-mannosidase activity represent a mixture of homozygous normal and carrier genotype distributions in a proportion determined by genotype frequency. A maximum likelihood approach employing data transformations for each genotype distribution and assuming a diallelic model of inheritance is described. Estimates of the transformation and genotype distribution parameters, gene frequency, genotype fitness and carrier probability were obtained simultaneously from a sample of 2,812 observations on U.S. purebred Salers cattle with enzyme activity, age, gender, month of pregnancy, month of testing, and parents identified. Transformations to normality were not required, estimated gene and carrier genotype frequencies of 0.074 and 0.148 were high, and the estimated relative fitness of heterozygotes was 1.36. The apparent overdominance in fitness may be due to a nonrandom sampling of progeny genotypes within families. The mean of plasma enzyme activity was higher for males than females, higher in winter months, lower in summer months and decreased with increased age. Estimates of carrier probabilities indicate that the test is most effective when animals are sampled as calves, although effectiveness of the plasma assay was less for males than females. Test effectiveness was enhanced through averaging repeated assays of enzyme activity on each animal. Our approach contributes to medical diagnostics in several ways. Rather than assume underlying normality for the distributions comprising the mixture, we estimate transformations to normality for each genotype distribution simultaneously with all other model parameters. This process also excludes potential biases due to data preadjustment for systematic effects. We also provide a method for partitioning phenotypic variation within each genotypic distribution which allows an assessment of the value of repeat measurements of the predictive variable for genotype assignment.

Pancytopenia in mannosidosis.

A case of autoimmune pancytopenia is described in a patient with mannosidosis who developed anti-platelet and anti-neutrophil antibodies and a low haptoglobin level. Bone marrow biopsy and 111InCl3 bone marrow scanning demonstrated hypoplasia of the marrow with a paucity of "storage cells." Pathogenic mechanisms and implications for other inborn errors of metabolism are discussed.

Retrovirus vector-mediated correction and cross-correction of lysosomal alpha-mannosidase deficiency in human and feline fibroblasts.

Lysosomal alpha-mannosidase (EC 3.2.1.24) is an exoglycosidase in the glycoprotein degradation pathway. A deficiency of this enzyme causes the lysosomal storage disease alpha-mannosidosis. Retrovirus vector transfer of a new human alpha-mannosidase cDNA resulted in high-level expression of alpha-mannosidase enzymatic activity in deficient human and feline fibroblasts. The expressed alpha-mannosidase had the same biochemical properties (thermal stability, pH profile, inhibitor/activator sensitivity) as the native enzyme expressed in normal cells. The transferred enzyme colocalized with a control lysosomal hydrolase in cell fractionation experiments. The vector-encoded enzyme also was released at high levels from the corrected cells, and was taken up by untreated mutant cells via the mannose 6-phosphate receptor-mediated endocytic pathway (cross-correction). It is envisioned that genetic correction of a subset of cells (e.g., hematopoietic stem cells) in patients will provide a source of corrective enzyme for other affected tissues in this multisystem disease. Development of a vector expressing high levels of alpha-mannosidase that cross-corrects mutant cells will enable somatic gene transfer experiments in the cat model of human alpha-mannosidosis.

Caprine beta-mannosidosis: clinical and pathological features.

Beta-mannosidosis, an inherited defect of glycoprotein catabolism associated with deficiency of tissue beta-mannosidase and accumulation of Man(beta 1-4)GlcNAc and Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc, appeared in four of 13 offspring of a single pair of clinically normal, related Nubian goats. Neurological examinations revealed that all four affected goats were unable to rise or walk. All had facial dysmorphism, dome-shaped skulls, small palpebral fissures, carpal contractures, hyperextension of the pastern joints, proximal muscle atrophy, intermittent ocular oscillations resembling pendular nystagmus, marked intention tremor, and deafness. With intensive care, three affected kids were hand-reared and then killed at 1, 7, and 21 days of age. Macroscopically, there were paucity of myelin in the cerebral and cerebellar hemispheres and ventricular dilatation. Microscopically, the extent and distribution of cytoplasmic vacuolation, myelin paucity, axonal spheroids, and filamentous expansions were evaluated in the cerebrum, cerebellum, brainstem, spinal cord, and peripheral nerves of the four affected kids and two age-matched, clinically normal kids. Widespread cytoplasmic vacuolation correlated with the previously reported accumulation of oligosaccharides in the brain and kidney and the deficiency of tissue beta-mannosidase. beta-Mannosidosis, not yet identified in man or other species, is characterized by distinctive neonatal clinical, pathological and biochemical features which differentiate it from the alpha-mannosidosis and other inherited diseases of glycoprotein catabolism.

Spinal autonomic neurons in Werdnig-Hoffmann disease, mannosidosis, and Hurler's syndrome: distribution of autonomic neurons in the sacral spinal cord.

In Werdnig-Hoffman disease, mannosidosis, and Hurler's syndrome, two groups of neurons (the Onuf's and intermediomedial nuclei) in the ventral horn of the mid-sacral region are found to share common selective sparing or vulnerability with the intermediolateral nuclei of the thoracolumbar and sacral regions of the spinal cord. This finding confirms the previous observations on the characteristic involvement or sparing in Fabry's disease (14), Shy-Drager syndrome (17), amyotrophic lateral sclerosis, anterior poliomyelitis, and neuronal intranuclear hyaline inclusion disease (15), and supports the assumption that the Onuf's and intermediomedial nuclei in the ventral horn represent autonomic neurons much as the thoracolumbar and sacral intermediolateral nuclei.

Mannosidosis: pathology of the nervous system.

In a girl with mannosidosis, who died at 3 1/2 years of age, histopathological and ultrastructural changes in the nervous system are described. A widespread neuronal storage evidenced by ballooning of the neuronal perikarya is the salient histological feature and the occurrence of electron-lucent storage vacuoloes in neurons, astrocytes, endothelial cells and pericytes is the most striking ultrastructural feature of mannosidosis in the nervous system. By virtue of the deficiency of acidic alpha-mannosidases A and B, the accumulation of mannose-containing oligosaccharides in tissues and the occurrence of storage vacuoles in various cells, mannoisidosis is similar to various neuronal storage diseases associated with lysosomal enzyme deficiencies. In mannosidosis, the storage vacuoles in the neural and visceral tissues are alike with little variation in details and contain chiefly loosely dispersed, finely reticulogranular material. The storage vacuoles in neurons in mannosidosis are, therefore, distinct from those in neurons in other lysosomal storage disease such as Pompe's disease, various lipidoses and mucopolysaccharidoses. However, they resemble closely the storage vacuoles in neurons in fucosidosis and those in liver cells in various mucopolysaccharidoses.

"Paramyxovirus-like" intranuclear inclusions occurring in the nervous system in diverse unrelated conditions.

Filamentous intranuclear inclusions similar to the "paramyxovirus-like" intranuclear inclusions described in active multiple sclerosis (MS) were observed incidentally in four patients who had diverse disorders such as rabies, mannosidosis, metachromatic leukodystrophy, cerebral aneurysm and ischemic infarcts. The observation is in support of the mounting evidence that the "virus-like" intranuclear inclusions are not specific for MS and may occur frequently in a variety of diverse conditions. Moreover, neither virological nor immunological evidence as to the viral nature of the intranuclear inclusions has been presented to date, and the intranuclear inclusions have been observed mostly in degenerating or autolyzed cells in biopsied or autopsied tissue samples. In view of all the circumstantial evidence, it is suggested that the intranuclear "paramyxovirus-like" inclusions may represent an alternation of nuclear chromatin common to antemortem degeneration and postmortem autolysis in a variety of cells, the nature of which remains to be elucidated.

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.

Mannosidosis in three brothers--a review of the literature.

Three brothers with mannosidosis were studied, and their clinical and biochemical manifestations are compared with those of 41 cases in the literature. All three boys have psychomotor and growth retardation, characteristic facies, recurrent respiratory infections, sensorineural deafness, craniosynostosis, protuberant abdomens, and thin limbs. Roentgenographic findings of mild dysostosis multiplex, thick calvaria, abnormally contoured vertebrae, coarse trabeculi and thin cortices are consistent with those of reported cases. The lymphocytes of peripheral blood and bone marrow are vacuolated. Alpha-mannosidase deficiency in leukocytes and cultured skin fibroblasts and glycoproteinuria have been documented. The biochemistry of this glycoproteinosis and the pitfalls in diagnosis, such as improper assay conditions of pH and substrate concentration, are discussed. Extrapolation of in vitro and animal model studies suggest that trace metal therapy may be more effective than attempts at enzyme replacement to treat this hereditary storage disease.

Mannosidosis. New clinical presentation, enzyme studied, and carbohydrate analysis.

Mannosidosis is a rare inborn error of metabolism characterized by deficiency of the lysosomal enzyme alpha-mannosidase and widespread storage of complex carbohydrate, which is enriched in mannose. Two affected unrelated males, aged 6 and 26 years, are reported. Both had a nonprogressive encephalopathy with moderately severe mental retardation. The older patient showed several unique features, including massive gingival hyperplasia associated with histiocytes containing large amounts of a material with the staining characteristics of glycoprotein. The best determinant of mannose storage proved to be the ratio of mannose to other carbohydrates in urinary polysaccharides. The enzyme deficiency in this disease is most convincingly demonstrated at pH values below 4.0. The ability of zinc to activate the mutant enzyme in vitro offers a possible mode of therapy for this disease. Retarded individuals with a Hurler-like appearance and gum hyperplasia of unknown cause should be screened for alpha-mannosidase deficiency.