Specificity of the mannosyltransferase which initiates outer chain formation in Saccharomyces cerevisiae.

The in vitro specificity of the alpha 1-6 mannosyltransferase that initiates outer chain formation in Saccharomyces cerevisiae (Romero and Herscovics, J. Biol. Chem., 264, 1946-1950, 1989) was reassessed by fast atom bombardment mass spectrometry (FAB-MS). A particulate fraction from the mnn1 mutant was incubated with GDP-mannose and either Man9GlcNAc (M9T) isolated from thyroglobulin or Man8GlcNAc (M8Y) obtained by treatment of the M9T with the yeast specific mannosidase. The Man10GlcNAc (M10Y) and Man9GlcNAc (M9Y) oligosaccharides thus obtained, and the substrate oligosaccharides, were peracetylated or perdeuteroacetylated and submitted to FAB-MS using meta-nitrobenzylalcohol as the matrix. The latter was chosen as the matrix because it enhances the abundance of high-mass-fragment ions of peracetylated oligosaccharides and thereby facilitates the assignment of branching patterns. The results indicate that the alpha 1-6 mannosyltransferase catalyses the addition of mannose to the alpha 1-3 mannose residue, and thus provide additional new evidence to support the revised structure of yeast mannoproteins proposed by Hernandez et al. (J. Biol. Chem., 264, 11849-11856, 1989). [formula: see text] where Gn is N-acetylglucosamine, M is mannose and M is mannose added by the enzyme.     

Structural fingerprinting of Asn-linked carbohydrates from specific attachment sites in glycoproteins by mass spectrometry: application to tissue plasminogen activator

A sensitive and specific strategy has been developed for determining the sites of attachment of Asn-linked carbohydrates in glycoproteins, and defining the compositions and molecular heterogeneity of carbohydrates at each specific attachment site. In this carbohydrate 'fingerprinting' strategy, potential glycopeptides are identified by comparing the high pressure liquid chromatography (HPLC) chromatograms of proteolytic digests of a glycoprotein obtained before and after digestion with a glycosidase, usually peptide:N-glycosidase F (PNGase F). The glycopeptide-containing HPLC fractions are analyzed by fast atom bombardment mass spectrometry (FAB MS) prior to and after digestion with PNGase F to identify the former glycosylation site peptide and its sequence location (Carr and Roberts, (1986) Anal. Biochem. 157, 396-406). Carbohydrates are extracted from these fractions as the peracetates which are then permethylated and analyzed by FAB MS. The spectra exhibit molecular weight-related ions for each of the parent oligosaccharides present in the fraction which provide composition in terms of hexose, deoxyhexose, N-acetylhexosamine and sialic acid. The relative ratios of these peaks reflect the relative abundances of the various carbohydrate homologs present in the mixture. The derivatives formed are directly amenable to methylation analysis for determination of linkage. This strategy enables the structural classes of carbohydrates at specific attachment sites to be determined using only a few nmol of glycoprotein. The carbohydrate fingerprinting strategy has been applied to a number of glycoproteins including tissue plasminogen activator, the results for which are described herein.     

Structure of sialylated fucosyl lactosaminoglycan isolated from human granulocytes

Sialylated fucosyl lactosaminoglycan was isolated from human neutrophilic granulocytes and its structure was elucidated. The lactosaminoglycan glycopeptides were digested by endo-beta-galactosidase and "the core portion" and released oligosaccharides were analyzed by permethylation, fast atom bombardment mass spectrometry, and exoglycosidases. In addition, lactosaminoglycan saccharides were obtained by hydrazinolysis and the structures of fractionated sialyl oligosaccharides were analyzed by fast atom bombardment mass spectrometry and permethylation coupled with exoglycosidase treatment. The structure of one of the major components was found to be: (Formula: see text). This structure is unique in that 1) four linear polylactosaminyl side chains are attached to the core portion, 2) the side chain arising from position 4 of 2,4-linked mannose contains one or more alpha 1----3 fucosyl residues, 3) the side chain arising from position 6 of 2,6-linked mannose is terminated with NeuNAc alpha 2----3Gal(Fuc alpha 1----3)GlcNAc, sialyl Lex, and 4) the side chain arising from position 2 of 2,4-linked mannose is terminated with sialic acid through alpha 2----6 linkage.     

Mass spectrometry of high-mannose oligosaccharides after trifluoroacetolysis and periodate oxidation.

High-mannose oligosaccharides were treated with a mixture of trifluoroacetic acid and trifluoroacetic anhydride under conditions where reducing terminal N-acetylglucosamine residues are specifically degraded. The resulting mannose-containing products were, after further chemical modifications, analyzed by mass spectrometry in fast atom bombardment and electron ionization modes. Binding positions between monosaccharide residues were deduced from mass spectra of peracetylated compounds, which, prior to the derivatization, had been subjected to periodate oxidation and borodeuteride reduction.     

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: II. The primary structures of the low molecular weight carbohydrate chains of the glycoproteins.

The primary structures of the O-glycosidically linked oligosaccharides isolated from glycoproteins GP I and GP II of Fusarium sp. M7-1 were established. The oligosaccharides released by alkaline borohydride treatment from the glycoproteins were purified by Bio-Gel P-4 and HPLC. This approach resulted in one monosaccharide and seven oligosaccharides. Their primary structures were resolved mainly by NMR spectrometry in combination with methylation mass spectrometry and fast atom bombardment mass spectrometry. The following structures have been determined. [formula: see text].     

Determination of the carbohydrate composition of mammalian glycoproteins by capillary gas chromatography/mass spectrometry

A technique to determine the carbohydrate composition of glycoproteins using capillary gas chromatography/mass spectrometry (electron impact) with selected ion monitoring is described. This method entails hydrolysis with methanolic-HCl followed by formation of trimethylsilyl methylglycoside derivatives, extraction of the carbohydrate derivatives into hexane, and GC/MS analysis. For those carbohydrates that are present in animal glycoproteins including fucose, mannose, galactose, glucosamine, galactosamine, and N-acetylneuraminic acid (sialic acid), the sensitivity of this assay was approximately 1-3 pmol and the assay was linear over a 100-fold range. The carbohydrate compositions determined on small quantities (1-10 pmol) of various glycoproteins including human transferrin and alpha-1 acid glycoprotein, fetuin, and ovalbumin were identical to their reported carbohydrate content and compositions. Major advantages of this technique include the time required to complete the sample preparation and analysis (less than 8 h), the sensitivity and specificity of the assay, and the fact that all carbohydrate moieties, including sialic acid, can be quantitated in a single hydrolysate of a glycoprotein.     

Carbohydrate mapping by mass spectrometry: a novel method for identifying attachment sites of Asn-linked sugars in glycoproteins

A new method is described for locating the specific sites of attachment of Asn-linked carbohydrates in glycoproteins. The molecular weights of peptides released from the glycoprotein with proteases of known specificity are determined by fast atom bombardment mass spectrometry and fitted to the known or DNA-derived sequence. Oligosaccharides attached to Asn are released either before or after proteolysis with a glycosidase, usually peptide: N-glycosidase F, an enzyme that cleaves the beta-aspartylglycosylamine linkage of all known types of Asn-linked sugars and converts the attachment-site Asn to Asp. New peaks appearing in the mass spectra after treatment with glycosidase correspond to formerly glycosylated sites. Conversely, signals which disappear after glycosidase treatment correspond to glycopeptides. The differences in mass between these sets of signals define the composition of the carbohydrate at the given site in terms of deoxyhexose, hexose, N-acetylhexosamine, and sialic acid content. The extent of glycosylation at a given site can be estimated from the ratio of the peak heights corresponding to the Asn- vs Asp-containing peptides which differ by 1 Da in mass. This rapid and sensitive (low nmol) technique is illustrated here for ribonuclease B and for tissue plasminogen activator, a multiply glycosylated glycoprotein.     

Glycoproteins of chicken liver nuclei cross-linked to DNA by cis-diamminedichloroplatinum

Glycoproteins which participate in DNA-protein cross-links induced by action of cis-diamminedichloroplatinum (cis-DDP) in intact nuclei of chicken liver were investigated. Digoxigenin-labelled lectins with different sugar specificity were used for detection and characterization of these glycoproteins. Our results showed the presence of glycoproteins bearing high mannose as well as complex type oligosaccharides in chicken liver nuclei. In most cases of complex oligosaccharides, sialic acid residues bound in alpha(2-6) but not in alpha(2-3) linkage were present.     

A novel sialylated N-acetylgalactosamine-containing oligosaccharide is the major complex-type structure present in Bowes melanoma tissue plasminogen activator.

We have employed fast atom bombardment mass spectrometry (FAB-MS) to screen the N-linked oligosaccharides of Bowes melanoma tissue plasminogen activator (mt-PA), and recombinant t-PAs produced by Chinese hamster ovary cells (rt-PA) and by a gene-enriched melanoma cell line (rmt-PA). These studies have confirmed the published structures for rt-PA, but are not in agreement with some of the structures reported for mt-PA. In the latter glycoprotein we have identified a novel structure as the major oligosaccharide attached to Asn-184 and Asn-448. This is a biantennary oligosaccharide consisting of a fucosylated trimannosyl core to which are attached two GalNAc(1----4)GlcNAc antennae, one of which carries a sialic acid linked at the 6-position of the GalNAc. Minor constituents are sialylated on both or neither antennae. The sialylated GalNAc moiety is unique in N-linked glycoproteins. The majority of complex structures in rmt-PA contain N-acetyllactosamine moieties at both the Asn-184 and Asn-448 sites with the novel oligosaccharide occurring as a minor component at the Asn-184 site. This study demonstrates the power of mass spectrometric strategies based on high-field two-sector FAB-MS for structure elucidations of natural and recombinant glycoproteins.     

Fucose containing oligosaccharides from human milk: I. Separation and identification of new constituents

Neutral oligosaccharides isolated from pooled human milk were subjected to fractionation on high-performance thin-layer chromatography (HPTLC) plates, Iatrobeads, and reverse-phase chromatography after borohydride reduction and peracetylation. By the combined HPLC and HPTLC separation a mixture of pooled human milk oligosaccharides was separated into 101 fractions. These fractions were characterized by field desorption or fast atom bombardment (FAB)-mass spectrometry. Each of the carbohydrate constituents, the peracetylated glucitol, the galactose, the glucosamine, and the fucose contribute specific mass increments to the molecular weight of the oligosaccharide. Therefore, the exact carbohydrate composition can be calculated from the molecular weight determined by mass spectrometry. Among the fractions obtained one trifucosyl-lacto-N-tetraose, five monofucosyl-, eleven difucosyl-, and nine trifucosyl-lacto-N-hexaoses, one monofucosyl-, eight difucosyl-, seven trifucosyl-, four tetrafucosyl-, and two pentafucosyl-lacto-N-octaoses, one trifucosyl-, and two difucosyl-lacto-N-decaoses could be identified. FAB spectra furnished additional data on structural features of the isolated oligosaccharides.     

Neutral oligosaccharides in the urine of a patient with glycogen storage disease type II

Neutral oligosaccharides were isolated from urine of an adult patient with glycogen storage disease type II, a deficiency of lysosomal acid alpha-glucosidase, by chromatography on columns of activated charcoal, Dowex 50 X 2 and Dowex 1 X 2. Total neutral oligosaccharides in the urine of the patient were increased about 5-fold as compared with those in normal controls. The most accumulated oligosaccharide was separated by Bio-Gel P-2 column chromatography, and finally purified by paper chromatography. Based on various studies, including carbohydrate analysis, chemical ionization mass spectrometry, fast atom bombardment mass spectrometry, degradation by glucoamylase and isopullulanase, and methylation analysis, the structure of this oligosaccharide was deduced to be Glc alpha 1----6Glc alpha 1----4Glc alpha 1----4Glc. This oligosaccharide appears to be accumulated in urine of the patient with acid alpha-glucosidase deficiency as an end product of the hydrolysis of glycogen.     

Structural studies on glycoprotein oligosaccharides of chromaffin granule membranes and dopamine beta-hydroxylase

Dopamine beta-hydroxylase present in the soluble matrix of bovine adrenal medullary chromaffin granules contains biantennary complex oligosaccharides and high-mannose oligosaccharides in a molar ratio of approximately 2:1. The high-mannose oligosaccharides contain an average of six mannose residues. The largest biantennary oligosaccharides (40% of the total) have two complete peripheral branches consisting of sialic acid-galactose-N-acetylglucosamine, but an equal proportion lack sialic acid on one branch and the remainder lack N-acetylglucosamine and/or galactose. Affinity chromatography on lentil lectin-agarose demonstrated that 84% of the dopamine beta-hydroxylase biantennary oligosaccharides are substituted by fucose on the core N-acetylglucosamine which is linked to asparagine. Based on carbohydrate concentration and the proportions of biantennary and high-mannose oligosaccharides, it would appear that the four dopamine beta-hydroxylase subunits of Mr congruent to 75,000 are not identical with respect to their oligosaccharide moieties. In chromaffin granule membranes, high-mannose and biantennary oligosaccharides comprise 20 and 35%, respectively, of the glycoprotein carbohydrate. Almost 40% is present in the form of large complex oligosaccharides with three or more antennas, less than 3% of which have both a core fucose residue and a 2,6-substituted alpha-linked mannose residue. Chromaffin granule membranes also contain a small proportion (approximately 6%) of O-glycosidically linked glycoprotein oligosaccharides which are predominantly monosialyl derivatives of galactosyl-N-acetylgalactosamine. The ratio of N-acetyl- to N-glycolylneuraminic acid in dopamine beta-hydroxylase and the glycoproteins of chromaffin granule membranes is approximately 1.5:1, which is within the same range as that previously found in membrane gangliosides and in the chromogranins isolated from the soluble granule matrix.     

The carbohydrate structures of a mouse monoclonal IgG antibody OKT3

A mouse monoclonal antibody OKT3, of IgG2a isotype, was isolated from hybridoma culture fluid. Sugar analysis showed the presence of sialic acid, galactose, mannose, fucose, and N-acetylglucosamine, i.e. sugars typical for N-glycosidically linked carbohydrate chains. The absence of N-acetylgalactosamine revealed that O-glycosidically linked carbohydrates were not present. The purified antibody was reduced, alkylated, and separated into heavy and light chains, and all carbohydrates were shown to be associated with the heavy chains. The N-linked carbohydrate chains were isolated as alditols using strong alkaline-borohydride degradation and further fractionated on a concanavalin A-Sepharose column and high performance ion exchange chromatography with pulsed amperometric detection. Structural analysis was carried out on the isolated oligosaccharide alditols by chemical analyses, fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. Triantennary and biantenary types of structures were found. The triantennary structures were present as trisialo and tetrasialo forms without fucose; the tetrasialo forms were shown to contain a sequence of Neu5Ac alpha 2-3Gal beta 1-3[Neu5Ac alpha 2-6]GlcNAc beta 1- on one of the branches. The biantennary structures were present as completely sialylated nonfucosylated species and as asialo-, agalacto-, and partially fucosylated structures.     

Glycoproteins of Sindbis Virus: Preliminary Characterization of the Oligosaccharides

The carbohydrate content of Sindbis virus was determined by gas chromatographic analysis. The two viral glycoproteins were found to be approximately 8% carbohydrate by weight. Mannose is the sugar present in the largest amount. Smaller amounts of glucosamine, galactose, sialic acid, and fucose were also detected. Each of the two viral glycoproteins appears to contain two structurally unrelated oligosaccharides. Two of the three Sindbis-specific glycoproteins found in infected chick cells were shown to contain short, unfinished oligosaccharides.     

Evidence for the presence of complex carbohydrates in Candida albicans cell wall glycoproteins

In order to test the hypothesis that cell wall glycoproteins of Candida albicans contained non-mannan oligosaccharides, the sugar composition of cell wall extracts and fractions of cell wall extracts was examined by means of fluorophore-assisted carbohydrate electrophoresis (FACE). In addition to the expected mannose, glucose, and N-acetyl-glucosamine, this analysis showed the presence of galactose, N-acetyl-galactosamine, fucose, and sialic acid and two unknown sugars. These sugars are also associated with complex oligosaccharides of mammalian glycoproteins. Presence of fucosylated cell wall components was further demonstrated by lectin-blotting analysis of cell wall extracts. Besides their structural role, complex carbohydrate structures on the surface of C. albicans may represent additional motifs through which interactions of this fungus with host cells and tissues could be established.     

Oligosaccharide sequence determination using B/E linked field scanning or tandem mass spectrometry of phosphatidylethanolamine derivatives

Product ion mass spectral data of [M + H]+ ions of oligosaccharides, mainly tetra- and pentasaccharides, as their dipalmitoyl phosphatidylethanolamine derivatives were obtained using both liquid secondary ion mass spectrometry with B/E linked scanning and fast atom bombardment ionization with collision-induced dissociation/tandem mass spectrometry. Both methods give similar positive product ion spectra of equivalent high sensitivity (detection limits of approximately 50 pmol) that principally contain glycosidic cleavage ions retaining the reducing end of the molecule from which monosaccharide sequence can be deduced. A series of ions from fission of the phosphate ester bond together with glycosidic cleavage are present in the tandem mass spectra and B/E linked scan spectra when helium collision gas is used. Monosaccharide linkage position of isomeric molecules is reflected in the intensity of glycosidic fragmentation, without retention of the oxygen atom, with decreasing cleavage in the order 1-3 greater than 1-4 greater than 1-6 linkage. Fucose and N-acetylhexosamines show an increased degree of fragmentation over hexose sugars. The application of product ion spectra of derivatized oligosaccharides is demonstrated for characterizing mixed samples and also the acquisition of spectra directly from the silica surface of high-performance thin-layer chromatography plates.     

Lectin affinity fractionation of asparagine-linked oligosaccharides from normal human and chronic leukemic leukocytes

Asparagine-linked oligosaccharides were isolated from normal and chronic leukemic leukocytes (normal neutrophils, normal lymphocytes, chronic myeloid, chronic lymphoid and hairy cell leukemic leukocytes) and analyzed by sequential lectin affinity column chromatography. The neutral and sialylated glycopeptides ranged in size from 1,800 to 4,000 da. on gel filtration. Sequential lectin affinity analysis was then used to fractionate the Asn-oligosaccharides into major structural classes of high mannose, hybrid, and bi-, tri- and tetraantennary complex structures. Using lectins of well defined specificity, the sequential chromatography provided a satisfactory means of assessing the overall glycopeptide profiles of the different leukocyte types. Results from 10 patient samples show that alterations in leukocyte Asn-oligosaccharides occur during leukemogenesis. Most notable was an average twofold increase in the relative amount of high mannose glycopeptides compared to complex glycopeptides for the leukemic cells. High mannose glycopeptides comprised 8.6 percent of the total lectin-adherent glycopeptides from leukemics, and 4.2 percent in the normals. In addition, carbohydrate analysis has revealed that the total amount of neutral hexose was markedly decreased in all leukemic samples. Leukemics ranged from 10.5 to 18.8, while normals ranged from 24.2 to 49.2 nanomole of hexose per 100 micrograms protein. The sialic acid content of the leukemic glycopeptides was relatively unchanged from that of normals, resulting in an apparent increase in the sialic acid: hexose ratio for all leukemic glycopeptides. The results suggest that in the leukemic cells, high mannose structures constitute a larger proportion of the total Asn-linked oligosaccharides, while the overall level of protein glycosylation is decreased. Complex multiantennary glycopeptides, when synthesized, tended to be more fully sialylated than their normal counterparts.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing.

The biosynthesis of the various types of N-linked oligosaccharide structures involves two series of reactions: 1) the formation of the lipid-linked saccharide precursor, Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol, by the stepwise addition of GlcNAc, mannose and glucose to dolichyl-P, and 2) the removal of glucose and mannose by membrane-bound glycosidases and the addition of GlcNAc, galactose, sialic acid, and fucose by Golgi-localized glycosyltransferases to produce different complex oligosaccharide structures. For most glycoproteins, the precise role of the carbohydrate is still not known, but specific N-linked oligosaccharide structures are key players in targeting of lysosomal hydrolases to the lysosomes, in the clearance of asialoglycoproteins from the serum, and in some cases of cell:cell adhesion. Furthermore, many glycoproteins have more than one N-linked oligosaccharide, and these oligosaccharides on the same protein frequently have different structures. Thus, one oligosaccharide may be of the high-mannose type whereas another may be a complex chain. One approach to determining the role of specific structures in glycoprotein function is to use inhibitors that block the modification reactions at different steps, causing the cell to produce glycoproteins with altered carbohydrate structures. The function of these glycoproteins can then be assessed. A number of alkaloid-like compounds have been identified that are specific inhibitors of the glucosidases and mannosidases involved in glycoprotein processing. These compounds cause the formation of glycoproteins with glucose-containing high mannose structures, or various high-mannose or hybrid chains, depending on the site of inhibition. These inhibitors have also been useful for studying the processing pathway and for comparing processing enzymes from different organisms.