Chemical structures of oligosaccharides in milk of the raccoon (<Emphasis Type="Italic">Procyon lotor</Emphasis>)

In this study on milk saccharides of the raccoon (Procyonidae: Carnivora), free lactose was found to be a minor constituent among a variety of neutral and acidic oligosaccharides, which predominated over lactose. The milk oligosaccharides were isolated from the carbohydrate fractions of each of four samples of raccoon milk and their chemical structures determined by ¹H-NMR and MALDI-TOF mass spectroscopies. The structures of the four neutral milk oligosaccharides were Fuc(α1–2)Gal(β1–4)Glc (2′-fucosyllactose), Fuc(α1–2)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (lacto-N-fucopentaose IV), Fuc(α1–2)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (fucosyl para lacto-N-neohexaose) and Fuc(α1–2)Gal(β1–4)GlcNAc(β1–3)[Fuc(α1–2)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (difucosyl lacto-N-neohexaose). No type I oligosaccharides, which contain Gal(β1–3)GlcNAc units, were detected, but type 2 saccharides, which contain Gal(β1–4)GlcNAc units were present. The monosaccharide compositions of two of the acidic oligosaccharides were [Neu5Ac]₁[Hex]₆[HexNAc]₄[deoxy Hex]₂, while those of another two were [Neu5Ac]₁[Hex]₈[HexNAc]₆[deoxy Hex]_3. These acidic oligosaccharides contained α(2–3) or α(2–6) linked Neu5Ac, non reducing α(1–2) linked Fuc, poly N-acetyllactosamine (Gal(β1–4)GlcNAc) and reducing lactose.     

Analysis of folate binding protein N-linked glycans by mass spectrometry

The folate binding protein (FBP), also known as the folate receptor (FR), is a glycoprotein which binds the vitamin folic acid and its analogues. FBP contains multiple N-glycosilation sites, is selectively expressed in tissues and body fluids, and mediates targeted therapies in cancer and inflammatory diseases. Much remains to be understood about the structure, composition, and the tissue specificities of N-glycans bound to FBP. Here, we performed structural characterization of N-linked glycans originating from bovine and human milk FBPs. The N-linked glycans were enzymatically released from FBPs, purified, and permethylated. Native and permethylated glycans were further analyzed by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS), while tandem MS (MS/MS) was used for their structural characterization. The assignment of putative glycan structures from MS and MS/MS data was achieved using Functional Glycomics glycan database and SimGlycan software, respectively. It was found that FBP from human milk contains putative structures that have composition consistent with high-mannose (Hex(5-6)HexNAc(2)) as well as hybrid and complex N-linked glycans (NeuAc(0-1)Fuc(0-3)Hex(3-6)HexNAc(3-5)). The FBP from bovine milk contains putative structures corresponding to high-mannose (Hex(4-9)HexNAc(2)) as well as hybrid and complex N-linked glycans (Hex(3-6)HexNAc(3-6)), but these glycans mostly do not contain fucose and sialic acid. Glycomic characterization of FBP provides valuable insight into the structure of this pharmacologically important glycoprotein and may have utility in tissue-selective drug targeting and as a biomarker.     

N-Glycosylation of the MUC1 mucin in epithelial cells and secretions

The MUC1 mucin is an important tumor-associated antigen that shows extensive glycosylation in vivo. The O-glycosylation of this molecule, which has been well characterized in many cell types and tissues, is important in conferring the unusual biochemical and biophysical properties on a mucin. N-Glycosylation is crucial to the folding, sorting, membrane trafficking, and secretion of many proteins. Here, we evaluated the N-glycosylation of MUC1 derived from two sources: endogenous MUC1 isolated from human milk and a recombinant epitope-tagged MUC1F overexpressed in Caco2 colon carcinoma cells. N-Glycans on purified MUC1F/MUC1 were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), gas chromatography-mass spectrometry (GC-MS), and CAD-ESI-MS/MS. The spectra indicate that MUC1F N-glycans have compositions consistent with high-mannose structures (Hex(5-9)HexNAc(2)) and complex/hybrid-type glycans (NeuAc(0-3)Fuc(0-3)Hex(3-8)HexNAc(3-7)). Many of the N-glycan structures are identical on MUC1F and native MUC1; however, a marked difference is seen between the N-glycans on membrane-bound and secreted forms of the native molecule.     

The sperm agglutination antigen-1 (SAGA-1) glycoforms of CD52 are O-glycosylated

CD52 is composed of a 12 amino acid peptide with N-linked glycans bound to the single potential glycosylation site at position 3, and a glycosylphosphatidylinositol-anchor attached at the C-terminus. Some glycoforms of this molecule expressed in the male reproductive tract are recognized by complement-dependent sperm-immobilizing antibodies in infertile patients making this antigen an important target for immunocontraception and fertility studies. Although the amount of posttranslational modification is already remarkable for such a small polypeptide, O-glycosylation of CD52 has additionally been implicated by several studies, but never rigorously characterized. In this report, we show clear evidence for the presence of O-glycans in CD52 preparations immunopurified using the murine S19 monoclonal antibody generated against sperm agglutination antigen-1 (SAGA-1), a male reproductive tract specific form of CD52. The O-glycans have been characterized by MALDI-TOF and tandem mass spectrometry after reductive elimination and permethylation. The data indicate that the major SAGA-1 O-glycans are core 1 and 2 mucin-type structures, with and without sialic acid (NeuAc(0-2)Hex(1-3)HexNAc(1-2)HexNAcitol). Minor fucosy- lated O-glycans are also present including some struc- tures with putative Le(y) epitopes (NeuAc(0-1)Fuc(1-3)Hex(1-2) HexNAc(0-1)HexNAcitol). Analysis of O-glycopeptides by tandem mass spectrometry provided an additional level of support for the O-glycosylation of SAGA-1. Elucidation of the O-glycosylation of SAGA-1 adds to the complexity of this molecule and may help to explain its biological activity.     

High-mannose glycans are elevated during breast cancer progression

Alteration in glycosylation has been observed in cancer. However, monitoring glycosylation changes during breast cancer progression is difficult in humans. In this study, we used a well-characterized transplantable breast tumor mouse model, the mouse mammary tumor virus-polyoma middle T antigen, to observe early changes in glycosylation. We have previously used the said mouse model to look at O-linked glycosylation changes with breast cancer. In this glycan biomarker discovery study, we examined N-linked glycan variations during breast cancer progression of the mouse model but this time doubling the number of mice and blood draw points. N-glycans from total mouse serum glycoproteins were profiled using matrix-assisted laser desorption/ionization Fourier transform-ion cyclotron resonance mass spectrometry at the onset, progression, and removal of mammary tumors. We observed four N-linked glycans, m/z 1339.480 (Hex(3)HexNAc), 1485.530 (Hex(3)HexNAc(4)Fuc), 1809.639 (Hex(5)HexNAc(4)Fuc), and 1905.630 (Man(9)), change in intensity in the cancer group but not in the control group. In a separate study, N-glycans from total human serum glycoproteins of breast cancer patients and controls were also profiled. Analysis of human sera using an internal standard showed the alteration of the low-abundant high-mannose glycans, m/z 1419.475, 1581.528, 1743.581, 1905.634 (Man(6-9)), in breast cancer patients. A key observation was the elevation of a high-mannose type glycan containing nine mannoses, Man(9), m/z 1905.630 in both mouse and human sera in the presence of breast cancer, suggesting an incompletion of the glycosylation process that normally trims back Man(9) to produce complex and hybrid type oligosaccharides.     

Strategy for the investigation of O-linked oligosaccharides from mucins based on the separation into neutral, sialic acid- and sulfate-containing species

A method for the separation of O-linked oligosaccharides into neutral, sialic acid-containing and sulfated species was applied to oligosaccharides released by alkaline borohydride from mucin glycopeptides from porcine small intestine. The released mixture of reduced oligosaccharides was applied to an anion exchange column, and the neutral oligosaccharides were collected as the unretarded fraction. A mixture of dimethyl sulfoxide and iodomethane was passed through the column to convert the sialic acid-containing oligosaccharides into methyl esters that were eluted and converted to methyl amides by methyl amine. Finally the sulfated oligosaccharide fraction was eluted with salt. The neutral and the derivatized sialic acid-containing oligosaccharides were analysed by gas chromatography-mass spectrometry after permethylation and the sulfated oligosaccharide fraction was analysed by high performance anion exchange chromatography.Abbreviations GC gas chromatography - GC/MS gas chromatography-mass spectrometry - HPAEC-PAD high performance anion exchange chromatography-pulsed amperometric detection - Hex hexose - HexNAc N-acetyl hexosamine - HexNAcol N-acetyl hexosaminitol - Fuc Fucose - NeuAc N-acetyl neuraminic acid - NeuGc N-glycolyl neuraminic acid     

Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry

High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.     

Characterization of glycosphingolipids from Schistosoma mansoni eggs carrying Fuc(alpha1-3)GalNAc-, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc- and Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc- (Lewis X) terminal structures.

The carbohydrate moieties of glycosphingolipids from eggs of the human parasite, Schistosoma mansoni, were enzymatically released, labelled with 2-aminopyridine (PA), fractionated and analysed by linkage analysis, partial hydrolysis, enzymatic cleavage, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano-electrospray ionization mass spectrometry. Apart from large, highly fucosylated structures with five to seven HexNAc residues, we found short, oligofucosylated species containing three to four HexNAc residues. Their structures have been determined as Fuc(alpha1-3)GalNAc(beta1-4)[ +/- Fuc (alpha1-3)]GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4) Glc-PA, Fuc(alpha1-3)GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-4) GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, and Fuc(alpha1-3) GalNAc(beta1-4)[ +/- Fuc(alpha1-2) +/- Fuc(alpha1-2)Fuc(alpha1-3)]Glc NAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA. The last structure exhibits a trifucosyl sidechain previously identified on the cercarial glycocalyx. These structures stress the importance of 3-fucosylated GalNAc as a terminal epitope in schistosome glycoconjugates. To what degree these glycans contribute to the pronounced antigenicity of S. mansoni egg glycolipids remains to be determined. In addition, we have identified the compounds GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3) GalNAc (beta1-4)Glc-PA, the latter of which is a Lewis X-pentasaccharide identical to that present on cercarial glycolipids, as well as Gal(beta1-3)GalNAc(1-4)Gal(1-4)Glc-PA, which corresponds to asialogangliotetraosylceramide and is most probably derived from the mammalian host.     

Structure elucidation of sulphated oligosaccharides from recombinant human tissue plasminogen activator expressed in mouse epithelial cells.

Sulphated N-linked carbohydrate chains isolated from recombinant human tissue plasminogen activator expressed in mouse epithelial (C127) cells were analysed as oligosaccharide alditols by methylation analysis, liquid secondary ion mass spectrometry, and one- and two-dimensional 1H-NMR spectroscopy. The results demonstrate that the major component has the following novel structure: NeuAc-alpha 2-6Gal beta 1-4GlcNAc beta 1-2[NeuAc alpha 2-3Gal beta 1- 4GlcNAc beta 1-4]-Man alpha 1-3[NeuAc alpha 2-3(SO4-6)Gal beta 1- 4-GlcNAc beta 1-2Man alpha 1-6]-Man beta 1-4GlcNAc beta 1- 4[Fuc alpha 1-6]GlcNAc-o1.     

Occurrence of tetra- and pentasaccharides with the sialyl-Le(a) structure in human milk

The occurrence of two novel oligosaccharides in human milk was investigated. These oligosaccharides were purified by affinity chromatography on a column of an immobilized monoclonal antibody, MSW 113. Structural studies, involving 500-MHz 1H NMR spectroscopy and fast atom bombardment-mass spectrometry, indicated the structures of these compounds to be NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNAc and NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNac beta 1----3Gal. This constitutes the first evidence for the occurrence of N-acetylglucosamine or galactose as the reducing-end residue of human milk oligosaccharides. These two oligosaccharides bound MSW 113 to nearly the same extent as sialyl-Lea hexasaccharide but to another sialyl-Le(a) structure-directed monoclonal antibody, NS-19-9, only weakly.     

Structural basis of the heterogeneity of asparagine-linked oligosaccharides of a Waldenstrom's macroglobulinemia immunoglobulin M

The extent of glycans heterogeneity in a pathological human immunoglobulin M ZAJ has been studied on oligosaccharides released by hydrazinolysis from the purified glycoprotein. After reduction with NaB3H4, asparagine-linked carbohydrate chains were separated by affinity chromatography on concanavalin A-Sepharose into oligomannosidic and N-acetyllactosaminic types. Glycans of the oligomannosidic type were further fractionated by HPLC and those of the N-acetyllactosamine type by preparative high-voltage electrophoresis. The primary structure of the main oligosaccharides was investigated on the basis of micro-methylation analysis, mass spectrometry and sequential exo-glycosidase digestion. Glycans of the oligomannosidic type varied in size from Man5GlcNAc2 to Man9GlcNAc2. N-Acetyllactosaminic glycans were found of the biantennary, bisected-biantennary and triantennary types. They presented a higher degree of heterogeneity due to the presence of a variable number of NeuAc and fucose residues. The new structures we report here were in addition to the major biantennary one we previously described on the basis of methylation analysis and 500 MHz 1H-NMR spectroscopy (Cahour, A., Debeire, P., Hartmann, L., Montreuil, J., Van Halbeek, H. and Vliegenthart, J.F.G. (1984) FEBS Lett. 170, 343-349): NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[Gal(beta 1-4)Glc-NAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)]Glc-NAc(beta 1-4) [Fuc(alpha 1-6)]GlcNAc.     

Structural studies on a binding site for Dolichos biflorus agglutinin in the small intestine of the mouse

Glycoproteins which bound to Dolichos biflorus agglutinin (DBA) were isolated from the small intestine of 129/Sv mice. Among oligosaccharides released from the carbohydrate moieties of the glycoproteins by endo-beta-galactosidase, the major one with N-acetylgalactosamine at the non-reducing end was isolated by QAE-Sephadex A-25 column chromatography. The structure of the oligosaccharide was elucidated to be GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc beta 1----3Gal by compositional analysis, methylation analysis before and after mild acid hydrolysis, sequential glycosidase digestion, secondary ion mass spectrometry (SIMS), and nuclear magnetic resonance spectroscopy. The SIMS signal of m/z 1,071 was consistent with the presence of the branched sequence, GalNAc(NeuAc)GalGlcNAc, and the signal was also detected in the high-molecular-weight fraction obtained after endo-beta-galactosidase digestion. The pentasaccharide identified here has the terminal structure of ganglioside GM2, and an apparently identical one has been identified as the epitope of blood group Sda and the DBA binding site in human T-H urinary glycoprotein. Thus, the present result has extended our knowledge of the biological meaning of the oligosaccharide structure and has established that GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc is a DBA binding site in the small intestine of the mouse.     

Structural studies of the sugar chains of human parotid alpha-amylase

Human parotid amylase can be separated into three families of isoenzymes (A', A, and B) by Sephadex G-75 column chromatography. Isoenzymes in family B were free from carbohydrate, while those in family A were all glycoproteins. The carbohydrate moieties of family A isoenzymes were released from their polypeptide portions by hydrazinolysis and labeled by reduction with NaB[3H]4. The yield of total radioactive oligosaccharides indicated that family A isoenzymes all contain single asparagine-linked sugar chains in one molecule. The radioactive oligosaccharides were fractionated into one acidic and two neutral oligosaccharide fractions by paper electrophoresis and paper chromatography. By sequential exoglycosidase digestion in combination with a methylation study, their structures were determined to be: Gal beta 1 leads to 4 (Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6 and 3[Gal beta 1 leads to 4 GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 and 6]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc Gal beta 1 leads to 4(Fuc alpha 1 leads to 3) GlcNAc beta 1 leads to 2Man alpha 1 leads to 6 (NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc.     

Structural characterization of fucose-containing oligosaccharides by high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Eight pyridylamino (PA) derivatives of fucose-containing oligosaccharides, which occur as free oligosaccharides in human milk and also are derived from glycosphingolipids, have been analyzed by high-performance liquid chromatography (HPLC) on normal-phase and reversed-phase columns, and by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Six out of eight PA-oligosaccharides were clearly separated by both normal- and reversed-phase HPLC at a column temperature of 40 degrees C, but two PA-oligosaccharides, lacto-N-fucopentaose II [Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-3Gal beta1-4GIcPA] and lacto-N-fucopentaose III [Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-3Gal beta1-4GIcPA], were not separated. The two unresolved PA-oligosaccharides were finally separated by reversed-phase HPLC at a column temperature of 11 degrees C. MALDI-TOF mass spectra of PA-oligosaccharides demonstrated pseudo-molecular ions as the predominant signals, therefore information about the molecular mass of each PA-oligosaccharide was easily obtained. Post-source decay (PSD) MALDI-TOF mass spectra of PA-oligosaccharides gave information about the carbohydrate sequences and carbohydrate species of each PA-oligosaccharide by detecting the ions responsible for the cleavage of the glycosidic bonds. The detection limits of the PA-oligosaccharides by HPLC, MALDI-TOF mass spectrometry, and PSD MALDI-TOF mass spectrometry were 20 fmol, 20 fmol, and 2 pmol, respectively. These results suggest that a system including HPLC and MALDI-TOF mass spectrometry or HPLC and PSD MALDI-TOF mass spectrometry is quite useful for the structural characterization of sub-pmol or pmol levels of fucose-containing oligosaccharides, and that these methods could be used for the analysis of various types of oligosaccharides derived from glycoproteins and glycosphingolipids.     

Sulfated N-linked carbohydrate chains in porcine thyroglobulin

N-linked carbohydrate chains of porcine thyroglobulin were released by the hydrazinolysis procedure. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis, the acidic fractions were further separated by high-performance liquid chromatography on Lichrosorb-NH2, and analyzed by 500-MHz 1H-NMR spectroscopy and, partially, by permethylation analysis. Of the acidic oligosaccharide-alditols, the following sulfated carbohydrate chains could be identified: NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3[(SO3Na----3)Gal beta 1----4GlcNAc beta1----2-Mana alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc-ol and NeuAc alpha 2----6Gal beta 1----4(SO3Na----)0-1 GlcNAc beta 1----2-Man alpha 1----3[NeuAc alpha 2----6Gal beta 1----4(SO3Na----6)1-0GlcNAc beta 1----2Man alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc- ol. The sulfated structural elements for porcine thyroglobulin form novel details of N-linked carbohydrate chains. They contribute to the fine structure of these oligosaccharides and are another type of expression of microheterogeneity.     

Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry. The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing beta1-2 xylosyl and/or alpha1-3 fucosyl residue(s) and occurrence of beta1-4GlcNAc residue in the insect glycoproteins. The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man(9 to approximately 4)GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3 GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1 Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3 Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of beta-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a beta1-4 linkage N-acetylglucosaminyl residue.     

Structural studies on the carbohydrate moieties of human liver alpha-L-fucosidase

alpha-L-Fucosidase was purified from human liver to apparent homogeneity and subjected to exhaustive digestion with Pronase. The resulting glycopeptides were isolated by gel filtration on Sephadex G-50 and further fractionated by Bio-Gel P-4 chromatography. Five glycopeptide fractions were obtained. The structures of the carbohydrate portions of all glycopeptide components were fully characterized by a combination of 500-MHz 1H NMR spectroscopy and carbohydrate composition analysis. Fraction I contained disialyl diantennary glycopeptides of the N-acetyllactosamine type. Fractions II and III contained predominantly mono(sialyl-N-acetyllactosaminyl) diantennary glycopeptides with the NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta(1----2) branch attached to alpha(1----3)-linked Man in II and to alpha(1----6)-linked Man in III. The N-acetyllactosamine-type glycopeptides in fractions I to III have a small portion (10-15%) of their Asn-linked GlcNAc residues substituted by additional alpha(1----6)-linked Fuc. Also, a minor portion of the NeuAc residues appeared to be attached to Gal in alpha(2----3) rather than alpha(2----6) linkage. Fraction IV contained a mixture of larger-size oligomannoside-type glycopeptides with a variable number (6 to 9) of Man residues. Smaller-size oligomannoside-type glycopeptides were found in fraction V, containing 3 or 5 Man residues; a small portion (10%) of the Man3GlcNAc2Asn component appeared to contain in addition a Fuc residue in alpha(1----6) linkage to the Asn-bound GlcNAc. The overall ratio of oligomannoside-type to N-acetyllactosamine-type carbohydrate structures was found to be 5:4. This article is the first account of the complete characterization of the oligomannoside-type structures in alpha-L-fucosidase; furthermore, the occurrence in alpha-L-fucosidase of mono(sialyl-N-acetyllactosaminyl) structures, Fuc-containing oligosaccharides, and NeuAc alpha(2----3) linked to Gal are reported for the first time.     

Oligosaccharide analyses of glycopeptides of horseradish peroxidase by thermal-assisted partial acid hydrolysis and mass spectrometry

Thermal-assisted partial acid hydrolysis of the carbohydrate moieties of N-glycosylated peptides of horseradish peroxidase (HRP) is used to generate oligosaccharide cleavage ladders. These ladders allow direct reading of components of the oligosaccharides by mass spectrometry. Acid hydrolysis performed with 1.4, 3.1, 4.5, or 6.7M trifluoroacetic acid at 37, 65, or 95 degrees C for 30min to 24h hydrolyzed mainly the oligosaccharide units of glycopeptides with least peptide bond or amino acid side chain hydrolysis. Tryptic N-glycosylated peptides from HRP with molecular weights of 2533, 2612, 3355, 3673, and 5647Da were used as test systems in these experiments. Data showed that the most labile group of oligosaccharides is the fucose (Fuc) and the majority of the end cleavage products are peptides with one or no N-acetylglucosamine (GlcNAc) residue linked to Asparagine (Asn). Additionally, the data agree with previous reports that glycopeptides 3355 and 3673Da carry an oligosaccharide (Xyl)Man3(Fuc)GlcNAc2, glycopeptide 5647Da carries two oligosaccharides (Xyl)Man3(Fuc)GlcNAc2, and glycopeptides 2612 and 2533Da carry (Xyl)Man3GlcNAc2 and (Fuc)GlcNAc, respectively. However, the glycosylation site of the 2612Da peptide at Asn286 is partially occupied. This method is particularly useful in identifying glycopeptides and obtaining monosaccharide compositions of glycopeptides.     

Characterization of O-glycosidically linked oligosaccharides of rat erythrocyte membrane sialoglycoproteins

The carbohydrate units of the rat erythrocyte membrane sialoglycoprotein rSGP-4 [Edge, A. S. B., & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705] have been characterized. All of the carbohydrate of this Mr 19,000 glycoprotein occurs in O-glycosidic linkage to the peptide; following alkaline borohydride treatment and chromatography on Bio-Gel P-2, sialic acid containing oligosaccharides terminating in N-acetylgalactosaminitol were obtained. Their structures were determined by compositional analysis, exoglycosidase digestions, alkaline sulfite degradation, and periodate oxidation. The oligosaccharides were characterized for molecular weight and linkage by direct chemical ionization and gas-liquid chromatography/mass spectrometry, respectively. The structures are proposed to be NeuAc alpha 2----3Gal beta 1----3GalNAc-ol, Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, and NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-ol. Two of the N-acetylglucosamine-containing hexasaccharides were present per molecule of rSGP-4 along with two trisaccharides and seven tetrasaccharides.