Fucose-containing oligosaccharides from human milk from a donor of blood group 0 Le(a) nonsecretor

The neutral oligosaccharides from the milk of a single donor with blood group 0, Lewis(a+b-) nonsecretor were separated into 18 fractions essentially according to the number of carbohydrate constituents using gel permeation chromatography on Biogel P-4 and Fractogel TSK HW-40. Further separation was achieved by HPLC and by HPTLC after reduction and peracetylation. The fractions obtained were analysed by FAB mass spectrometry with and without derivatization and by one- and two-dimensional proton NMR. Besides the already described 3-fucosidolactose, fucopentaose II (2), fucopentaose III (6) and difucohexaose II (4) the following fucosylated oligosaccharides could be identified. Among the higher oligosaccharides a branched lacto-N-decaose (12) was obtained in pure form after removal of the fucose residues by mild acid treatment.     

Application of liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin

High-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS) were applied to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin (EPO) used as a model of the sialylated glycoprotein. N-linked oligosaccharides were released from recombinant human EPO expressed in Chinese hamster ovary cells enzymatically and reduced with NaBH(4). Many different sialylated oligosaccharides of EPO were separated and characterized by LC/MS equipped with a graphitized carbon column (GCC). Glycosylation sites and the preliminary glycosylation pattern at each glycosylation site were determined by LC/MS of endoproteinase Glu-C-digested EPO. The detailed site-specific carbohydrate heterogeneity caused by the differences in the molecular weight, branch, linkage, and sequence was elucidated by GCC-LC/MS of the N-linked oligosaccharides released from the isolated glycopeptides. Structural details of the isomers were analyzed by LC/MS/MS, and it was indicated that di- and trisialylated tetraantennary oligosaccharides are attached to Asn24, 38, and 83, whereas their isomers, di- and trisialylated triantennary oligosaccharides containing N-acetyllactosamines, are combined with Asn24. Our method is useful for the determination of glycosylation sites, the site-specific carbohydrate heterogeneity of glycoproteins, and the carbohydrate structure.     

Structural analysis of the N-linked oligosaccharides from murine glycophorin

Glycophorins, isolated from BALB/c mouse erythrocytes, were degraded under mild and strong reductive alkaline conditions and the N-linked oligosaccharides were isolated as alditols. The oligosaccharide alditols were fractionated and purified using gel filtration, concanavalin A-Sepharose affinity chromatography, and high-performance ion-exchange chromatography. Structural analysis was carried out by chemical analyses, periodate oxidation in combination with fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. The results revealed the presence of sialylated biantennary, triantennary, and tetraantennary complex type oligosaccharides, all fucosylated at the innermost N-acetylglucosamine residue. The tri- and tetraantennary oligosaccharide-containing fractions also contained species elongated by one and/or two N-acetyllactosamine (-3Gal beta 1-4GlcNAc beta 1-) sequences. The N-linked oligosaccharides were shown to be combined only with one (the low molecular weight) of the two mouse glycophorins.     

Structural characterization of multibranched oligosaccharides from seal milk by a combination of off-line high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and sequential exoglycosidase digestion

A complex mixture of diverse oligosaccharides related to the carbohydrates in glycoconjugates involved in various biological events is found in animal milk/colostrum and has been challenging targets for separation and structural studies. In the current study, we isolated oligosaccharides having high molecular masses (MW ∼ 3800) from the milk samples of bearded and hooded seals and analyzed their structures by off-line normal-phase-high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight (NP-HPLC-MALDI-TOF) mass spectrometry (MS) by combination with sequential exoglycosidase digestion. Initially, a mixture of oligosaccharides from the seal milk was reductively aminated with 2-aminobenzoic acid and analyzed by a combination of HPLC and MALDI-TOF MS. From MS data, these oligosaccharides contained different numbers of lactosamine units attached to the nonreducing lactose (Galβ1-4Glc) and fucose residue. The isolated oligosaccharides were sequentially digested with exoglycosidases and characterized by MALDI-TOF MS. The data revealed that oligosaccharides from both seal species were composed from lacto-N-neohexaose (LNnH, Galβ1-4GlcNAcβ1-6[Galβ1-4GlcNAcβ1-3]Galβ1-4Glc) as the common core structure, and most of them contained Fucα1-2 residues at the nonreducing ends. Furthermore, the oligosaccharides from both samples contained multibranched oligosaccharides having two Galβ1-4GlcNAc (N-acetyllactosamine, LacNAc) residues on the Galβ1-4GlcNAcβ1-3 branch or both branches of LNnH. Elongation of the chains was observed at 3-OH positions of Gal residues, but most of the internal Gal residues were also substituted with an N-acetyllactosamine at the 6-OH position.     

Structural analysis of five new monosialylated oligosaccharides from human milk.

The total monosialylated oligosaccharide fraction from pooled human milk was isolated by gel filtration and ion-exchange chromatography. Further separation by HPLC using a mobile phase containing an ion-pairing reagent of triethylamine gave five new monosialylated oligosaccharides. Structural analysis was carried out by chemical analyses, fast atom bombardment mass spectrometry, and 500-MHz NMR spectroscopy. Combined structural data revealed the following new structures: [formula: see text]     

Identification and characterization of glycosylation sites in human serum clusterin.

Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the alpha subunit contains 0-30% carbohydrate and the beta subunit contains 27-30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the alpha subunit (alpha 64N, alpha 81N, alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site beta 64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site beta 147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated alpha and beta subunits, including all Ser- and Thr-containing peptides. No evidence was found for the presence of O-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.     

Incorporation of orally applied (13)C-galactose into milk lactose and oligosaccharides

Biosynthesis and functions of human milk oligosaccharides (HMO) are not well known. A typical housekeeping enzyme, beta1,4-galactosyltransferase, links galactose to glucose to form lactose which is then used as backbone for the assembly of HMO. We investigated whether milk lactose and HMO may be labeled in vivo by an orally given (13)C-galactose bolus. Eleven exclusively breastfeeding mothers were given a (13)C-galactose bolus at the end of their breakfast. Milk and urine samples from each nursing up to 36 h were analyzed for carbohydrate composition by high-performance thin-layer chromatography, high-pH anion-exchange chromatography, and fast atom bombardment mass spectrometry. (13)C enrichment of milk fractions, urinary carbohydrates, lactose, and oligosaccharides as well as of breath CO(2) was determined by isotope ratio mass spectrometry. Up to 10% of the orally given galactose bolus was directly transported to the mammary gland and incorporated into milk components. Characteristic for most milk samples was the appearance of two (13)C-peaks, the first immediately after the (13)C-bolus was taken and the second on the next morning. The highest (13)C enrichment was found in lactose followed by neutral and acidic oligosaccharides. In breath samples, the (13)C-excretion followed the same pattern as in milk. (13)C nuclear magnetic resonance of isolated lactose revealed (13)C only at C(1)-atom of galactose and C(1)-atom of glucose. This label was without any exception at the same position as the (13)C-label of the orally applied galactose. Neutral and acidic HMO can easily be (13)C-labeled in vivo which facilitates investigations of their metabolic fate in infants.     

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le^x(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal, expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.Abbreviations DCl direct chemical ionization - FAB tastatiom bombardment - GC gas chromatography - GSLs glycosphingolipids - MS mass spectrometry - SSEA-1 stage specific embryonic antigen-1 - TLC thin layer chromatographys     

Structural classification of carbohydrates in glycoproteins by mass spectrometry and high-performance anion-exchange chromatography

A general strategy has been developed for determining the structural class (oligomannose, hybrid, complex), branching types (biantennary, triantennary, etc.), and molecular microheterogeneity of N-linked oligosaccharides at specific attachment sites in glycoproteins. This methodology combines mass spectrometry and high-performance anion-exchange chromatography with pulsed amperometric detection to take advantage of their high sensitivity and the capability for analysis of complex mixtures of oligosaccharides. Glycopeptides are identified and isolated by comparative HPLC mapping of proteolytic digests of the protein prior to, and after, enzymatic release of carbohydrates. Oligosaccharides are enzymatically released from each isolated glycopeptide, and the attachment site peptide is identified by fast atom bombardment mass spectrometry (FAB-MS) of the mixture. Part of each reaction mixture is then permethylated and analyzed by FAB-MS to identify the composition and molecular heterogeneity of the carbohydrate moiety. Fragment ions in the FAB mass spectra are useful for detecting specific structural features such as polylactosamine units and bisecting N-acetylhexosamine residues, and for locating inner-core deoxyhexose residues. Methylation analysis of these fractions provides the linkages of monomers. Based on the FAB-MS and methylation analysis data, the structural classes of carbohydrates at each attachment site can be proposed. The remaining portions of released carbohydrates from specific attachment sites are preoperatively fractionated by high-performance anion-exchange chromatography, permethylated, and analyzed by FAB-MS. These analyses yield the charge state and composition of each peak in the chromatographic map, and provide semiquantitative information regarding the relative amounts of each molecular species. Analytically useful data may be obtained with as little as 10 pmol of derivatized carbohydrate, and fmol sensitivity has been achieved. The combined carbohydrate mapping and structural fingerprinting procedures are illustrated for a recombinant form of the CD4 receptor glycoprotein.     

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.     

Structures of the oligosaccharides isolated from milk of the platypus

Eight major oligosaccharides were isolated from platypus milk. By sequential exoglycosidase digestion and methylation study, their structures were elucidated as shown in Fig. 9 of this paper. The characteristics feature of the platypus milk oligosaccharides is that lacto-N-neotetraose and lacto-N-neohexaose are the major cores in contrast to human milk oligosaccharides in which lacto-N-tetraose and lacto-N-hexaose are found as the major core.     

Isolation and identification of a high molecular weight protein in sow milk

A high molecular weight protein (HMWP) was isolated and purified from sow milk, and some of its biochemical characteristics and biological functions were identified. The origin of HMWP was also investigated. The molecular weight of HMWP was determined to be about 115 000 and 114 800 by SDS-PAGE and gel filtration, respectively. The sequence of 10 amino acids in N-terminal of HMWP was Ala-Leu-Val-Gln-Ser-Cys-Leu-Asn-Leu-Val. The sequence was blasted against GenBank. No protein showed significant similarity with this sequence suggesting the HMWP may be novel. The result of liquid chromatography mass spectrometry (LC-MS) also proved HMWP could be a novel protein. By amino acid assay, HMWP was rich in glutamate (including glutamine), cysteine, glycine, aspartic acid (including asparagines) and proline. The content of hydrophobic amino acids (Ala, Val, Leu, Ile, Met, Phe and Pro) was lower at 18.59% of the total amino acids suggesting HMWP has high solubility in water. Western blots of lectins were used to identify the kinds of carbohydrate residues attached to HMWP qualitatively. The result showed that HMWP was a kind of glycoprotein containing N-acetylneuraminic acid (NeuNAc), mannose (Man) and/or N-acetylglucosamine (GlcNAc). By isoelectric focusing, HMWP pI was found to be 5.1. Compared with milk fat globule membrane protein (MFGMP) isolated from the sow milk in SDS-PAGE, MFGMP did not contain HMWP. HMWP was assumed to be a secretory milk protein. HMWP was not found in bovine, goat, rabbit or human milk in SDS-PAGE gel suggesting HMWP may be unique to sow milk. By Western blot, HMWP could be detected in sow milk, not in sow serum, which suggests it is synthesized and secreted by the mammary gland. HMWP concentrations in sows milk were the lowest in the first day of lactation, rose significantly during lactation 1 to 7 days. The HMWP content of sows milk remained relatively constant ((1.95±0.13) g/l) during lactation 7 to 20 days. HMWP significantly inhibited Escherichia coli in a dose related manner in vitro. Overall, HMWP could be a novel sow milk protein with implications for the mammary gland and the piglet.     

The carbohydrate of human thrombin: Structural analysis of glycoprotein oligosaccharides by mass spectrometry

The carbohydrate structure of human thrombin has been determined by direct probe mass spectrometry of the oligosaccharides released by trifluoroacetolysis from the asialo glycoprotein. The free oligosaccharides were studied as permethylated and N-trifluoroacetylated oligosaccharide alditols. The structure was confirmed by sequential exoglycosidase digestion of intact thrombin and sugar and methylation analysis of the oligosaccharides by gas-liquid chromatography-mass spectrometry. The results indicate the following structure:

with Fuc present on only about 50% of the oligosaccharides.     

Oligosaccharide alterations of rat glioblast membrane-bound glycoproteins during differentiation induced by glia maturation factor

Differentiation of normal glioblasts was induced by glia maturation factor (GMF), and the structural change in the oligosaccharide chains of the plasmalemmal glycoproteins was investigated. After the glycopeptides obtained by trypsin treatment of the intact cells had been digested with pronase, the resulting glycopeptides were separated into 4 fractions by gel filtration. The first 2 fractions were found to contain mainly N-glycosidically linked glycopeptides, and the last 2, O-linked oligosaccharides. There were a variety of N-linked oligosaccharides whose apparent molecular weights were greater than that of isomaltoheptaose. As compared to those, O-linked oligosaccharides were fewer in type and lower in molecular weight. The N-linked oligosaccharides corresponding to isomaltohepta- decaose and larger saccharide chains augmented in differentiated glioblasts, whereas the N-linked oligosaccharides smaller than isomaltoheptade- caose decreased. The turnover rate of the high molecular weight oligosaccharides was faster than that of other membrane oligosaccharides, and was accelerated by GMF treatment. The content of an O-linked oligosaccharide fraction increased after GMF treatment.     

Structural studies of the acidic oligosaccharide units from bovine glycophorin

The O-glycosidically-linked carbohydrate units of glycophorin from bovine erythrocyte membrane were released by alkaline borohydride treatment. These oligosaccharides were separated into the neutral fractions and the acidic fractions by ion-exchange chromatography followed by gel filtration. The two acidic fractions (fractions 10 and 13) which have the smallest molecular weight in acidic oligosaccharides, were further purified by gel filtration on Bio-Gel P-4 column. Two acidic oligosaccharides (fractions 10-I and 10-II), heptasaccharides, were separated by gel filtration on a Bio-Gel P-4 column from fraction 10. These structures were determined by methylation analyses, nitrous acid deamination after hydrazinolysis and Smith degradation after desialylation. In addition, the structures were also analyzed by direct-probe mass spectrometry of the permethylated derivatives before and after desialylation. These studies indicated that one of them (fraction 10-I) was NeuNGcα(2→3)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→3) GalNAcol and another heptasaccharide (fraction 10-II) was Galβ(1→4)GlcNAcβ(1→3)Galβ(1→3) [NeuNGcα(2→3)Galβ(1→4)GlcNAcβ(1→6)]GalNAcol. Athough another acidic fraction (fraction 13) was obtained as a single peak on a Bio-Gel P-4 column, it appeared to be the mixture of a heptasaccharide, NeuNGcα(2→3)Galβ(1→4)GlcNAcβ(1→3 or 6)[Galβ(1→4)GlcNAcβ(1→6 or 3)]Galβ(1→3)GalNAcol and an oligosaccharide similar to fraction 10-II, by analysis of two products obtained by Smith degradation after desialylation.     

Trace amounts of ganglioside GM1 in human milk inhibit enterotoxins from Vibrio cholerae and Escherichia coli

Gangliosides were isolated from human milk fat and purified by silica gel column chromatography and high performance liquid chromatography (HPLC). Low amounts of the ganglioside GM1, detected by high performance thin layer chromatography (HPTLC)-immunoassay, were found in all fractions with enterotoxin-inhibitory activity, while fractions without GM1 were inactive. It is concluded that GM1 is responsible for enterotoxin-inhibitory activity in the ganglioside fraction from human milk.     

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.     

Rhizoferrin — a novel siderophore from the fungusRhizopus microsporus var.rhizopodiformis

Summary From a strain ofRhizopus microsporus var.rhizopodiformis a novel siderophore, named rhizoferrin, was isolated by ion-exchange column chromatography, gel filtration and preparative HPLC. Hydrolysis with 6 M HCl and subsequent gas chromatography/mass spectrometry (GUMS) of the esterified/trifluoroacetylated derivatives indicated that citric acid and diaminobutane were the only constituents. From positive fastatom-bombardment (FAB) and ion-spray tandem mass spectrometry, a molecular mass of 436 Da and the assignment of several daughter ion fragments could be obtained, which indicated the presence of two citric acid residues and one diaminobutane residue. NMR studies finally confirmedN ¹,N ⁴-bis(1-oxo-3-hydroxy-3,4-dicarboxybutyl)-diaminobutane as the structure of rhizoferrin. The iron-binding property was demonstrated on chromeazurol S plates and its siderophore activity was confirmed by iron transport measurements in young mycelia ofR. microsporus. While rhizoferrin and also ferrioxamines B and E proved to be effective siderophores, coprogen was a poor siderophore in this fungus.     

Structural characterization and in vitro inhibitory activities in P-selectin-mediated leukocyte adhesion of polysaccharide fractions isolated from the roots of Physalis alkekengi

Selectin-mediated leukocyte initial attachment and rolling over vessel endothelial surface are crucial steps for inflammatory responses. As P-selectin is a promising target for anti-inflammation therapeutic strategy, recent works have focused on searching for more potent and non-toxic P-selectin antagonists among various natural carbohydrate products. Here, we isolated three water-soluble polysaccharide fractions (PPS-1, PPS-2 and PPS-3) from the roots of Physalis alkekengi by DEAE-cellulose and Sephacryl S-200 chromatography. Their physicochemical and structural characterizations were determined by chemical methods, GC (gas chromatography), HPLC (high performance liquid chromatography), FT-IR (Fourier transform infrared spectrometry), partial acid hydrolysis, methylation and GC-MS (gas chromatography-mass spectrometry) analyses. The inhibitory capacity of the polysaccharide fractions in P-selectin-mediated leukocyte adhesion was evaluated by flow cytometric, static adhesion and laminar flow assays. Results showed that different polysaccharide fractions possess distinct physicochemical and structural properties, including carbohydrate, protein and uronic acid contents, molecular weight, monosaccharide composition and glycosidic linkage type. Among the polysaccharide fractions, PPS-2 could effectively block the interaction between P-selectin and its native ligand.