A mucus-secreting human colonic cancer cell line. Purification and partial characterization of the secreted mucins.

A stably differentiated clonal derivative (Cl.16E) of the human colonic adenocarcinoma cell line HT29 secretes in culture high-Mr glycoproteins that were purified from the serum-free conditioned medium by preparative SDS/polyacrylamide-gel electrophoresis. Analysis of the oligosaccharides released from the [3H]glucosamine-labelled high-Mr glycoproteins by alkaline-borohydride treatment showed that this material consisted of O-linked oligosaccharides (without any detectable N-linked oligosaccharides) that were eluted as three fractions from Bio-Gel P-6 columns. The main oligosaccharide fraction obtained after such treatment and desialylation was eluted together with a six-unit glucose polymer from a Bio-Gel P-4 column. Polyclonal antibodies were raised against the high-Mr glycoproteins, and in immunoblot analysis they reacted specifically with the high-Mr glycoproteins present in the conditioned medium. Furthermore, immunohistochemical staining of sections in paraffin wax revealed that these antibodies labelled normal human gastrointestinal mucins. We conclude that (1) the high-Mr glycoproteins prepared by SDS/polyacrylamide-gel electrophoresis are pure mucus glycoproteins on the basis of sensitivity to alkaline-borohydride treatment, monosaccharide composition and immunochemical and immunohistological findings, and (2) these mucins have antigenic determinants in common with the normal human gastrointestinal mucins.     

Separation and analysis of charged isomers of monoclonal immunoglobulin G by ceramic hydroxyapatite chromatography

Analysis of monoclonal antibody (MAb) heterogeneity caused by posttranslational modifications is important for pharmaceutical quality assurance of antibody drugs. In this study, by employing small ceramic hydroxyapatite (HAp) particles that were self-manufactured to have a 2.5-µm diameter, we attempted to separate and analyze MAb isomers. The MAb without N-linked oligosaccharides could be separated by 2.5-µm HAp chromatography as well as MAb with N-linked oligosaccharides. Hence, a variety of N-linked oligosaccharides do not appear to be involved in the separation mechanism of HAp chromatography. However, there is a difference in retention time between MAb with and without N-linked oligosaccharides, meaning that the presence of N-glycan could influence the retention time of HAp chromatography. Subsequently, the MAb fractions separated by 2.5-µm HAp chromatography were analyzed by isoelectric focusing, and seven isomers of the MAb having different isoelectric points (pI) were identified. The MAb isomers were eluted in order of lower pI isomers with sodium phosphate buffer, and enzyme-linked immunosorbent assay indicated the immunoreactivity of the fraction including the lowest pI isomers to be remarkably reduced. This study yielded details of the separation behavior of HAp chromatography owing to 2.5-µm HAp particles.     

Structural study of N-linked oligosaccharides of human intercellular adhesion molecule-3 (CD50).

The N-linked oligosaccharides were released from purified human intercellular adhesion molecule (ICAM)-3 by hydrazinolysis. Approximately 6 mol of oligosaccharides were released from 1 mol of ICAM-3. The oligosaccharides reduced with NaB[3H]4 were separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial lectin column chromatography followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endo-glycosidase digestion and by methylation analysis revealed that N-linked oligosaccharides of ICAM-3 are mainly of tri- and tetra-antennary complex-type, about 60% of which contain two to three poly N-acetyllactosamine chains terminated with the type 1 structure and those without the type 1 structure per oligosaccharide. In addition, a small amount of the high mannose-type oligosaccharide with six alpha-mannose residues, which could act as a ligand for the dendritic cell-specific ICAM-3 grabbing nonintegrin, was detected.     

Analyses of N-linked oligosaccharides using a two-dimensional mapping technique

We propose a two-dimensional sugar map method for the simple, reproducible, and sensitive analysis of the structures of N-linked oligosaccharides. The structure of an unknown oligosaccharide can be characterized from its position on the map. The data base for the sugar map is prepared by the use of 113 kinds of standard oligosaccharides, 58 of whose structures have been confirmed by 1H NMR spectroscopy. The present method involves six steps, (i) preparation of oligosaccharides from glycopeptides by N-oligosaccharide glycopeptidase (almond) digestion, (ii) derivatization of the reducing ends of oligosaccharides with a fluorescent reagent, 2-amino-pyridine, by using sodium cyanoborohydride, (iii) separation of oligosaccharide derivatives by high-performance liquid chromatography with an ODS-silica column, (iv) analysis of the size of each separated oligosaccharide on an amide-silica column, (v) plotting of the elution position of a sample on the two-dimensional sugar map obtained for the standard oligosaccharides, and (vi) structural analysis of the oligosaccharides by a combination of sequential exoglycosidase digestion and the steps (iii-v). The present method was applied to the identification of the structures of oligosaccharides in hen ovalbumin. It was found that two unusual oligosaccharides that have not yet been reported exist in ovalbumin.     

Carbohydrate components of lipovitellin of the sand crab Emerita asiatica

Lipovitellin II (Lv II), the major yolk protein of the anomuran crab Emerita asiatica, was purified using heparin-sepharose affinity column chromatography. The purified Lv II was a glycoprotein as it was stainable with periodic acid-Schiff's reagent. Quantitative analysis of sugars showed the presence of fucose, mannose, galactosamine, N-linked oligosaccharides, as well as O-linked oligosaccharides containing N-acetyl hexosamine as the terminal residue. The amount of N-linked oligosaccharides is higher than that of the O-linked oligosaccharides. Biogel P-4 column chromatographic separation of the radiolabeled oligosaccharides of Lv II showed the presence of five different O-linked oligosaccharides and four different N-linked oligosaccharide species. HPTLC separation of the neoglycolipids prepared from the O-linked oligosaccharides also showed the presence of five different O-linked oligosaccharide species. N-linked oligosaccharides contain significant quantities of mannose. Unisil column chromatographic purification in conjunction with HPTLC separation revealed three neutral glycolipid species such as monoglycosylceramide, diglycosylceramide, and triglycosylceramide in the Lv II. The functional significance of these carbohydrate components of the major yolk protein during embryogenesis of the sand crab is discussed.     

Chromatographic behaviour of rat-liver monophosphoinositide

1. Chromatography of rat-liver lipids on a column of silicic acid or a mixture of silicic acid and Hyflo Super-Cel, with chloroform–methanol mixtures, gave monophosphoinositide-containing fractions which were invariably contaminated by the presence of nitrogen-containing phospholipids. The behaviour of the inositide was extremely sensitive to column loading and the results with different batches of silicic acid were not reproducible. 2. However, when chromatography on an alumina column was used, the solvent system chloroform–methanol–water (23:23:4, by vol.) completely eluted the neutral lipids, choline-containing phospholipids and phosphatidylethanolamine. An increase of the water content of the solvent to 14% (by vol.) then led to the elution of the monophosphoinositide component, now free from nitrogen-containing phospholipids, but still contaminated by the presence of a phospholipid, which from its properties was taken to be polyglycerophosphatide. 3. Most of the polyglycerophosphatide could be removed from a rat-liver lipid extract by silicic acid chromatography with chloroform–methanol (19:1, v/v). The other phospholipids were then eluted and applied to an alumina column, whereby a monophosphoinositide fraction of much greater purity was obtained. 4. Further purification of the monophosphoinositide was achieved by chromatography on a mixture of silicic acid and cellulose powder. The final product was virtually pure by thin-layer chromatography and gave the expected analysis for monophosphoinositide.     

高速逆流色谱分离制备甘草中的甘草苷和芒柄花苷

应用高速逆流色谱分离制备甘草中的甘草苷和芒柄花苷。将甘草乙酸乙酯提取物经聚酰胺柱粗分后,30%乙醇洗脱物用高速逆流色谱进一步分离,所用两相溶剂系统为乙酸乙酯-水(5∶5,v/v),转速850 rpm,流速2.0 mL/min,检测波长254 nm,从50 mg30%乙醇洗脱物中得到甘草苷8.7 mg、芒柄花苷4.2 mg,纯度分别为99.5%和97.3%。所得产物的结构经核磁共振谱(NMR)鉴定。利用该方法可以对甘草中的甘草苷和芒柄花苷进行快速的分离和纯化。     

Two-dimensional mapping by the high-performance liquid chromatography of oligosaccharides released from glycosphingolipids by endoglycoceramidase

A two-dimensional sugar mapping method has been developed by which sensitive, reproducible, and simple analysis can be carried out on the structures and compositions of oligosaccharides released from glycosphingolipids by endoglycoceramidase. The oligosaccharides were labeled quantitatively with an ultraviolet-absorbing compound, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-oligosaccharides were separated first on an amide-silica column and then on a C4-silica column by high-performance liquid chromatography. The acidic ABEE-oligosaccharides were eluted as a group at the start of the chromatography while the neutral ABEE-oligosaccharides were separated according to size and structure on an amide-silica column using an eluent without salt. The acidic oligosaccharides were separated according to size and structure when rechromatographed on the same column using an eluent containing KH2PO4. NeuAc-containing ABEE-oligosaccharides were extensively separated from the corresponding NeuGc derivatives. The ABEE-oligosaccharides separated on an amide-silica column were then chromatographed on a column of C4-silica on which lactotriose and neolacto-series oligosaccharides were clearly shown to be separated from the others. On the basis of the retention times of the individual ABEE-oligosaccharides on two separate columns, 9 neutral and 15 acidic oligosaccharides derived from glycosphingolipid standards were two-dimensionally mapped without overlapping. The gangliosides of a human chondrosarcoma tissue and glycosphingolipids of tumor tissue of FBJ virus-transformed murine osteosarcoma cells were analyzed by this method in conjunction with exoglycosidase treatment. At least 11 species of glycosphingolipids were identified in both cases.     

Structural analysis of sulfated N-linked oligosaccharides in erythropoietin.

We previously demonstrated that high-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) is useful for the structural analysis of carbohydrates in glycoproteins. Using LC/MS with GCC, sulfated N-linked oligosaccharides were found in erythropoietin (EPO) expressed in baby hamster kidney cells. Sulfation occurs in a part of the N-linked oligosaccharides in the EPO. Sulfated monosaccharide residue in the sulfated N-linked oligosaccharide was determined by exoglycosidase digestion followed by sugar mapping by LC/MS. The linkage position and branch-location of the sulfate group in the tetraantennary oligosaccharide were analyzed by (1)H-nuclear magnetic resonance. It was suggested that sulfation occurs on the C-6 position of GlcNAc located in the GlcNAcbeta1-4Manalpha1-3 branch.     

Chromatography of permethylated oligosaccharide alditols

A new chromatographic method which enables the separation of permethylated oligosaccharide alditols has been developed. The method entails chromatography on precoated silica gel plates using benzene-methanol (16:1, v/v or 10:1 v/v) as developing solvent. Separations of disaccharides were obtained on the basis of glycosidic linkage and anomeric configuration; the method can accomodate oligosaccharides containing up to 15 glycose units. The combined use of thin-layer chromatography and gas-liquid chromatography provides a powerful approach for the characterization of oligosaccharides. Retention indices are given of permethylated oligosaccharide alditols on a fused-silica capillary column bonded with DB-1.     

Transfection with fragments of the adenovirus 12 gene induces tumorigenicity-associated alteration of N-linked sugar chains in rat cells

N-linked sugar chains of rat 3Y1 cells and their poorly tumorigenic (E1Y cells) and highly tumorigenic (CY1 cells) transformants carrying various adenovirus 12 gene fragments were quantitatively released as oligosaccharides from their membrane preparations by hydrazinolysis. After being fractionated by a series of immobilized lectin column chromatography, structures of oligosaccharides in each fraction were studied by sequential glycosidase digestion in combination with methylation analysis. All cells contain bi-, tri-, and tetraantennary complex-type oligosaccharides as well as high mannose-type oligosaccharides in different molar ratio. Expression of 2,4-branched triantennary oligosaccharides increased in both transformed cells. However, their contents were rather higher in poorly tumorigenic E1Y cells than in highly tumorigenic CY1 cells. In contrast, the increase of 2,6-branched triantennary and tetraantennary oligosaccharides was positively correlated to the tumorigenic potential of the transformed cells. The data indicate that glycosylation of cellular proteins is differently affected by the expression of specific regions of the adenovirus genome, and the combined action of E1 and E4 gene products is important for the expression of the GlcNAc beta 1----6Man group associated with tumorigenicity of the cells.     

Structural analysis of the asparagine-linked glycan units of the ZP2 and ZP3 glycoproteins from mouse zona pellucida

Zona pellucida (ZP), the extracellular glycocalyx that surrounds the mammalian egg plasma membrane, is a relatively simple structure consisting of three to four glycoproteins. In the mouse, the ZP is composed of three glycoproteins, namely ZP1 (200 kDa), ZP2 (120 kDa), and ZP3 (83 kDa). Extensive studies in this species have resulted in the identification of primary (mZP3) and secondary (mZP2) binding sites for spermatozoa. The two zona components are highly glycosylated containing N-linked and O-linked glycan units. In an attempt to characterize N-linked glycan units, mZP2 and mZP3 were purified and the N-linked carbohydrate chains were released by exhaustive digestion with N-glycanase. The released oligosaccharides (OSs) were radiolabeled by reduction with NaB3H4 and resolved by gel filtration on a column of Bio-Gel P-4. The OSs separated into several peaks indicating the presence of a variety of N-linked glycans. Interestingly, the radioactive peaks resolved from mZP2 and mZP3 were quite different, a result suggesting qualitative and quantitative differences in the glycans. The [SH]-labeled glycans present in mZP2 and mZP3 were pooled separately and fractionated by serial lectin chromatography. Experimental evidence included in this report strongly suggests that mZP3 (but not mZP2) contains polylactosaminyl glycan with terminal, nonreducing alpha-galactosyl residues. The mZP3 glycans eluted from the immobilized lectin columns were further characterized by lectin and sizing column chromatography before or after digestion with endo-/ exo-glycohydrolases. Data revealed the presence of a variety of OSs, including poly-N-acetyllactosaminyl, bi-, tri-, and tetraantennary complex-type, and high-mannose-type glycans. Taken together, these results provide additional evidence on the complex nature of the glycan chains present on mZP glycoconjugates.     

Analysis of N-glycosylation of phospholipase A2 from venom of individual bees by microbore high-performance liquid chromatography-electrospray mass spectrometry using an ion trap mass spectrometer

The N-linked oligosaccharides were released from the phospholipase A2 (PLA) with glycopeptidases and reductively aminated with the chromophore, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-labeled oligosaccharides were separated by microbore high-performance liquid chromatography (micro-HPLC) using a reversed-phase column and analyzed by electrospray mass spectrometry. Differentiation between alpha-1,3 and alpha-1,6 core-fucosylated glycans was achieved by comparison the glycans released by glycopeptidases peptide-N-glycanase A (PNase A) and peptide-N-glycanase F (PNase F). All N-linked oligosaccharides except 3B and 3C could be identified in this approach. The analysis of PLA oligosaccharides from the venom of individual bees indicated that glycosylation patterns between the younger and the older bees were similar.     

Comparative structural study of N-linked oligosaccharides of urinary and recombinant erythropoietins

The structures of the N-linked oligosaccharides of the urinary erythropoietin (u-EPO) purified from urine of aplastic anemic patients were analyzed and compared with those for recombinant erythropoietin (r-EPO) prepared with baby hamster kidney (BHK) cells. Asparagine-linked neutral oligosaccharides were released from each EPO protein by N-oligosaccharide glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high-performance liquid chromatography (HPLC) on an ODS silica column. More than 8 and 13 kinds of oligosaccharide fractions for u-EPO and r-EPO (BHK), respectively, were completely separated by the one-step HPLC procedure. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amide-silica column. Furthermore, high-resolution proton nuclear magnetic resonance (1H NMR) spectroscopy and methylation analyses were carried out in the case of r-EPO (BHK).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

1H-NMR structural determination of the N-linked carbohydrate chains on glycopeptides obtained from the bean lectin phytohemagglutinin.

Phytohemagglutinin, the lectin of the common bean Phaseolus vulgaris, is a N-linked glycoprotein with one high-mannose-type and one xylose-containing oligosaccharide side chain per polypeptide. The high-mannose-type glycan is attached to Asn12 and the complex-type glycan to Asn60 [Sturm, A. & Chrispeels, M. J. (1986) Plant Physiol. 81, 320-322]. The structures of the oligosaccharides were elucidated from two glycopeptides obtained from the lectin by Pronase digestion, affinity chromatography on concanavalin-A--Sepharose and gel-filtration chromatography on a column of BioGel P-4. The N-linked glycan structures were investigated by 500-MHz 1H-NMR spectroscopy and were established to be: [formula; see text]     

Heparin-derived oligosaccharides: affinity for acidic fibroblast growth factor and effect on its growth-promoting activity for human endothelial cells

The minimal structural requirements for the interaction of heparin with acidic fibroblast growth factor (aFGF) were investigated. Oligosaccharides (tetra- to decasaccharides) obtained by nitrous acid depolymerisation of standard heparin were separated by affinity chromatography on Sepharose-immobilised aFGF. The shortest fragment retained by the affinity column at 0.2 M NaCl and eluted at 1 M NaCl was a "regular" hexasaccharide, a trimer of the most abundant disaccharide sequence in heparin. More complex octa- and decasaccharides were also retained by the column. The oligosaccharides eluted by 1 M NaCl from the affinity column ("high-affinity" oligosaccharides) and those washed from the column at 0.2 M NaCl ("low-affinity" oligosaccharides) were compared for their capacity to protect aFGF from proteolysis and to potentiate its mitogenic activity. At a low ionic strength, all oligosaccharides tested, except the "regular" disaccharide, protected aFGF against trypsin and collagenase digestion. At higher ionic strength (greater than 0.2 M NaCl), only high-affinity oligosaccharides showed a protective effect. The high-affinity oligosaccharides (hexa- to decasaccharides) potentiated the mitogenic activity of aFGF, as measured by [3H]thymidine incorporation into DNA of human fibroblasts. The effect of the oligosaccharides on human endothelial cell proliferation was more complex: inhibition of proliferation was observed in the presence of serum and low concentrations of aFGF (1-5 ng/ml) and potentiation in the presence of higher concentrations of aFGF. The potentiating effect increased as a function of molecular size of the heparin fragments and, for a given size, as a function of the anionic charge of the oligosaccharide. Our results suggest that inhibition of cell proliferation by heparin may result from interference with an autocrine basic FGF-like activity.     

Lectin affinity chromatography of proteins bearing O-linked oligosaccharides: application of jacalin-agarose.

The lectin jacalin immobilized on agarose was found to bind a variety of glycoproteins known to contain typical O-linked oligosaccharides, including human IgA, C1 inhibitor, chorionic gonadotropin, plasminogen, bovine protein Z, bovine coagulation factor X, and fetuin. These proteins were eluted from columns of jacalin-agarose specifically by alpha-galactopyranosides such as melibiose and alpha-methylgalactopyranoside but not by lactose or other sugars. Treatment of asialofetuin with endo--alpha--N--acetylgalactosaminidase eliminated its affinity for the lectin column, and other proteins known to contain only N-linked oligosaccharides such as ovalbumin, transferrin, and alpha 1-acid glycoprotein were not retained by the lectin. Binding of proteins with O-linked oligosaccharides to the lectin column did not require divalent cations and was affected little by changes in pH and ionic strength over a wide range. Virtually all of the glycosidically linked oligosaccharides of fetuin, chorionic gonadotropin, and plasminogen are known to be sialated. Thus, binding of these glycoproteins to jacalin, which is known to have affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides of these proteins, was not prevented by the presence of sialic acids. Affinity of oligosaccharides for jacalin did appear to be reduced by occurrence of sialic acids as it was found that higher concentrations of melibiose were required to elute asialofetuin than fetuin from jacalin-agarose. Results of the present study indicate that affinity chromatography using this lectin is a widely applicable technique for identifying and purifying proteins bearing O-linked oligosaccharides.     

视黄酸对人肝癌细胞表面N糖链类型及天线数的影响

本文采用系列凝集素柱层析法,并配合外切糖苷酶处理研究了在视黄酸(RA)作用1—5天过程中人肝癌细胞株SMMC-7721细胞表面N糖链结构的变化。结果表明,RA促进3~H-甘露糖(Man)参入细胞表面N糖链,使高甘露糖型N糖链的百分比下降,复杂型百分比上升,并促进二天线N糖链的生物合成,使多天线特别是四天线和C_2,C_(21)b三天线N糖链的合成减少。结果提示,N糖链结构的这些变化可能是RA诱导SMMC-7721细胞向正常方向分化的结果。     

Separation of chloroplast polar lipids and measurement of galactolipid metabolism by high-performance liquid chromatography

Procedures are described for the separation of polar lipids from plant chloroplasts by high-performance liquid chromatography, using a polar-modified silica column. Glycolipids and phospholipids were eluted with a gradient of 2-propanol/n-hexane (80:55, v/v) and 2-propanol/n-hexane/water/methanol (80:55:15:10, v/v). The lipids were detected by uv absorbance at 202 nm. Diacylglycerol and mono-, di-, and trigalactosyldiacylglycerol and phosphatidylcholine were separated on a LiChrosorb NH2 column (7-microns particles, Merck, FRG), but acidic lipids were retained. These lipids could be quantified from their 202-nm absorbance recording. The absorption coefficients obtained depended on the mean number of double bonds in the different lipid classes. The separation was applied for a rapid monitoring of the lipid composition in thylakoids and in fractionated inner and outer envelopes. The activities of galactosyltransferases involved in galactolipid metabolism, UDPGal:diacylglycerol galactosyltransferase and galactolipid:galactolipid galactosyltransferase, could be measured quantitatively in specific assays for both enzymes.