Synthesis and evaluation of affinity adsorbents for glycoproteins: an artificial lectin

A combination of rational design based on mimicking natural protein–carbohydrate interactions and solid-phase combinatorial chemistry has led to the identification of an affinity ligand which displays selectivity for the mannose moiety of glycoproteins. The ligand, denoted 18/18 and comprising a triazine scaffold bis-substituted with 5-aminoindan, has been synthesised in solution, characterised by TLC, ¹H-NMR and MS. When immobilised to amine-derivatised agarose at concentrations >24 μmol/g moist weight gel, ligand 18/18 selectively binds glucose oxidase. The adsorbed enzyme was quantitatively eluted with 0.5 M α- -methyl-mannoside and to a lesser extent with the equivalent glucoside. An investigation of the comparative retention times of saccharidic solutes showed that significant retardation was observed for α- -mannose, mannobiose and mannan, with little or no evidence for selective retention of other saccharides, with the exception of α- -fucose. Interestingly, α- -fucose and α- -mannose share an identical configuration of the hydroxyl groups on C-2, C-3 and C-4. Analysis of Scatchard plots from partition equilibrium studies on the interaction of glucose oxidase and the p-nitrophenyl-glycosides of -mannose, -glucose, -fucose and -galactose with immobilised 18/18 establish that the affinity constants (K_AX) for the enzyme, the glycosides of mannose, glucose and fucose, and the p-nitrophenyl-galactoside are 4.3×10⁵ M⁻¹, 1.9×10⁴ M⁻¹ and 1.2×10⁴ M⁻¹ respectively. ¹H-NMR studies on the interaction of α- -methyl-mannoside with ligand 18/18 in solution confirm the involvement of the hydroxyl group in the C-2 position. Molecular modelling suggests the formation of four hydrogen bonds between the hydroxyl groups at positions C-2, C-3 and C-4 of α- -methyl-mannoside and the bridging and ring nitrogen atoms of the triazine scaffold, with aromatic stacking of a second ligand against the carbohydrate face. The greater specificity of ligand 18/18 for mannose and glucose than for galactose parallels that exhibited by concanavalin A.     

Chromatography of dansyl-peptides from tryptic digests of electrophoretically separated proteins.

With the aid of DANS-Cl it is possible to detect peptides down to 10^?12 g. From electrophoresis runs, such amounts of proteins could be separated with a described electrophoresis apparatus. After tryptic digestion and dansylation the obtained peptides were run in a two dimensional chromatography system on 5 × 5 cm polyamide plates. The solvents were different concentrations of formic acid and benzene: acetic acid mixtures. The results show the reproducibility and specificity of this method for handling nanomole amounts of peptides.     

Studies on glycopeptides derived from acidic glycoproteins of guinea-pig serum

1. Fraction I, a fraction containing acidic glycoproteins, isolated from guinea-pig serum, was digested with Pronase after removal of sialic acid and a major and a minor glycopeptide fraction were isolated by chromatography with Sephadex G-25 and G-50. 2. The major fraction was examined by various methods and shown to contain several glycopeptides. Estimates of molecular weight of the glycopeptide fractions were obtained. Although some variation appeared to occur, the glycopeptides were not grossly heterogeneous with respect to size. An average prosthetic group was estimated to contain about 15 sugar residues. 3. Aspartic acid was the principal amino acid present in the fractions and in all subfractions of the major fraction investigated. Where examined, ammonia was liberated on acid hydrolysis in approximately equimolar amounts to the aspartic acid present. The carbohydrate composition of the fractions was also determined. 4. The glycopeptides showed relatively little degradation in alkaline solution. 5. These results suggest that an N-acylglycosylamine bond involving aspartic acid forms the major type of linkage between carbohydrate and polypeptide. The isolation of a compound with the composition and chromatographic properties of 2-acetamido-1-(l-beta-aspartamido)-1,2-dideoxy-beta-d-glucose supports this view, and indicates that N-acetylglucosamine is the sugar involved in at least many linkages. 6. Fraction I contains some glycoproteins that are susceptible to Pronase and one or more others that resist digestion before the removal of sialic acid. A brief examination revealed some similarities between prosthetic groups derived from both kinds of glycoprotein.     

A method for the identification of glycoproteins from human serum by a combination of lectin affinity chromatography along with anion exchange and Cu-IMAC selection of tryptic peptides

This paper reports a method for identifying glycoproteins from human serum. Glycoproteins were selected with a concanavalin A (Con A) lectin column and then tryptically digested prior to sequential chromatographic selection of acidic and histidine containing peptides. Acidic peptides were selected with a strong anion exchange (SAX) column. Peptides captured by the SAX columns were then released and histidine-containing peptides in the mixture selected with a copper loaded immobilized metal affinity chromatography (Cu-IMAC) column. This serial chromatographic selection process reduced the complexity of proteolytic digests by more than an order of magnitude. Peptides selected by this serial process were then fractionated by reversed-phase chromatography (RPC) and identified by tandem mass spectrometry. The method was initially validated using human transferrin before application to human serum. The results show that all the peptides identified except one contained histidine and acidic amino acids.     

基于凝集素-糖蛋白特异结合的特性筛选与鉴定空肠弯曲杆菌NCTC11168株糖蛋白

[目的]从空肠弯曲杆菌NCTC11168菌株中筛选糖蛋白.[方法]本实验基于凝集素-糖蛋白特异结合的特性,先用免疫印迹的方法确定空肠弯曲杆菌内糖蛋白能与凝集素SBA特异结合,再用包被有凝集素SBA的磁珠捕获样品中的潜在糖蛋白,通过N-乙酰半乳糖胺竞争性洗脱及双向电泳分离,最后利用串联质谱鉴定捕获的糖蛋白.[结果]质谱分析共鉴定到22种空肠弯曲杆菌蛋白,其中至少5种为已报道糖蛋白,包括Cj0633在内的17种为迄今为止未知的潜在糖蛋白.[结论]这种糖蛋白筛选策略可用于细菌糖蛋白的分离鉴定.     

Microwave-assisted glycosylation for the synthesis of glycopeptides

An efficient one-step synthesis of O-linked glycosylamino acids is described. This methodology converts commercially available peracetylated mono- and disaccharides activated by cheap and environmentally safe FeCl(3) under microwave irradiation with Fmoc-Ser-OBn to the corresponding beta-glycosides in short reaction times and moderate yields.     

Composition and Subcellular Distribution of Glycoproteins and Glycosaminoglycans Undergoing Axonal Transport in Garfish Olfactory Nerves

The study examined the subcellular distribution of [3H]glucosamine-labeled glycoconjugates undergoing axonal transport in 100,000 x g soluble and two membranous subfractions of the garfish olfactory nerve. Analysis was made of intact glycoconjugates and of glycopeptides and glycosaminoglycans derived from these molecules by limit protease digestion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed labeling of a variety of high-molecular-weight molecules with a lower molecular weight distribution in the soluble fraction than in the membranous fractions. Following protease digestion, nearly two-thirds of transported radioactivity in glycopeptides was recovered in the plasma membrane-enriched subfraction, with the remainder equally divided between soluble and higher density membrane fraction. Comparison of the distribution of glycopeptide radioactivity and chemically assayed hexosamine revealed transport labeling of a large variety of different-sized neutral and acidic glycopeptides in all subfractions. Transport labeling of most glycoprotein carbohydrate chains was in proportion of their hexosamine content. Transported glycosaminoglycan label was most heavily concentrated in the plasma membrane fraction, whereas hexosamine was most concentrated in the higher density membrane fraction. The labeling pattern suggested both transported and nontransported pools of these molecules. The specific glycosaminoglycans chondroitin sulfate and heparan sulfate were recovered in all subfractions, whereas hyaluronic acid was confined to the soluble fraction.     

Selection of affinity ligands for protein purification from proteolytic digests

A simple method for the selection of affinity ligands from proteolytic digests by affinity chromatography is proposed. A small proportion of the peptides in the trypsin digest of bovine serum albumin (BSA) or the pepsin digest of cytochrome are retarded on insulin-immobilised or HSA (human serum albumin)-immobilised affinity columns, respectively. The peptides in these selected fractions can be immobilised onto solid phases and used in affinity chromatography procedures for the purification of insulin or HSA. © Rapid Science Ltd. 1998     

Selective isolation and identification of N-terminal blocked peptides from tryptic protein digests.

A method for the easy isolation and direct sequencing of N-terminally blocked peptide in proteins refractory to N-terminal sequencing was developed. It is based essentially on tandem enzymatic treatments of the protein with trypsin and carboxypeptidase B, and selective isolation of the Nalpha-blocked peptide using ion-exchange chromatography. The chromatographic step was optimized for picomole amounts of sample and very short elution times by placing a thin layer of the resin over the membrane of an ultrafiltration tube. The isolated fraction can be analyzed directly using MALDI or ESI mass spectrometry. The method was applied to several recombinant and natural N-terminal acetylated proteins. A critical discussion on the intrinsic limitations of the method is also given.     

Uronic acid-containing glycopeptides from Fusarium oxysporum: Possible significance as chemotypes

Hot aqueous extraction of mycelia of Fusarium oxysporum, followed by fractionation on an anionic resin column gave glycopeptides FL-2 and FL-3. Methylation analysis and 1D and 2D NMR data demonstrated β-d-Manp units and partial hydrolysis gave -d-GlcpA(1→2)-d-Gal, arising from β-d-Galf-containing groups. Both are chemotaxonomic markers of Fusarium spp. FL-3 contained 2,6-di-O-substituted Manp, as well as 2,6-di-O-substituted Galf units, raising the possibility that the former are main-chain constituents, as well as the expected latter structure. The carbohydrate structures of FL-2 and FL-3 differ from those of previously examined polysaccharides of Fusarium spp., which are in turn all different from each other, so that they can serve as fingerprints. Possible variations in their main-chain structures can occur as well as those of their side-chains.     

Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides

Two LC-ESI-MS methods for the analysis of antibody glycosylation are presented. In the first approach, tryptic glycopeptides are separated by RP chromatography and analyzed by ESI-MS. This "glycopeptide strategy" allows a protein- and subclass-specific quantitation of both neutral and sialylated glycan structures. Additional information about under- or deglycosylation and the protein backbone, e.g., termini, can be extracted from the same data. In the second LC-ESI-MS method, released oligosaccharides are separated on porous graphitic carbon (PGC). A complete structural assignment of neutral and sialylated oligosaccharides occurring on antibodies is thereby achieved in one chromatographic run. The two methods were applied to polyclonal human IgG, to commercial mAb expressed in CHO cells (Rituximab, Xolair, and Herceptin), in SP2/0 (Erbitux and Remicade) or NS0 cells (Zenapax) and the anti-HIV antibody 4E10 produced either in CHO cells or in a human cell line. Both methods require comparably little sample preparation and can be applied to SDS-PAGE bands. They both outperform non-MS methods in terms of reliability of peak assignment and MALDI-MS of underivatized glycans with regard to the recording of sialylated structures. Regarding fast and yet detailed structural assignment, LC-MS on graphitic carbon supersedes all other current methods.     

Release of glycopeptides and mucopolysaccharides from ascites hepatoma cells by tryptic treatment.

Two types of ascites hepatoma cells, AH 66 and AH 130 FN, were treated with trypsin to observe the release of complex carbohydrates constituting the plasma membranes. From AH 66 cells, mucopolysaccharide (heparan sulfate) was preferentially released. From AH 130 FN cells, N-glycosidic glycopeptides were preferentially released whereas no mucopolysaccharide (chondroitin sulfate A) was released.     

Separation of glycopeptides derived from brain glycoproteins by gel filtration.

Glycopeptides obtained from rat brain by proteolytic digestion with papain have been separated from glycosaminoglycans by means of gel filtration. The glycosaminoglycans appear in the void volume, whereas the glycopeptides are retarded. Glycopeptides of groups A+B (Brunngraber et al., 1973) (MW = 3800-500) C+D (MW = 2000) which were partially resolved by the method, were identified in the elution profile. Nucleic acids, also solubilized by papain, are eluted together with the glycosaminoglycans.     

Characterization of glycoprotein digests with hydrophilic interaction chromatography and mass spectrometry

A new hydrophilic interaction chromatography (HILIC) column packed with amide 1.7 μm sorbent was applied to the characterization of glycoprotein digests. Due to the impact of the hydrophilic carbohydrate moiety, glycopeptides were more strongly retained on the column and separated from the remaining nonglycosylated peptides present in the digest. The glycoforms of the same parent peptide were also chromatographically resolved and analyzed using ultraviolet and mass spectrometry detectors. The HILIC method was applied to glyco-profiling of a therapeutic monoclonal antibody and proteins with several N-linked and O-linked glycosylation sites. For characterization of complex proteins with multiple glycosylation sites we utilized 2D LC, where RP separation dimension was used for isolation of glycopeptides and HILIC for resolution of peptide glycoforms. The analysis of site-specific glycan microheterogeneity was illustrated for the CD44 fusion protein.     

Porcine oocyte zona pellucida M(r) 55,000 glycoproteins: identification of O-glycosylated domains.

The distribution of O-linked oligosaccharides on the M(r) 55,000 glycoproteins, ZP3 alpha and ZP3 beta, of the porcine oocyte zona pellucida was examined. Purified preparations of endo-beta-galactosidase digested ZP3 alpha and ZP3 beta were reduced and carboxamidomethylated and digested with trypsin. When the trypsin digests were mapped by HPLC, each glycoprotein yielded only one N-acetylgalactosamine containing glycopeptide. Purification of the O-glycopeptides was achieved by a two-step protocol. Tryptic digests were applied to jacalin-agarose and specifically-bound O-glycopeptides (alpha OGP and beta OGP) were eluted with buffer containing 50 mM alpha-methylgalactoside as the haptenic sugar. Further purification of each O-glycopeptide was accomplished by reverse phase HPLC. Purified O-glycopeptides were characterized with respect to amino acid and carbohydrate compositions and sequenced by automated Edman degradation; alpha OGP was a 41-residue glycopeptide with three O-linked sugar chains. Sequence comparisons revealed a 75% identity between alpha OGP and a corresponding segment of rabbit rec55 zona protein; beta OGP was a 25-residue glycopeptide characterized by the presence of one N-linked and five O-linked sugar chains and a trypsin-resistant internal arginine residue. Sequence alignments revealed an 80% or greater identity between beta OGP and internal peptides of mouse, hamster and human ZP3 zona proteins. These studies demonstrate that in the case of ZP3 alpha and ZP3 beta, the pig homologues of rabbit rec55 and mouse ZP3, respectively, O-linked oligosaccharides are confined within delimited domains rather than widely dispersed on the polypeptide backbone. Such clustering of O-linked oligosaccharides may represent an essential determinant of the structure and biological activity of zona proteins.     

Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with α-methylmannoside, constitute about 25–30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chrolide columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with α-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.     

Chemical studies on the cysteine and terminal peptides in tryptic digests of actin

1. On exhaustive digestion of carboxymethylated actin in 6m-urea solutions with carboxypeptidase A, 1 mole of phenylalanine was liberated/43000g. of protein. At a lower urea concentration and in the absence of urea, carboxymethyl-cysteine (CMCys) was also liberated. 2. Three cysteine-containing peptides were identified by the study of peptide ;maps' of tryptic digests of actin treated with thiol reagents. 3. The three peptides, each containing one residue of CMCys, were isolated from tryptic digests of carboxymethylated actin by ion-exchange chromatography. 4. One of these peptides was possibly the N-terminal peptide and contained about 17-18 residues; another was CMCys-Asp-Ile-Asp-Ile-Arg; the other, CMCys-Phe, was the C-terminal tryptic peptide. 5. The chemical evidence suggests that the actin molecule consists of a single polypeptide chain of molecular weight about 44000.