Proteomics of glycoproteins based on affinity selection of glycopeptides from tryptic digests

Identification of glycoproteins in complex mixtures derived from either human blood serum or a cancer cell line was achieved in a process involving the steps of (1) reduction and alkylation, (2) proteolysis of all proteins in the mixture with trypsin, (3) affinity chromatographic selection of the glycopeptides with an immobilized lectin, (4) direct transfer of the glycopeptide fraction to a reversed-phase liquid chromatography (RPLC) column and further fractionation by gradient elution, (5) matrix-assisted laser desorption ionization mass spectrometry of individual fractions collected from the RPLC column, and (6) peptide identification based on a database search. The types of glycoproteins analyzed were; (1) N-type glycoproteins of known primary structure, (2) N-type glycoproteins of unknown structure, and (3) O-type glycoproteins glycosylated with a single N-acetylglucosamine. Identification of peptides from complex mixtures was greatly facilitated by either C-terminal sequencing with a carboxypeptidase mixture or by comparing chromatographic behavior and mass to standards, as in the case of a known protein. In addition, deglycosylation of peptides with N glycosidase F was necessary to identify N-type glycoproteins of unknown structure. The strength of this approach is that it is fast and targets specific molecular species or classes of glycoproteins for identification. The weakness is that it does not discriminate between glycoforms.     

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls—fucosylated and sialylated human lactoferrin glycopeptides—and negative controls—high mannose glycopeptides from Saccharomyces cerevisiae—that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.     

Cold stress changes the concanavalin A-positive glycosylation pattern of proteins expressed in the basal parts of rice leaf sheaths

Post-translational modifications such as glycosylation are important for changing the properties and functions of proteins. To analyze the importance of glycosylation during cold stress in rice, a proteomics approach was used. Proteins extracted from the basal part of rice leaf sheaths were separated by two-dimensional polyacrylamide gel electrophoresis, and subjected to lectin blot analysis using concanavalin A. From a total of 250 detected proteins, 22 reacted with the lectin, suggesting that they were N-glycosylated proteins. To determine how N-glycosylation of these proteins is affected by cold stress, rice seedlings were incubated at 5°C for 48 h, and proteins extracted from the basal parts of leaf sheaths were analyzed by the lectin blot assay. Cold stress changed the reactivity toward the lectin for 12 of the 22 glycoproteins. The identity of the 12 proteins was determined by protein sequencing and mass spectrometry with the majority of these glycoproteins being categorized as involved in energy production. Furthermore, calreticulin, one of the 12 glycoproteins, was also phosphorylated as a result of cold stress. These results indicate that cold stress of the basal parts of rice leaf sheaths changes the glycosylation and phosphorylation profiles of calreticulin, a key protein that regulates the quality control of other proteins.     

Selective isolation and analysis of glycoprotein fractions and their glycomes from hepatocellular carcinoma sera

As one of the most important post‐translational modifications, the discovery, isolation, and identification of glycoproteins are becoming increasingly important. In this study, a Con A‐magnetic particle conjugate‐based method was utilized to selectively isolate the glycoproteins and their glycomes from the healthy donor and hepatocellular carcinoma (HCC) case sera. The isolated glycoproteins and their N‐linked glycans were identified by LC‐ESI‐MS/MS and MALDI‐TOF/TOF‐MS, respectively. A total of 93 glycoproteins from the healthy donors and 85 glycoproteins from the HCC cases were identified. There were 34 different glycoproteins shown between the healthy donors (21/34) and the HCC cases (13/34). Twenty‐eight glycans from the healthy donors and 30 glycans from the HCC cases were detected and there were 22 different glycans shown between the healthy donors (10/22) and HCC cases (12/22). Among these glycoproteins, 50 were known to be N‐linked glycoproteins and three novel glycopeptides from two predicted potential glycoproteins were discovered. Moreover, lectin blotting, Western blotting and lectin/glyco‐antibody microarrays were applied to definitely elucidate the change of selective protein expressions and their glycosylation levels, the results indicated that the differences of the identified glycoproteins between the healthy donors and HCC cases were caused by the change of both protein expression and their glycosylation levels.     

Non-Reducing Terminal Fucose within N-Linked Glycan Plays a Significant Role in the Recognition of Human Milk Lactoferrin by the 1CF11 Monoclonal Antibody

We have recently demonstrated that the 1CF11 monoclonal antibody bound human milk lactoferrin (hLf) through the recognition of two distinct portions of the molecule, namely the N-glycan-relevant and -irrelevant structural elements. In this present study, we prepared four immunoreactive peptide fractions containing N-linked glycan from tryptic digests of reduced and alkylated hLf by using a concanavalin A lectin column and reverse-phase HPLC. Deglycosylation of these fractions and a competitive binding assay using fucosylated oligosaccharides revealed that the non-reducing terminal fucose residue in N-linked glycan(s) played a significant role in recognizing the N-glycan-relevant element in hLf by 1CF11.     

A lectin from the Asian horseshoe crabTachypleus tridentatus: purification,specificity and interaction with tumour cells

A lectin from the haemolymph of the Asian horseshoe crabTachypleus tridentatus was purified to homogeneity by affinity chromatography on Sepharose 4B-boundN-acetylneuraminic acid. The specificity of this lectin was studied by haemagglutination inhibition with sialic acid analogues,N-acetylhexosamines and glycoproteins. For the interaction with the agglutinin theN-acetyl group and the glyceryl side chain ofN-acetylneuraminic acid are important, while presence of an aglycon, specially an -glycosidically linked lactose increases affinity to the lectin. The strongest glycoprotein inhibitors were ovine as well as bovine submaxillary mucin andCollocalia mucin, all beingO-chain glycoproteins but carrying completely different carbohydrate chains. The majority ofN-chain proteins were inactive. As the lectin agglutinates human erythrocytes, but not the murine lymphoma lines Eb and ESb or the human colon carcinoma HT 29, these cancer cells apparently lack the Tachypleus tridentatus agglutinin-receptor which is present on red cells andO-chain glycoproteins.Abbreviations TTA Tachypleus tridentatus agglutinin - SDS sodium dodecyl sulfate - BSM bovine sub-maxillary mucin - VCS Vibrio cholerae sialidase - OSM ovine submaxillary mucin - WGA Wheat germ agglutinin - NeuAc N-acetylneuraminic acid.     

Lectin binding and uptake and glycoprotein characterization of isolated porcine aortic endothelial and smooth muscle cells

Endothelial and smooth muscle cells were isolated from porcine aorta and kept in short-term culture. To determine the terminal carbohydate composition of the plasma membranes from both cell populations, the cells were incubated with a panel of fluorescein-labelled lectins. Both cell populations shared a number of terminal carbohydrates, but the N-galactosamine specific lectin Wistaria floribunda agglutinin labelled only the endothelial cells. A lectin which selectively labelled smooth muscle cells was not found. Western blot analysis of isolated endothelial cell membrane glycoproteins indicated that most membrane glycoproteins are labelled by Wistaria floribunda agglutinin.     

Synthesis of Diaryl Sulphonates and N-Acyl-aryl Sulphonamides as Potential Fungicides

A novel lectin (CBA) was isolated from the green alga, Codium barbatum, by conventional chromatographic methods. The hemagglutination-inhibition profile with sugars and glycoproteins indicated that CBA had preferential affinity for complex type N-glycans but not for monosaccharides, unlike the other known Codium lectins specific for N-acetylgalactosamine. CBA consisted of an SS-linked homodimer of a 9257-Da polypeptide containing seven cysteine residues, all of which were involved in disulfide linkages. The cDNA of the CBA subunit coded a polypeptide (105 amino acids) including the signal peptide of 17 residues. The calculated molecular mass from the deduced sequence was 9705 Da, implying that the four C-terminal amino acids of the CBA proprotein subunit were post-translationally truncated to afford the mature subunit (84 amino acids). No significantly similar sequences were found during an in silico search, indicating CBA to be a novel protein. CBA is the first Codium lectin whose primary structure has been elucidated.     

Carbohydrate‐binding agents act as potent trypanocidals that elicit modifications in VSG glycosylation and reduced virulence in Trypanosoma brucei

The surface of Trypanosoma brucei is covered by a dense coat of glycosylphosphatidylinositol‐anchored glycoproteins. The major component is the variant surface glycoprotein (VSG) which is glycosylated by both paucimannose and oligomannose N‐glycans. Surface glycans are poorly accessible and killing mediated by peptide lectin–VSG complexes is hindered by active endocytosis. However, contrary to previous observations, here we show that high‐affinity carbohydrate binding agents bind to surface glycoproteins and abrogate growth of T. brucei bloodstream forms. Specifically, binding of the mannose‐specific Hippeastrum hybrid agglutinin (HHA) resulted in profound perturbations in endocytosis and parasite lysis. Prolonged exposure to HHA led to the loss of triantennary oligomannose structures in surface glycoproteins as a result of genetic rearrangements that abolished expression of the oligosaccharyltransferase TbSTT3B gene and yielded novel chimeric enzymes. Mutant parasites exhibited markedly reduced infectivity thus demonstrating the importance of specific glycosylation patterns in parasite virulence.     

N-glycosylation potential of maize: the human lactoferrin used as a model

In order to determine the N-glycosylation potential of maize, a monocotyledon expression system for the production of recombinant glycoproteins, human lactoferrin was used as a model. The human lactoferrin coding sequence was inserted into the pUC18 plasmid under control of the wheat glutenin promoter. Maize was stably transformed and recombinant lactoferrin was purified from the fourth generation seeds. Glycosylation was analysed by gas chromatography, lectin detection, glycosidase digestions and mass spectrometry. The results indicated that both N-glycosylation sites of recombinant lactoferrin are mainly substituted by typical plant paucimannose-type glycans, with 1,2-xylose and 1,3-linked fucose at the proximal N-acetylglucosamine, and that complex-type glycans with Lewis^a determinants are not present in maize recombinant lactoferrin.     

Chemo-enzymatic synthesis of poly-<Emphasis Type="Italic">N</Emphasis>-acetyllactosamine (poly-LacNAc) structures and their characterization for CGL2-galectin-mediated binding of ECM glycoproteins to biomaterial surfaces

Poly-N-acetyllactosamine (poly-LacNAc) structures have been identified as important ligands for galectin-mediated cell adhesion to extra-cellular matrix (ECM) proteins. We here present the biofunctionalization of surfaces with poly-LacNAc structures and subsequent binding of ECM glycoproteins. First, we synthesized β-GlcNAc glycosides carrying a linker for controlled coupling onto chemically functionalized surfaces. Then we produced poly-LacNAc structures with defined lengths using human β1,4-galactosyltransferase-1 and β1,3-N-acetylglucosaminyltransferase from Helicobacter pylori. These compounds were also used for kinetic characterization of glycosyltransferases and lectin binding assays. A mixture of poly-LacNAc-structures covalently coupled to functionalized microtiter plates were identified for best binding to our model galectin His₆CGL2. We further demonstrate for the first time that these poly-LacNAc surfaces are suitable for further galectin-mediated binding of the ECM glycoproteins laminin and fibronectin. This new technology should facilitate cell adhesion to biofunctionalized surfaces by imitating the natural ECM microenvironment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural Analysis of N-Glycans of Storage Glycoproteins in Soybean (Glycine max. L) Seed

The structures of N-linked sugar chains (N-glycans) of storage glycoproteins in soybean seeds have been identified. Eight pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the storage glycoproteins by reverse-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were first identified by two-dimensional PA-sugar chain mapping and ion-spray mass analysis, considering the results of sugar composition analysis or sequential exoglycosidase digestion. The deduced structures were further analyzed by ion-spray tandem mass spectrometry and 500 MHz ¹H-NMR spectrometry. The eight structures fell into two categories; the major class (96.6%) was a typical high mannose-type, the minor class was a xylose containing-type (Man₃Xyl₁GlcNAc₂, Man₃Fuc₁Xyl₁GlcNAc₂; 3.4%).     

Fine sugar specificity of the mistletoe (Viscum album) lectin I

The behaviour ofN-acetyllactosamine-type oligosaccharides and glycopeptides on a column of mistletoe lectin I (MLI) immobilized on Sepharose 4B was examined. The immobilized lectin does not show any affinity for asialo-N-glycosylpeptides and related oligosaccharides, which possess one to four unmaskedN-acetyllactosamine sequences. However, substitution of at least one of theN-acetyllactosamine sequences by sialic acid residues, either at O-3 or O-6 of galactose, slightly enhances the affinity of the lectin. Such sialylatedN-glycosylpeptides or oligosaccharides are eluted from the lectin column by the starting buffer as retarded fractions. Surprisingly, the affinity of the immobilized MLI is higher for P1 antigen-containing glycopeptide isolated from turtle-dove ovomucoid and for glycopeptides from bovine thyroglobulin containing terminal non-reducing Gal1–3Gal sequences. These structures are strongly bound on the lectin column and their elution is obtained with 0.15M galactose in the starting buffer.In memory of Hartmut Franz.     

Approaches to the study of N-linked glycoproteins in human plasma using lectin affinity chromatography and nano-HPLC coupled to electrospray linear ion trap--Fourier transform mass spectrometry

In this publication, we will describe the combination of lectin affinity chromatography with nano high performance liquid chromatography (HPLC) coupled to a linear ion trap Fourier transform mass spectrometer (capillary LC-LTQ/FTMS) to characterize N-linked glycosylation structures in human plasma proteins. We used a well-characterized glycoprotein, tissue plasminogen activator (rt-PA), which is present at low levels in blood, as a standard to determine the dynamic range of this approach. N-linked glycopeptides derived from rt-PA could be characterized at a ratio of 1:200 in human plasma (rtPA: total plasma protein, w/w) by accurate mass measurement in the FTMS and fragmentation (MS(n)) in the linear ion trap. We demonstrated that this platform has the potential to characterize the general N-linked glycosylation structures of abundant glycoproteins present in human plasma without the requirement for antibody-based purification, or additional carbohydrate analytical protocols. This conclusion was supported by the determination of carbohydrate structures for three glycoproteins, IgG, haptoglobin, and alpha-1-acid glycoprotein, at their natural levels in a human plasma sample, but only after the lectin enrichment step.     

The Cell Wall of Fusarium oxysporum

Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50–60% of the total mass of the wall. X-ray diffraction studies showed the presence of α-1,3-glucan in the alkali-soluble cell wall fraction and of β-1,3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan.     

Structures and contribution to the antigenicity of oligosaccharides of Japanese cedar (Cryptomeria japonica) pollen allergenCry j I: relationship between the structures and antigenic epitopes of plantN-linked complex-type glycans

The oligosaccharide structures ofCry j I, a major allergenic glycoprotein ofCryptomeria japonica (Japanese cedar, sugi), were analysed by 400 MHz¹H-NMR and two-dimensional sugar mapping analyses. The four major fractions comprised a series of biantennary complex type N-linked oligosaccharides that share a fucose/xylose-containing core and glucosamine branches including a novel structure with a nongalactosylated fucosylglucosamine branch.Rabbit polyclonal anti-Cry j I IgG antibodies cross-reacted with three different plant glycoproteins having the same or shorter N-linked oligosaccharides asCry j I. ELISA and ELISA inhibition studies with intact glycoproteins, glycopeptides and peptides indicated that both anti-Cry j I IgGs and anti-Sophora japonica bark lectin II (B-SJA-II) IgGs included oligosaccharide-specific antibodies with different specificities, and that the epitopic structures against anti-Cry j I IgGs include a branch containing 1–6 linked fucose and a core containing fucose/xylose, while those against anti-B-SJA-II IgGs include nonreducing terminal mannose residues. The cross-reactivities of human allergic sera to miraculin andClerodendron Trichotomum lectin (CTA) were low, and inhibition studies suggested that the oligosaccharides onCry j I contribute little or only conformationally to the reactivity of specific IgE antibodies.Abbreviations Cry j I a major allergenic glycoprotein ofCryptomeria japonica - B-SJA-II Sophora japonica bark lectin II - CTA Clerodendron trichotomum lectin - TFMS trifluoromethanesulfonic acid - HRP horseradish peroxidase     

Characterization of N- and O-linked glycosylation changes in milk of the tammar wallaby (Macropus eugenii) over lactation

As one of several biologically active compounds in milk, glycoproteins have been indicated to be involved in the protection of newborns from bacterial infection. As much of the physical and immune development of the tammar wallaby (Macropus eugenii) young occurs during the early phases of lactation and not in utero, the tammar is a model species for the characterization of potential developmental support agents provided by maternal milk. In the present study, the N- and O-linked glycans from tammar wallaby milk glycoproteins from six individuals at different lactation time points were subjected to glycomics analyses using porous graphitized carbon liquid chromatography electrospray ionization mass spectrometry. Structural characterization identified a diverse range of glycan structures on wallaby milk glycoproteins including sialylated, sulphated, core fucosylated and O-fucosylated structures. 30 % of N-linked structures contained a core (α1-6) fucose. Several of these structures may play roles in development, and exhibit statistically significant temporal changes over the lactation period. The N-glycome was found to contain structures with NeuGc residues, while in contrast the O-glycome did not. O-fucosylated structures were identified in the early stages of lactation indicating a potential role in the early stages of development of the pouch young. Overall the results suggest that wallaby milk contains structures known to have developmental and immunological significance in human milk and reproduction in other animals, highlighting the importance of glycoproteins in milk.     

Purification and Characterization of a Lectin from Crotalaria paulina Seeds

A lectin was purified from Crotalaria paulina seeds by ion-exchange and FPLC molecular exclusion chromatography. CrpL had an apparent molecular mass of 30 kDa, as determined by SDS-PAGE under non-reducing and reducing conditions. CrpL effectively agglutinated human and cow erythrocytes, and this activity was not affected by 20 mM EDTA, showing no dependence of divalent cations. Hemagglutination was inhibited by N-acetyl- D-galactosamine, D-galactose and was also inhibited by glycoproteins, fetuin and asialofetuin. The N-terminal amino acid sequence of CrpL was identical to those of other lectins from the genus Crotalaria, and amino acid composition showed high amounts of Asx and Glx, and was rich in Gly, Ala and Ser, as also reported for lectins from other Crotalaria species. CrpL inhibited the growth of Xanthomonas axonopodis pv. phaseoli and Xanthomonas axonopodis pv. passiflorae, suggesting a role of this lectin in the defense of seeds against bacterial infections.     

Exposure of Trypanosoma brucei to an N-acetylglucosamine-Binding Lectin Induces VSG Switching and Glycosylation Defects Resulting in Reduced Infectivity

Trypanosoma brucei variant surface glycoproteins (VSG) are glycosylated by both paucimannose and oligomannose structures which are involved in the formation of a protective barrier against the immune system. Here, we report that the stinging nettle lectin (UDA), with predominant N-acetylglucosamine-binding specificity, interacts with glycosylated VSGs and kills parasites by provoking defects in endocytosis together with impaired cytokinesis. Prolonged exposure to UDA induced parasite resistance based on a diminished capacity to bind the lectin due to an enrichment of biantennary paucimannose and a reduction of triantennary oligomannose structures. Two molecular mechanisms involved in resistance were identified: VSG switching and modifications in N-glycan composition. Glycosylation defects were correlated with the down-regulation of the TbSTT3A and/or TbSTT3B genes (coding for oligosaccharyltransferases A and B, respectively) responsible for glycan specificity. Furthermore, UDA-resistant trypanosomes exhibited severely impaired infectivity indicating that the resistant phenotype entails a substantial fitness cost. The results obtained further support the modification of surface glycan composition resulting from down-regulation of the genes coding for oligosaccharyltransferases as a general resistance mechanism in response to prolonged exposure to carbohydrate-binding agents.     

Subcellular characterization of glycoproteins in the principal cells of human gallbladder

Gallbladder mucus is mainly composed of glycoproteins, which seem to play a critical role in cholesterol nucleation during gallstone formation. The biosynthetic pathway and sequential processing as well as the characterization of the oligosaccharide sidechains of human gallbladder secretory glycoproteins have not been completely defined. The aim of the present study is the subcellular characterization of the glycoproteins in the principal cells of human gallbladder. Principal cells of normal human gallbladder were studied by means of a variety of cytochemical techniques, including lectin histochemistry, enzyme and chemical treatments, immunocytochemistry and lectin-gold technology. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid residues were detected in mucous granules, Golgi apparatus and apical membrane of principal cells. Mannose residues were only observed in dense bodies. Oligosaccharide side-chains of the glycoproteins contained in the biliary mucus are synthesized in the Golgi apparatus of the principal cells of the gallbladder epithelium and are also contained in the mucous granules of these cells. Terminal N-acetylneuraminic acid(2-3)galactose(1-3)N-acetylgalactosamine, N-acetylneuraminic acid(2-3)galactose(1-4)N-acetylglucosamine and galactose(1-4)N-acetylglucosamine sequences are contained in the oligosaccharide chains of gallbladder mucus glycoproteins. The dense bodies detected in the cytoplasm of the principal cells contained N-linked glycoproteins. Mucin-type O-linked glycoproteins were the main components of the mucous granules although some N-linked chains were also detected.