Glycoprotein Glycans Research Articles

Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline

Background An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the transglycosylation activity of endo-β- N-acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N-acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one. Methods We carried out the transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk. Results Endo-M-N175Q showed efficient transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield. Conclusions Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed transglycosylation. General significance Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.

Towards Glycoengineering in Archaea: Replacement of Haloferax volcanii AglD with Homologous Glycosyltransferases from Other Halophilic Archaea

Like eukarya and bacteria, archaea also perform N-glycosylation. However, the N-linked glycans of archaeal glycoproteins present a variety not seen elsewhere. Archaea accordingly rely on N-glycosylation pathways likely involving a broad range of species-specific enzymes. To harness the enormous applied potential of such diversity for the generation of glycoproteins bearing tailored N-linked glycans, the development of an appropriate archaeal glycoengineering platform is required. With a sequenced genome, a relatively well-defined N-glycosylation pathway, and molecular tools for gene manipulation, the haloarchaeon Haloferax volcanii (Hfx. volcanii) represents a promising candidate. Accordingly, cells lacking AglD, a glycosyltransferase involved in adding the final hexose of a pentasaccharide N-linked to the surface (S)-layer glycoprotein, were transformed to express AglD homologues from other haloarchaea. The introduction of nonnative versions of AglD led to the appearance of an S-layer glycoprotein similar to the protein from the native strain. Indeed, mass spectrometry confirmed that AglD and its homologues introduce the final hexose to the N-linked S-layer glycoprotein pentasaccharide. Heterologously expressed haloarchaeal AglD homologues contributed to N-glycosylation in Hfx. volcanii despite an apparent lack of AglD function in those haloarchaea from where the introduced homologues came. For example, although functional in Hfx. volcanii, no transcription of the Halobacterium salinarum aglD homologue, OE1482, was detected in cells of the native host grown under various conditions. Thus, at least one AglD homologue works more readily in Hfx. volcanii than in the native host. These results warrant the continued assessment of Hfx. volcanii as a glycosylation "workshop."

N-linked glycan analysis of glycoproteins secreted from rice cell suspension cultures under sugar starvation

Recently, the rice α-amylase 3D promoter system activated under sucrose-starved conditions has emerged as an alternative system for the production of useful recombinant proteins. However, the influence of N-linked glycan structures related to sugar starvation conditions has not yet been investigated. In this study, we determined the N-linked glycans of glycoproteins and human granulocyte-macrophage colony stimulating factor secreted from rice cell suspension cultures under sugar starvation conditions. The N-glycan patterns obtained from glycoproteins grown in growth medium with sucrose (+S) were different from those obtained under sucrose starvation (−S). The mass spectra of +S glycans exhibited eleven N-glycan patterns. Among these, the most abundant glycans were GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc (27.4% of total), followed by Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, GlcNAcβ1-2Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, and Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, each of which represented 15–17% of the total. Except for Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, all +S N-glycans were complex-types containing β1,3-galactose or α1,4-fucose. The five N-glycans that disappeared under −S conditions were GlcNAcβ1-2Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4GlcNAc, GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4GlcNAc, Galβ1-3GlcNAcβ1-2Manα1-6(Galβ1-3GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-6(Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4GlcNAc, and Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-6(Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc. The N-glycan pattern of glycoproteins included hGM-CSF derived from rice suspension cell culture, and all N-glycans contained plant-specific β1,2-xylose, core α1,3-fucose, and some Lewis a epitopes. High-mannose type N-glycans were not detected under either condition (−S or +S) among the secreted glycoproteins.

Thermal decomposition of β- d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy- d-hexopyranoses under neutral conditions

β- d-Galactopyranosyl-(1→3)-2-acetamido-2-deoxy- d-glucose (LNB) and β- d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy- d-galactose (GNB) decompose rapidly upon heating into d-galactose and mono-dehydrated derivatives of the corresponding 2-acetamido-2-deoxy- d-hexoses, including 2-acetamido-2,3-dideoxy-hex-2-enofuranoses and bicyclic 2-acetamido-3,6-anhydro-2-deoxy-hexofuranoses. The decomposition is conducted under neutral conditions where glycosyl linkages are generally believed to be stable. The half-lives of LNB and GNB were 8.1 min and 20 min, respectively, at 90 °C and pH 7.5. The pH dependency of decomposition rates suggests that the instabilities are an extension of the conditions for the peeling reaction, often observed with glycans of O-linked glycoproteins under alkaline conditions. Such decomposition under the neutral conditions is commonly observed with 3-O-linked reducing aldoses.

Biotechnological production and applications of N-acetyl-d-neuraminic acid: current state and perspectives

N-Acetyl-D-neuraminic acid (Neu5Ac) and its derivates are a very important group of biomolecules because these sugars occupy the terminal positions in numerous macromolecules, such as the glycans of glycoproteins, and are involved in many biological and pathological phenomena. The synthesis and applications of Neu5Ac are attracting much interest due to the potential applications of this compound in the pharmaceutical industry, such as in the synthesis of the anti-flu drug zanamivir. In this review article, we discuss existing knowledge on the biotechnological production and applications of Neu5Ac and also propose some guidelines for future studies.

In Situ trans Ligands of CD22 Identified by Glycan-Protein Photocross-linking-enabled Proteomics

CD22, a regulator of B-cell signaling, is a siglec that recognizes the sequence NeuAcalpha2-6Gal on glycoprotein glycans as ligands. CD22 interactions with glycoproteins on the same cell (in cis) and apposing cells (in trans) modulate its activity in B-cell receptor signaling. Although CD22 predominantly recognizes neighboring CD22 molecules as cis ligands on B-cells, little is known about the trans ligands on apposing cells. We conducted a proteomics scale study to identify candidate trans ligands of CD22 on B-cells by UV photocross-linking CD22-Fc chimera bound to B-cell glycoproteins engineered to carry sialic acids with a 9-aryl azide moiety. Using mass spectrometry-based quantitative proteomics to analyze the cross-linked products, 27 glycoproteins were identified as candidate trans ligands. Next, CD22 expressed on the surface of one cell was photocross-linked to glycoproteins on apposing B-cells followed by immunochemical analysis of the products with antibodies to the candidate ligands. Of the many candidate ligands, only the B-cell receptor IgM was found to be a major in situ trans ligand of CD22 that is selectively redistributed to the site of cell contact upon interaction with CD22 on the apposing cell.

Evaluation of the effect of the immunopurification‐based procedures on the CZE‐UV and CZE‐ESI‐TOF‐MS determination of isoforms of intact α‐1‐acid glycoprotein from human serum

Differences in alpha-1-acid glycoprotein (AGP) peptidic and glycan moieties originate several isoforms, whose modifications have been related to different pathophysiological situations. Differences in the isoforms of AGP existing in serum of individuals suffering from different diseases compared to healthy ones could be potentially used as biomarkers. CZE has been proven to be a useful technique for the analysis of glycoprotein isoforms. However, direct CZE analysis of AGP isoforms in serum samples needs efficient purification methods that allow the protein analysis. In this work two new and fast methods to purify AGP from human serum are evaluated in regard to their effect on the determination of isoforms of the intact glycoprotein by CZE-UV and by a developed CZE-ESI-TOF-MS method. Both preparation methods, which differ in the pre-treatment of the sample prior to an anti-AGP immunochromatographic step are shown to be adequate to analyze isoforms of intact AGP. Comparison of both purification methods by CZE-UV and CZE-ESI-TOF-MS indicates that serum AGP purified without acidic precipitation as pre-treatment is more adequate due to AGP higher yield, which leads to better CZE-Mass spectra. Both CZE methods show no indication that acidic precipitation influences the glycosylation (including sialylation) of AGP.

Quantitation of Saccharide Compositions of O-glycans by Mass Spectrometry of Glycopeptides and Its Application to Rheumatoid Arthritis

Profiling of oligosaccharide structures is widely utilized for both identification and evaluation of glycobiomarkers, and site-specific profiling of N-linked glycans of glycoproteins is conducted by mass spectrometry of glycopeptides. However, our knowledge of mucin-type O-glycans including site occupancy and profile variance, as well as attachment sites, is quite limited. Saccharide compositions and site-occupancy of O-glycans were calculated from the signal intensity of glycopeptide ions in the mass spectra and tandem mass spectra from electron transfer dissociation. The results for two major plasma glycoproteins, IgA1 and hemopexin, representing clustered and scattered O-glycan attachments, respectively, indicated that the variability in modifications among individuals is so small as to justify rigorous standards enabling reliable detection of disease-related alterations. Indeed, this method revealed a novel abnormality associated with rheumatoid arthritis: a significant decrease in the N-acetylgalactosamine content of IgA1 O-glycans, indicating that the glycosylation abnormality is not limited to hypogalactosylation of IgG N-glycans in chronic inflammatory conditions.

Glycophenotype of prostatic carcinomas

The factors that affect the progression of prostatic carcinoma are poorly understood, but it is known that carbohydrate antigens on the tumour cell surface play a role in the transforming and metastatic processes. The present report aimed to perform a comparative, lectin-histochemical study of benign and carcinomatous prostates, using a battery of 15 lectins, in combination with monoclonal antibodies against Lewis antigens, and a semi quantitative study, to investigate the changes in glycosylation patterns that occur in prostatic carcinoma. Blocks from 27 necropsy cases of prostatic carcinoma were sectioned and stained with H&E, 15 biotinylated lectins chosen to probe for a wide range of oligosaccharide sequences within several categories of glycoprotein glycans, using a lectin-biotin avidin–peroxidase method, and monoclonal antibodies against Lewisa, sialyl Lewisa and sialyl Lewisx antigens. The glycophenotype of prostatic carcinoma differed from that of the noncancerous prostate in revealing more intense staining with the following lectins (AAA, UEA-1, DBA, WFA, VVA, HPA, BSA-1B4, MPA, ECA, AHA and CTA), while the binding patterns of (GNA and NPA) were almost similar in both prostatic carcinoma and the noncancerous prostate. Lewis antigens are found to be expressed in prostatic carcinomas but not in the noncancerous prostate. The observations of this study suggest that the gylcophenotype of transformed prostatic cells was modified. It showed a moderate increase in, and changing patterns of, fucosylation and galactosylation, increased branching of side chains and sharp rise in 2 deoxy, 2 acetamido galactosylation and masking process by sialylation, especially by α2–3 and α2–6 linkages. O-glycans seems to play an important role in the glycosylation patterns found in prostate carcinoma cells.

Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus

Carbohydrate polymers are medically and industrially important. The S-layer of many Gram-positive organisms comprises protein and carbohydrate polymers and forms an almost paracrystalline array on the cell surface. Not only is this array important for the bacteria but it has potential application in the manufacture of commercially important polysaccharides and glycoconjugates as well. The S-layer glycoprotein glycan from Geobacillus stearothermophilus NRS 2004/3a is mainly composed of repeating units of three rhamnose sugars linked by α-1,3-, α-1,2-, and β-1,2-linkages. The formation of the β-1,2-linkage is catalysed by the enzyme WsaF. The rational use of this system is hampered by the fact that WsaF and other enzymes in the pathway share very little homology to other enzymes. We report the structural and biochemical characterisation of WsaF, the first such rhamnosyltransferase to be characterised. Structural work was aided by the surface entropy reduction method. The enzyme has two domains, the N-terminal domain, which binds the acceptor (the growing rhamnan chain), and the C-terminal domain, which binds the substrate (dTDP-β-l-rhamnose). The structure of WsaF bound to dTDP and dTDP-β-l-rhamnose coupled to biochemical analysis identifies the residues that underlie catalysis and substrate recognition. We have constructed and tested by site-directed mutagenesis a model for acceptor recognition.

Definitive evidence that a single N-glycan among three glycans on inducible costimulator is required for proper protein trafficking and ligand binding

Glycosylation is a widespread post-translational modification found in glycoproteins. Glycans play key roles in protein folding, quality control in the endoplasmic reticulum (ER) and protein trafficking within cells. However, it remains unclear whether all positions of protein glycosylation are involved in glycan functions, or if specific positions have individual roles. Here we demonstrate the integral involvement of a specific N-glycan from amongst the three glycans present on inducible costimulator (ICOS), a T-cell costimulatory molecule, in proper protein folding and intracellular trafficking to the cell surface membrane. We found that glycosylation-defective mutant proteins lacking N-glycan at amino-acid position 89 (N89), but not proteins lacking either N23 or N110, were retained within the cell and were not detected on the cell surface membrane. Additional evidence suggested that N89 glycosylation was indirectly involved in ICOS ligand binding. These data suggest that amongst the three putative ICOS glycosylation sites, N89 is required for proper ICOS protein folding in the ER, intracellular trafficking and ligand binding activity. This study represents a substantial contribution to the current mechanistic understanding of the necessity and potential functions of a specific N-glycan among the multiple glycans of glycoproteins.

Synthesis of Bivalent Lactosides Based on Terephthalamide, N,N′-Diglucosylterephthalamide, and Glycophane Scaffolds and Assessment of Their Inhibitory Capacity on Medically Relevant Lectins

Glycan recognition by lectins initiates clinically relevant processes such as toxin binding or tumor spread. Thus, the development of potent inhibitors has a medical perspective. Toward this goal, we report the synthesis of both rigid and flexible bivalent lactosides on scaffolds that include secondary and tertiary terephthalamides and N,N'-diglucosylterephthalamides. Construction of these compounds involved Schmidt-Michel glycosidation, and amide coupling or Ugi reactions of relevant glycosyl amines in key steps. A glycocluster based on a rigid glycophane was also prepared from coupling of a glucuronic acid derivative and p-xylylenediamine with subsequent ring-closing metathesis. Finally, a more flexible bivalent lactoside was produced from lactosyl azide with use of the copper-catalyzed azide-alkyne cycloaddition. Distances between lactose residues were analyzed computationally as were their orientations for the relatively rigid subset of compounds. Distinct spacing properties were revealed by varying the structure of the scaffold or by varying the location of the lactose residue on the scaffold. To relate these features to bioactivity a plant toxin and human adhesion/growth-regulatory galectins were used as sensors in three types of assay, i.e. measuring agglutination of erythrocytes, binding to glycans of a surface-immobilized glycoprotein, or binding to human cells. Methodologically, the common hemeagglutination assay was found to be considerably less sensitive than both solid-phase and cell assays. The bivalent compounds were less effective at interfering with glycoprotein binding to the plant toxin than to human lectins. Significantly, a constrained compound was identified that displayed selectivity in its inhibitory potency between galectin-3 and its proteolytically processed form. Conversely, compounds with a high degree of flexibility showed notable ability to protect human cells from plant toxin binding. The applied conjugation chemistry thus is compatible with the long-term aim to produce potent and selective lectin inhibitors.

Nano-LC and HPLC-Chip–ESI–MS: An Emerging Technique for Glycobioanalysis

In less than 5 years, an impressive number of methods based on nano-LC and HPLC-chip coupled online to MS were developed and implemented to comprehensively address structural heterogeneity of glycoconjugates and glycans in biological matrices. C18, graphitized carbon and amide-based stationary phases were adapted to nanoflow level and on chip format, leading to improved sensitivity of structural analysis and superior level of information on highly complex glycan and glycoconjugate mixtures. This review offers a summary of the recent progress in the application of nano-LC and HPLC-chip-MS in glycoanalytics of glycopeptides, glycoprotein glycans, glycosaminoglycans, oligosaccharides and glycosphingolipids.

Protective effect of N-glycan bisecting GlcNAc residues on -amyloid production in Alzheimer's disease

Alteration of glycoprotein glycans often changes various properties of the target glycoprotein and contributes to a wide variety of diseases. Here, we focused on the N-glycans of amyloid precursor protein whose cleaved fragment, beta-amyloid, is thought to cause much of the pathology of Alzheimer's disease (AD). We previously determined the N-glycan structures of normal and mutant amyloid precursor proteins (the Swedish type and the London type). In comparison with normal amyloid precursor protein, mutant amyloid precursor proteins had higher contents of bisecting GlcNAc residues. Because N-acetylglucosaminyltransferase III (GnT-III) is the glycosyltransferase responsible for synthesizing a bisecting GlcNAc residue, the current report measured GnT-III mRNA expression levels in the brains of AD patients. Interestingly, GnT-III mRNA expression was increased in AD brains. Furthermore, beta-amyloid treatment increased GnT-III mRNA expression in Neuro2a mouse neuroblastoma cells. We then examined the influence of bisecting GlcNAc on the production of beta-amyloid. Both beta-amyloid 40 and beta-amyloid 42 were significantly decreased in GnT-III-transfected cells. When secretase activities were analyzed in GnT-III transfectant cells, alpha-secretase activity was increased. Taken together, these results suggest that upregulation of GnT-III in AD brains may represent an adaptive response to protect them from additional beta-amyloid production.

Development of fully functional proteins with novel glycosylation via enzymatic glycan trimming

Recombinant glycoproteins present unique challenges to biopharmaceutical development, especially when efficacy is affected by glycosylation. In these cases, optimizing the protein's glycosylation is necessary, but difficult, since the glycan structures cannot be genetically encoded, and glycosylation in nonhuman cell lines can be very different from human glycosylation profiles. We are exploring a potential solution to this problem by designing enzymatic glycan optimization methods to produce proteins with useful glycan compositions. To demonstrate viability of this new approach to generating glycoprotein-based pharmaceuticals, the N-linked glycans of a model glycoprotein, ribonuclease B (RNase B), were modified using an α-mannosidase to produce a new glycoprotein with different glycan structures. The secondary structure of the native and modified glycoproteins was retained, as monitored using circular dichroism. An assay was also developed using an RNA substrate to verify that RNase B had indeed retained its function after being subjected to the necessary glycan modification conditions. This is the first study that verifies both activity and secondary structure of a glycoprotein after enzymatic glycan trimming for use in biopharmaceutical development methods. The evidence of preserved structure and function for a modified glycoprotein indicates that extracellular enzymatic modification methods could be implemented in producing designer glycoproteins. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2581–2591, 2009

The N-glycome of human embryonic stem cells

BackgroundComplex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses.ResultsThe data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Lex and H type 2 antennae in sialylated complex-type N-glycans.ConclusionThe N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.

Conotruncal heart defects in three patients with congenital disorder of glycosylation type Ia (CDG Ia)

Congenital heart defects (CHDs) are a group of structural abnormalities of the heart that have a combined incidence of approximately 1% in humans. It is estimated that 4–5% of CHDs...

Structural and Functional Studies of QdtC: An N-Acetyltransferase Required for the Biosynthesis of dTDP-3-Acetamido-3,6-dideoxy-α-d-glucose

3-Acetamido-3,6-dideoxy-alpha-D-glucose or Quip3NAc is an unusual dideoxy sugar found in the O-antigens of various Gram-negative bacteria and in the S-layer glycoprotein glycans of some Gram-positive bacteria. It is produced in these organisms as a dTDP-linked sugar, with five enzymes ultimately required for its biosynthesis. The focus of this investigation is on the enzyme QdtC, a CoA-dependent N-acetyltransferase that catalyzes the last step in the Quip3NAc biosynthetic pathway. For this analysis, three crystal structures were determined: the wild-type enzyme in the presence of acetyl-CoA and two ternary complexes of the enzyme with CoA and either dTDP-D-Quip3N or dTDP-3-amino-3,6-didexoy-alpha-D-galactose (dTDP-D-Fucp3N). Each subunit of the trimeric enzyme is dominated by a left-handed beta-helix motif with 11 turns. The three active sites are located at the subunit-subunit interfaces, and the two dTDP-sugar ligands employed in this study bind to the protein in nearly identical manners. Those residues responsible for anchoring the hexose moieties of the dTDP-sugars to the protein include Glu 141, Asn 159, and Asp 160 from one subunit and His 134 from another subunit. To probe the roles of various amino acid residues in the catalytic mechanism of the enzyme, 10 site-directed mutant proteins were constructed and their kinetic parameters measured. On the basis of these data, a catalytic mechanism is proposed for QdtC in which the acetylation of the sugar amino group does not require a catalytic base provided by the protein. Rather, the sulfur of CoA functions as the ultimate proton acceptor.

The Determination of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) Types of Sialic Acids in Hematopoietic Organ of The Silkworm, Bombyx mori L. (Lepidoptera: Bombycidae

Summary In insects, for a long time, it was generally accepted that members of insect do not have sialic acids which are typical terminal glycans of cells surface glycolipids and glycoproteins, and thus especially important in cell interactions and signaling events. Here, it was reported the occurrence of sialic acid known the most common in all organisms (Neu5Ac and Neu5Gc) in hematopoietic organ of larvae of the silkworm, Bombyx mori. Histochemical and cytochemical analysis of hematopeietic organ sections with the FITC- and Gold-conjugated lectins (Limulus polyphemus, LPA) showed the presence of sialic acids in hematopoietic organ. The results provide further evidence for the existence of sialic acids in insects.

Focused Differential Glycan Analysis with the Platform Antibody-assisted Lectin Profiling for Glycan-related Biomarker Verification

Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery.