Oligosaccharides Of Glycoproteins Research Articles

Site‐Specific Protein Modification with Reducing Carbohydrates

AbstractProtein glycosylation plays critical roles in many biological processes. However, the fundamental study and application of glycobiology are hindered by the heterogeneousness of oligosaccharides in natural glycoproteins and the difficulty in constructing glycoproteins of human design. Herein, we describe a semisynthetic method to site‐specifically modify proteins with reducing carbohydrates. The method involves the genetic incorporation of a side‐chain‐esterified aspartate, which was subsequently quantitatively converted into alanine‐β‐hydrazide (Aβz), and chemoselective conjugation of Aβz with a range of readily available reducing carbohydrates. The resulting Aβz‐linked GlcNAc is a close mimic of native N‐GlcNAc and could be installed on various proteins, including IL‐17A and RNase A. Notably, Aβz‐linked GlcNAc on proteins reacted with biantennary oligosaccharide oxazoline derivatives through endoglycosidase‐catalyzed transglycosylation reactions to enable the assembly of homogeneous glycans on proteins.

Site-Specific Protein Modification with Reducing Carbohydrates.

Protein glycosylation plays critical roles in many biological processes. However, the fundamental study and application of glycobiology are hindered by the heterogeneousness of oligosaccharides in natural glycoproteins and the difficulty in constructing glycoproteins of human design. Herein, we describe a semisynthetic method to site-specifically modify proteins with reducing carbohydrates. The method involves the genetic incorporation of a side-chain-esterified aspartate, which was subsequently quantitatively converted into alanine-β-hydrazide (Aβz), and chemoselective conjugation of Aβz with a range of readily available reducing carbohydrates. The resulting Aβz-linked GlcNAc is a close mimic of native N-GlcNAc and could be installed on various proteins, including IL-17A and RNase A. Notably, Aβz-linked GlcNAc on proteins reacted with biantennary oligosaccharide oxazoline derivatives through endoglycosidase-catalyzed transglycosylation reactions to enable the assembly of homogeneous glycans on proteins.

Overexpression of cell-wall GPI-anchored proteins restores cell growth of N-glycosylation-defective och1 mutants in Schizosaccharomyces pombe.

The glycoproteins of yeast contain a large outer chain on N-linked oligosaccharides; therefore, yeast is not suitable for producing therapeutic glycoproteins for human use. Using a deletion mutant strain of α1,6-mannosyltransferase (och1Δ), we previously produced humanized N-glycans in fission yeast; however, the Schizosaccharomyces pombe och1Δ cells displayed a growth delay even during vegetative growth, resulting in reduced productivity of heterologous proteins. To overcome this problem, here we performed a genome-wide screen for genes that would suppress the growth defect of temperature-sensitive och1Δ cells. Using a genomic library coupled with screening of 18,000 transformants, we identified two genes (pwp1+, SPBC1E8.05), both encoding GPI-anchored proteins, that increased the growth rate of och1Δ cells, lacking the outer chain. We further showed that a high copy number of the genes was needed to improve the growth rate. Mutational analysis of Pwp1p revealed that the GPI-anchored region of Pwp1p is important in attenuating the growth defect. Analysis of disruptants of pwp1+ and SPBC1E8.05 showed that neither gene was essential for cell viability; however, both mutants were sensitive β-glucanase, suggesting that Pwp1p and the protein encoded by SPBC1E8.05 non-enzymatically support β-glucan on the cell-surface of S. pombe. Collectively, our work not only sheds light on the functional relationships between GPI-anchored proteins and N-linked oligosaccharides of glycoproteins in S. pombe, but also supports the application of S. pombe to the production of human glycoprotein. KEY POINTS: • We screened for genes that suppress the growth defect of fission yeast och1Δ cells. • Appropriate expression of GPI-anchored proteins alleviates the growth delay of och1Δ cells. • The GPI-anchor domain of Pwp1p is important for suppressing the growth defect of och1Δ cells.

Substrate specificities of α1,2- and α1,3-galactosyltransferases and characterization of Gmh1p and Otg1p in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe, α1,2- and α1,3-linked D-galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases. Although the galactomannans are important for cell-cell communication in S. pombe (e.g., in nonsexual aggregation), the mechanisms underlying galactosylation in cells remain unclear. Schizosaccharomyces pombe has 10 galactosyltransferase-related genes: seven belonging to glycosyltransferase (GT) family 34 and three belonging GT family 8. Disruption of all 10 α-galactosyltransferases (strain Δ10GalT) has been shown to result in a complete lack of α-Gal residues. Here, we have investigated the function and substrate specificities of galactosyltransferases in S pombe by using strains expressing single α-galactosyltransferases in the Δ10GalT background. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides showed that two GT family 34 α1,2-galactosyltransferases (Gma12p and Gmh6p) and two GT family 8 α1,3-galactosyltransferases (Otg2p and Otg3p) are involved in galactosylation of O-linked oligosaccharide. Moreover, 1H-NMR of N-glycans revealed that three GT family 34 α1,2-galactosyltransferases (Gmh1p, Gmh2p and Gmh3p) are required for the galactosylation of N-linked oligosaccharides. Furthermore, HPLC and lectin-blot analysis revealed that Otg1p showed α1,3-galactosyltransferase activity under conditions of co-expression with Gmh6p, indicating that α-1,2-linked galactose is required for the galactosylation activity of Otg1p in S. pombe. In conclusion, eight galactosyltransferases have been shown to have activity in S. pombe with different substrate specificities. These findings will be useful for genetically tailoring the galactosylation of both N- and O-glycans in fission yeast.

NUP62: the target of an anti-sperm auto-monoclonal antibody during testicular development.

Ts4, an autosperm-monoclonal antibody (mAb), reacts with a specific oligosaccharide (OS) of glycoproteins containing bisecting N-acetylglucosamine residues. Ts4 reactivity was observed against epididymal spermatozoa, testicular germ cells, and the early embryo, but not against major organs in adult mice. In mature testis, Ts4 exhibits immunoreactivity with a germ cell-specific glycoprotein, TEX101, whereas the mAb immunoreacts with alpha-N-acetylglucosaminidase in the acrosomal region of cauda epididymal spermatozoa. Thus, Ts4 seems to react against different molecules throughout spermiogenesis via binding to its OS epitope. Since the Ts4-epitope OS is observed only in reproduction-related regions, the Ts4-reactive OS may play a role in the reproductive process. The aim of this study is to investigate the characteristics of the Ts4-reactive molecule(s) during testicular development. Ts4 reactivity was observed in testes from the prenatal period; however, its distribution changed according to the stage of maturation and was identical to that of the adult testes after 29-day-postpartum (dpp). Ts4 immunoreactivity was detected against a protein with 63 kDa in testis from 1 to 29 dpp. In contrast, Ts4 showed reactivity against some other glycoproteins after 29 dpp, including TEX101 at the 5-week-old stage and onward. To identify the Ts4-reactive 63 kDa molecule, we identified NUP62 as the target of Ts4 in 22 dpp testis using liquid chromatography-tandem mass spectrometry analysis. Because NUP62 has been known to play active roles in a variety of cellular processes including mitosis and cell migration, the bisecting GlcNAc recognized by Ts4 on NUP62 may play a role in regulating the early development of germ cells in male gonadal organs.

Differential expression of N-linked oligosaccharides in methotrexate-resistant primary central nervous system lymphoma cells

BackgroundOligosaccharides of glycoprotein, particularly negatively-charged sialylated N-glycans, on the surface of lymphomas play important roles in cell–cell interactions and bind immunoglobulin-like lectins, causing inflammatory responses and bioregulation. However, their characterizations have largely been unknown in central nervous system (CNS) lymphoma.MethodsHere, we investigated expression patterns of N-linked oligosaccharides of glycoproteins in cells derived from CNS lymphomas and clinical specimens.ResultsWe first generated methotrexate (MTX)-resistant cells derived from HKBML and TK as CNS lymphoma, and RAJI as non-CNS lymphoma and determined N-linked oligosaccharide structures in these cells and other non-CNS lymphoma-derived cells including A4/FUK, OYB, and HBL1. Major components of the total oligosaccharides were high-mannose type N-glycans, whose level increased in MTX-resistant HKBML and TK but decreased in MTX-resistant RAJI. We also detected sialylated biantennary galactosylated N-glycans with α1,6-fucosylation, A2G2F, and A2G2FB from HKBML, TK, and RAJI. Sialylated A4G4F was specifically isolated from RAJI. However, the ratios of these sialylated N-glycans slightly decreased against MTX-resistant compared to non-resistant cells. Interestingly, almost all complex-type oligosaccharides were α2,6-sialylated.DiscussionThis is the first study for the expression profile of N-oligosaccharides on MTX-resistant primary CNS lymphoma-derived cells HKBML and TK, and tumor tissues resected from patients with CNS lymphoma,ConclusionThese results propose a possibility that the differential expression of high-mannose types and sialylated A2G2F, A2G2FB, and A4G4F on the surface of CNS lymphomas may provide a hint for targets for diagnoses and treatments of the oligosaccharide type-specific lymphomas.

Characterization of MiFUT11 from Mangifera indica L.: A functional core α1,3-fucosyltransferase potentially involved in the biosynthesis of immunogenic carbohydrates in mango fruit

In higher plants, asparagine-linked oligosaccharides (N-glycans) in glycoproteins carry unique carbohydrate epitopes, namely, a core α1,3-fucose and/or a β1,2-xylose, which are common determinants responsible for the cross-reactivity of plant glycoproteins due to their strong immunogenicity. While these determinants and the relevant genes have been well characterized for herbaceous plants, information concerning whether many food plants cross-react with airborne pollens is not available. In this paper, we report on the characterization of a novel core α1,3-fucosyltransferase gene identified from Mangifera indica L., one of the major plants potentially related to food allergy. Based on sequence information of plant homologues, we amplified a candidate cDNA (MiFUT11) from pericarp tissue. An in vitro assay demonstrated that the recombinant MiFUT11 protein transfers a fucose unit onto both non-fucosylated and core α1,6-fucosylated oligosaccharides. A glycoform analysis using MALDI-TOF mass spectrometry showed that the introduction of the MiFUT11 cDNA increased the production of a core α1,3- and α1,6-fucosylated pauci-mannosidic oligosaccharide in Spodoptera Sf21 cells. Our findings suggest that MiFUT11 is a functional core α1,3-fucosyltransferase gene that is involved in the assembly of cross-reactive N-glycans in mango fruit.

Trichophyton rubrum LysM proteins bind to fungal cell wall chitin and to the N-linked oligosaccharides present on human skin glycoproteins.

The Trichophyton rubrum genome contains six proteins containing two or more lysin M (LysM) domains. We have characterized two of these proteins, LysM1 and LysM2, and demonstrated that these proteins have the capacity to bind two substrates, chitin and N-linked oligosaccharides associated with human skin glycoproteins. We have characterized the individual LysM domains in LysM1, and shown that the protein contains two functional LysM domains. Each of these domains can bind to chitin, to N-linked oligosaccharides in human skin glycoproteins, and to N-linked oligosaccharides on fungal glycoproteins. We hypothesize that LysM proteins could provide the pathogen with three important functions. First, the T. rubrum LysM proteins could shield host cell wall chitin from the human immune system. Second, the LysM proteins could shield the pathogen’s glycoproteins from host degradation and immune surveillance. Third, the LysM proteins could help facilitate pathogen adhesion to human skin.

2-3H]Mannose-labeling and Analysis of N-linked Oligosaccharides.

Modifications of N-linked oligosaccharides of glycoproteins soon after their biosynthesis correlate to glycoprotein folding status. These alterations can be detected in a sensitive way by pulse-chase analysis of [2-3H]mannose-labeled glycoproteins, with enzymatic removal of labeled N-glycans, separation according to size by HPLC and radioactive detection in a scintillation counter.

Highly sensitive derivatization reagents possessing positively charged structures for the determination of oligosaccharides in glycoproteins by high-performance liquid chromatography electrospray ionization tandem mass spectrometry

We have developed three kinds of novel derivatization reagents (4-CEBTPP, 4-CBBTPP, 5-COTPP) with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge for resolution of the oligosaccharides in glycoprotein using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS). The synthesized reagents reacted with the sialylglycosylamine of the sialylglycopeptide after treatment by PNGase F. The final derivatives were analyzed by ESI–MS and sensitively detected in the selected reaction monitoring (SRM) mode. Furthermore, the limits of detection (S/N=3) on the SRM chromatograms were at the fmol level (30fmol). Therefore, we used the limit of detection of the reagent products detected by the SRM and evaluated the utility of each reagent. Among the reagents, the positively charged 4-CEBTPP derivative’s peak area was the highest; 4-CEBTPP with a positively charged structure showed about a 20 times greater sensitivity for the glycosylamine of the SGP product compared to the conventional fluorescence reagent, Fmoc-Cl. In addition, various fragment ions based on the carbohydrate units also appeared in the MS/MS spectra. Among the fragment ions, m/z 627.37 (CE=40eV) corresponding to 4-CEBTPP-GlcNAc and m/z 120.09 (CE=100eV) corresponding to 4-CEBTPP are the most important ones for identifying the oligosaccharide. 4-CEBTPP-SGA was easily identified by the selected-ion chromatogram in the product ion scan (m/z 120.09) and in the precursor ion scan (m/z 627.37) by MS/MS detection. The derivatized analytes have a high ionization efficiency and they are detected with a high sensitivity in the electrospray ionization. The novel derivatization reagent with a multi-function provided a higher sensitivity for the oligosaccharide analysis, as well as a better specificity and feasibility. Furthermore, several oligosaccharides in fetuin and ribonuclease B were successfully identified by the proposed procedure.

Studies on the Precise Chemical Synthesis of Human Glycoproteins

Abstract Our research group demonstrated the semi-synthesis of complex-type biantennary sialyloligosaccharide-library by means of branch-specific glycosidase and several sialyltransferase reactions toward complex-type H2N-Asn-(biantennary compex-type sialyloligosaccharide)-COOH isolated from egg yolk at over a gram scale. This methodology yielded 35 kinds of homogeneous complex-type oligosaccharides. Using this oligosaccharide library, an efficient Boc solid phase peptide synthesis (SPPS) of acid labile sialylglycopeptide-α-thioester was demonstrated. Our research group showed that the sialoside in which the carboxylic acid is protected with a phenacyl group is stable under strong acidic conditions, and this critical finding enabled us to demonstrate the first Boc-SPPS for the synthesis of a sialylglycopeptide-α-thioester. Using both a synthetic method of sialylglycopeptide-α-thioester and native chemical ligation (NCL), our research group synthesized several glycoproteins such as chemokine MCP-3, three kinds of erythropoietin analogues, and interferon-ß. Our research group also demonstrated a unique synthesis that was an intentional synthesis of homogeneous misfolded glycoproteins bearing M9-high-mannose-type oligosaccharide. This unique synthetic misfolded-glycoprotein is useful for the purpose of uncovering the mechanism of molecular recognition in glycoprotein quality control (GQC) in the endoplasmic reticulum (ER). This account discusses the function of the oligosaccharides of glycoproteins based on our research findings.

Expression of Fucosyltransferase 8 Is Associated with an Unfavorable Clinical Outcome in Non-Small Cell Lung Cancers

Objecitive: Fucosyltransferase 8 (FUT8), the only enzyme responsible for the core α1,6-fucosylation of asparagine-linked oligosaccharides of glycoproteins, is a vital enzyme in cancer development and progression. We examined FUT8 expression in non-small cell lung cancers (NSCLCs) to analyze its clinical significance. We also examined the expression of guanosine diphosphate-mannose-4,6-dehydratase (GMD), which is imperative for the synthesis of fucosylated oligosaccharides. Methods: Using immunohistochemistry, we evaluated the expression of FUT8 and GMD in relation to patient survival and prognosis in potentially curatively resected NSCLCs. Results: High expression of FUT8 was found in 67 of 129 NSCLCs (51.9%) and was significantly found in non-squamous cell carcinomas (p = 0.008). High expression of FUT8 was associated with poor survival (p = 0.03) and was also a significant and independent unfavorable prognostic factor in patients with potentially curatively resected NSCLCs (p = 0.047). High expression of GMD was significantly associated with high FUT8 expression (p = 0.04). Conclusions: High expression of FUT8 is associated with an unfavorable clinical outcome in patients with potentially curatively resected NSCLCs, suggesting that FUT8 can be a prognostic factor. The analysis of FUT8 expression and its core fucosylated products may provide new insights for the therapeutic targets of NSCLCs.

Determination of major sialylated N-glycans and identification of branched sialylated N-glycans that dynamically change their content during development in the mouse cerebral cortex.

Oligosaccharides of glycoproteins expressed on the cell surface play important roles in cell-cell interactions, particularly sialylated N-glycans having a negative charge, which interact with sialic acid-binding immunoglobulin-like lectins (siglecs). The entire structure of sialylated N-glycans expressed in the mouse brain, particularly the linkage type of sialic acid residues attached to the backbone N-glycans, has not yet been elucidated. An improved method to analyze pyridylaminated sugar chains using high performance liquid chromatography (HPLC) was developed to determine the entire structure of sialylated N-linked sugar chains expressed in the adult and developing mouse cerebral cortices. Three classes of sialylated sugar chains were prevalent: 1) N-glycans containing α(2-3)-sialyl linkages on a type 2 antennary (Galβ(1-4)GlcNAc), 2) sialylated N-glycans with α(2-6)-sialyl linkages on a type 2 antennary, and 3) a branched sialylated N-glycan with a [Galβ(1-3){NeuAcα(2-6)}GlcNAc-] structure, which was absent at embryonic day 12 but then increased during development. This branched type sialylated N-glycan structure comprised approximately 2 % of the total N-glycans in the adult brain. Some N-glycans (containing type 2 antennary) were found to change their type of sialic acid linkage from α(2-6)-Gal to α(2-3)-Gal. Thus, the linkages and expression levels of sialylated N-glycans change dramatically during brain development.Electronic supplementary materialThe online version of this article (doi:10.1007/s10719-014-9566-2) contains supplementary material, which is available to authorized users.

The Involvement of CD146 and Its Novel Ligand Galectin-1 in Apoptotic Regulation of Endothelial Cells

CD146 is a highly glycosylated junctional adhesion molecule, expressed on human vascular endothelial cells and involved in the control of vessel integrity. Galectin-1 is a lectin produced by vascular cells that can binds N- and O-linked oligosaccharides of cell membrane glycoproteins. Because both CD146 and Galectin-1 are involved in modulation of cell apoptosis, we hypothesized that Galectin-1 could interact with CD146, leading to functional consequences in endothelial cell apoptosis. We first characterized CD146 glycosylations and showed that it is mainly composed of N-glycans able to establish interactions with Galectin-1. We demonstrated a sugar-dependent binding of recombinant CD146 to Galectin-1 using both ELISA and Biacore assays. This interaction is direct, with a K(D) of 3.10(-7) M, and specific as CD146 binds to Galectin-1 and not to Galectin-2. Moreover, co-immunoprecipitation experiments showed that Galectin-1 interacts with endogenous CD146 that is highly expressed by HUVEC. We observed a Galectin-1-induced HUVEC apoptosis in a dose-dependent manner as demonstrated by Annexin-V/7AAD staining. Interestingly, both down-regulation of CD146 cell surface expression using siRNA and antibody-mediated blockade of CD146 increase this apoptosis. Altogether, our results identify Galectin-1 as a novel ligand for CD146 and this interaction protects, in vitro, endothelial cells against apoptosis induced by Galectin-1.

Sialyl Lewis x expression in cervical scrapes of premalignant lesions

Sialylated oligosaccharides of glycoproteins and glycolipids have been implicated in tumour progression and metastases. Altered expression of glycosidic antigens has been reported in cervical cancer. In cervix premalignant lesions, an increased expression of sialic acid has been reported. In the present study we determined the expression profiles of the glycosidic antigens Tn, sialyl Tn (sTn), Lewis a (Lea), sialyl Lewis a (sLea), Lewis x (Lex) and sialyl Lewis x (sLex) in cervical scrapes with cytological diagnoses of normal, low-grade squamous intraepithelial lesions (LGSIL) and highgrade squamous intraepithelial lesions (HGSIL). Cervical scrapings were collected to detect tumour antigens expressions by flow cytometry using monoclonal antibodies. Cytometry analysis of Tn, sTn, Lea and Lex did not reveal differences at the expression level among groups. The number of positive cells to sLea antigen increased in the HGSIL group with respect to the normal group (p=0.0495). The number of positive cells to sLex antigen in the samples increased with respect to the grade of squamous intraepithelial lesion (SIL) (p less than 0.001, Mann-Whitney U test). The intensity of expression of this antigen increased in the HGSIL samples with respect to normal samples (p less than 0.0068). sLex antigen could be a candidate to be used as biomarker for the early diagnosis of cervical cancer.

Reconstructed glycan profile for evaluation of operating status of the endoplasmic reticulum glycoprotein quality control

Glycoprotein oligosaccharides function as tags for protein quality control in the endoplasmic reticulum (ER). Since most of proteins are glycosylated and function only after they are properly folded, glycoprotein glycan profiles in the ER might be useful to analyze various cellular status including diseases. Here, we examined whether ER glycan-processing profiles in diabetic rats and osteoporotic mice as models might have different cellular status from those of normal controls. Direct analysis of glycoprotein-processing profiles in the ER is often hampered by glycoforms that are retro-translocated to the ER from other cellular compartments. Moreover, when we focus on the mixture of glycoproteins as the processing substrates, the glycan-processing efficiencies are influenced by the aglycon states including their polypeptide folding. To overcome this problem, we reconstructed glycan profiles using ER extracts as an enzymatic source and synthetic glycoprotein mimetic having homogeneous aglycon as a substrate, resulted in disease-specific glycan profiles. To understand such differences, we also analyzed the activity, and expression level, of each glycan-related enzyme. These glycan profiles are expected to be useful indexes for operational status of the ER glycoprotein quality control, and may also give information to classify some diseases.

N-linked oligosaccharides of glycoproteins are altered during mouse aging and are possible regulators of the IGF-1 signaling pathway

N-linked oligosaccharides of glycoproteins are altered during mouse aging and are possible regulators of the IGF-1 signaling pathway

Heterologous protein expression in Pichia thermomethanolica BCC16875, a thermotolerant methylotrophic yeast and characterization of N-linked glycosylation in secreted protein

This study describes Pichia thermomethanolica BCC16875, a new methylotrophic yeast host for heterologous expression. Both methanol-inducible alcohol oxidase (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoters from Pichia pastoris were shown to drive efficient gene expression in this host. Recombinant phytase and xylanase were expressed from both promoters as secreted proteins, with the former showing different patterns of N-glycosylation dependent on the promoter used and culture medium. In addition, growth temperature also had an effect on N-glycan modification of cell wall mannoproteins. The major glycoprotein oligosaccharide species produced from P.thermomethanolica BCC16875 is Man(8-12) GlcNAc(2) , which is similar to that from other methylotrophs. Moreover, mannosylphosphate and α-1,6- and α-1,2-linked mannose modifications of heterologous secreted protein were also detected. The attainably high level of protein production in complement to distinctive thermotolerance rarely found in other industrial yeasts makes this microorganism an attractive host for large-scale fermentation.

Cleavage of ST6Gal I by Radiation-Induced BACE1 Inhibits Golgi-Anchored ST6Gal I-Mediated Sialylation of Integrin β1 and Migration in Colon Cancer Cells

BackgroundPreviously, we found that β-galactoside α2,6-sialyltransferase (ST6Gal I), an enzyme that adds sialic acids to N-linked oligosaccharides of glycoproteins and is frequently overexpressed in cancer cells, is up-regulated by ionizing radiation (IR) and cleaved to a form possessing catalytic activity comparable to that of the Golgi-localized enzyme. Moreover, this soluble form is secreted into the culture media. Induction of ST6Gal I significantly increased the migration of colon cancer cells via sialylation of integrin β1. Here, we further investigated the mechanisms underlying ST6Gal I cleavage, solubilization and release from cells, and addressed its functions, focusing primarily on cancer cell migration.MethodsWe performed immunoblotting and lectin affinity assay to analyze the expression of ST6 Gal I and level of sialylated integrin β1. After ionizing radiation, migration of cells was measured by in vitro migration assay. α2, 6 sialylation level of cell surface was analyzed by flow cytometry. Cell culture media were concentrated and then analyzed for soluble ST6Gal I levels using an α2, 6 sialyltransferase sandwich ELISA.ResultWe found that ST6Gal I was cleaved by BACE1 (β-site amyloid precursor protein-cleaving enzyme), which was specifically overexpressed in response to IR. The soluble form of ST6Gal I, which also has sialyltransferase enzymatic activity, was cleaved from the Golgi membrane and then released into the culture media. Both non-cleaved and cleaved forms of ST6Gal I significantly increased colon cancer cell migration in a sialylation-dependent manner. The pro-migratory effect of the non-cleaved form of ST6Gal I was dependent on integrin β1 sialylation, whereas that of the cleaved form of ST6Gal I was not, suggesting that other intracellular sialylated molecules apart from cell surface molecules such as integrin β1 might be involved in mediating the pro-migratory effects of the soluble form of ST6Gal I. Moreover, production of soluble form ST6Gal I by BACE 1 inhibited integrin β1 sialylation and migration by Golgi-anchored form of ST6Gal I.ConclusionsOur results suggest that soluble ST6Gal I, possibly in cooperation with the Golgi-bound form, may participate in cancer progression and metastasis prior to being secreted from cancer cells.

Highly sensitive capillary electrophoresis analysis of N‐linked oligosaccharides in glycoproteins following fluorescence derivatization with rhodamine 110 and laser‐induced fluorescence detection

We describe a highly sensitive CE with laser-induced fluorescence (LIF) detection for the analysis of N-linked oligosaccharides in glycoproteins using rhodamine 110 as a fluorescence derivatization reagent. One CE separation is performed using a fused-silica capillary and neutral pH buffer conditions and allows for the separation of sialo-oligosaccharides according to the number of sialic acids. An alternate separation is performed using the same capillary and acidic pH buffer conditions, enabling the separation of asialo-oligosaccharides according to their sizes. The derivatization and separation conditions for the analysis of sialo- and asialo-oligosaccharides were optimized. Furthermore, we applied the proposed method for the analyses of N-linked sialo- and asialo-oligosaccharides in glycoproteins (ribonuclease B, fetuin, and recombinant human erythropoietin).