Kazuaki Kakehi

Comparison of Methods for Profiling O-glycosylation: HUPO Human Disease Glycomics/Proteome Initiative Multi-Institutional Study of IgA1

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiatives activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

Hyphenated technique for releasing and MALDI MS analysis of O-glycans in mucin-type glycoprotein samples.

We developed an automatic apparatus for the release of O-glycans from mucin-type glycoproteins and proteoglycans (Matsuno, Y.-k.; Yamada, K.; Tanabe, A.; Kinoshita, M.; Maruyama, S.-z.; Osaka, Y.-s.; Masuko, T.; Kakehi, K. Anal. Biochem. 2007, 363, 245-257. Yamada, K.; Hyodo, S.; Matsuno, Y. K.; Kinoshita, M.; Maruyama, S. Z.; Osaka, Y. S.; Casal, E.; Lee, Y. C.; Kakehi, K. Anal. Biochem. 2007, 371, 52-61). The method allows rapid release of O-glycans as the reducing form within 10 min. In the present study, we connected the device to a MALDI-TOF MS spotter and achieved routine analysis of O-glycans in biological samples for clinical use after in situ derivatization of the released O-glycans with phenylhydrazine. We applied the method to the analysis of O-glycans expressed on MKN45 cells derived from human stomach cancer cells and found that MKN45 cells expressed characteristic trisialo-polylactosamine-type glycans as reported previously (Yamada, K.; Kinoshita, M.; Hayakawa, T.; Nakaya, S.; Kakehi, K. J. Proteome Res. 2009, 8, 521-537). We also applied the method to the analysis of O-glycans in serum samples. The present technique is the first attempt to use MS measurement for routine clinical diagnostic works.

Charge heterogeneity of a therapeutic monoclonal antibody conjugated with a cytotoxic antitumor antibiotic, calicheamicin.

A robust and highly reproducible capillary isoelectric focusing (cIEF) method for the evaluation of charge heterogeneity of monoclonal antibody (mAb) pharmaceutical which contains covalently bound antitumor compounds was developed using a combination of commercially available dimethylpolysiloxane-coated capillary and carrier ampholyte. In order to optimize major analytical parameters for robust mobilization, experimental responses from three pI markers were selected. The optimized method gave excellent repeatability and intermediate precision in estimated pI values of charge variants with relative standard deviations (RSDs) of not more than 0.06% and 0.95%, respectively, when using IgG(4) as a model. Furthermore, RSDs of charge variant compositions were less than 5.0%. These results suggest that the proposed method can be a powerful tool for reproducible evaluation of charge variants of both naked mAbs and their conjugates with high resolution, and it is applicable to quality testing and detailed characterization in the pharmaceutical industry. In addition, it should be noticed that the method provided non-linear pH gradient within the tested ranges, from pI 9.50 to 3.78, and the pH gradient caused the inconsistency of estimated pI ranges between cIEF and gel IEF. This result indicates that selecting appropriate pI markers based on the target pI ranges of charge variants for each mAb related pharmaceutical is highly recommended for the precise determination of pI values.

The Cell Surface Protein Gene ecm33+ Is a Target of the Two Transcription Factors Atf1 and Mbx1 and Negatively Regulates Pmk1 MAPK Cell Integrity Signaling in Fission Yeast

We identified and characterized ecm33+, which encodes a GPI-anchored cell surface protein as a transcriptional target of Atf1 and Mbx1. Here, we show that Ecm33 is involved in the negative regulation of Pmk1 MAPK signaling and demonstrates real-time monitoring of the activation of the cell integrity MAPK signaling pathway.

Comparative studies on the structural features of O-glycans between leukemia and epithelial cell lines.

Recently, we developed an automated apparatus for rapid releasing of O-glycans from mucin-type glycoproteins and proteoglycans ( Anal. Biochem. 2007 , 362 , 245 - 251 ; 2007 , 371 , 52 - 61 ). In the present paper, we released O-glycans from some leukemia and epithelial cells using the apparatus, and compared the profiles of O-glycans among these cells after fluorescent labeling of the released glycans with 2-aminobenzoic acid. The fluorescent labeled glycans were analyzed using a combination of HPLC and off-line MALDI-(QIT)TOF mass spectrometry We found that leukemia cells generally showed simple glycan profiles and commonly contained sialyl-T (NeuAcalpha2-3Galbeta1-3GalNAc) and disialyl-T (NeuAcalpha2-3Galbeta1-3(NeuAcalpha2-6)GalNAc) antigens as major O-glycans. In contrast, epithelial cancer cell lines usually showed extremely complex profiles. We found that polylactosamine-type O-glycans were abundantly present in MKN45 cells. Especially, we found characteristic glycans, of which Galbeta1-3 residue of core1 structure is modified with biantennary polylactosamine units. In contrast, this cell line did not contain polylactosamine-type N-glycans ( J. Proteome Res. 2006 , 5 , 88 - 97 ). These results suggest that the different biosynthetic pathways for N- and O-glycans are proposed. The method presented here will accelerate the speed for comprehensive analysis of O-glycans in biological samples and will be a powerful tool for clinical/biochemical analysis in cancer biology.

Structural characterization of multibranched oligosaccharides from seal milk by a combination of off-line high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and sequential exoglycosidase digestion.

A complex mixture of diverse oligosaccharides related to the carbohydrates in glycoconjugates involved in various biological events is found in animal milk/colostrum and has been challenging targets for separation and structural studies. In the current study, we isolated oligosaccharides having high molecular masses (MW approximately 3800) from the milk samples of bearded and hooded seals and analyzed their structures by off-line normal-phase-high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight (NP-HPLC-MALDI-TOF) mass spectrometry (MS) by combination with sequential exoglycosidase digestion. Initially, a mixture of oligosaccharides from the seal milk was reductively aminated with 2-aminobenzoic acid and analyzed by a combination of HPLC and MALDI-TOF MS. From MS data, these oligosaccharides contained different numbers of lactosamine units attached to the nonreducing lactose (Galbeta1-4Glc) and fucose residue. The isolated oligosaccharides were sequentially digested with exoglycosidases and characterized by MALDI-TOF MS. The data revealed that oligosaccharides from both seal species were composed from lacto-N-neohexaose (LNnH, Galbeta1-4GlcNAcbeta1-6[Galbeta1-4GlcNAcbeta1-3]Galbeta1-4Glc) as the common core structure, and most of them contained Fucalpha1-2 residues at the nonreducing ends. Furthermore, the oligosaccharides from both samples contained multibranched oligosaccharides having two Galbeta1-4GlcNAc (N-acetyllactosamine, LacNAc) residues on the Galbeta1-4GlcNAcbeta1-3 branch or both branches of LNnH. Elongation of the chains was observed at 3-OH positions of Gal residues, but most of the internal Gal residues were also substituted with an N-acetyllactosamine at the 6-OH position.

Determination of Tn antigen released from cultured cancer cells by capillary electrophoresis

An incomplete elongation of O-glycans in mucins has been found in epithelial cancers, leading to the expression of shorter carbohydrate structures such as Tn antigen (GalNAc-O-Ser/Thr), which has been reported to be one of the most specific human cancer-associated structures. However, there have been no appropriate physicochemical methods for the determination of Tn antigen in biological samples. In the present paper, we developed a capillary electrophoresis method for the determination of Tn antigen, and applied the method to the analysis of the expressed Tn antigen on some leukemia and epithelial cancer cells.

Determination of sulfate ester content in sulfated oligo- and poly-saccharides by capillary electrophoresis with indirect UV detection

Carbohydrates having sulfate groups such as glycosaminoglycans and chemically synthesized sucrose sulfate show interesting and important biological activities. We adapted CE with indirect UV detection technique to the determination of sulfate ester in sulfated carbohydrates, which were previously hydrolyzed with HCl. The liberated sulfate ion was analyzed using a background electrolyte consisting of triethanolamine-buffered chromate with hexamethonium bromide. Sulfate contents of glucose 3-sulfate and sucrose octasulfate used as a model were in good agreement with theoretical values (accuracy, 95.9-96.7 and 97.4-101.9%, respectively), and relative standard deviation values run-to-run were 0.977 and 1.90%, respectively. We applied the method to the determination of the sulfate contents of some glycosaminoglycan samples and showed that the contents were in good agreement with those calculated from sulfur content.

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.

Capillary lectin-affinity electrophoresis for glycan analysis.

Glycosylation is one of the most important post-translational events for proteins, affecting their functions in health and disease, and plays significant roles in various information trafficking for intercellular and intracellular biological events. The glycans which show such important effects are generally present as quite complex mixtures in minute amounts. The approach described here makes it possible to profile glycans for the analysis of post-translational modification of proteins with carbohydrates. The method is based on high-resolution separation of fluorescent-labeled carbohydrates by capillary electrophoresis with laser-induced fluorescent detection in the presence of carbohydrate-binding proteins at different concentrations. The technique affords simultaneous determination of glycans having similar structures even in complex mixtures.