Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines : HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.

[Structural analysis of carboxymethyl cellulose used as an antiadhesive material for surgical wound healing].

Carboxymethyl cellulose (CMC) is one of the most important cellulose derivatives and used in the fields of food, pharmaceuticals, cosmetics, and paint. Fibrous CMC is used an antiadhesive material to prevent postoperative wound adhesions. The degree of substitution and distribution of the substituent (i.e., the carboxymethyl group) are the most important parameters for the function of CMC. Thus, CMC used for antiadhesive material must be carefully evaluated, because the CMC product is retained in patients' bodies over the long term. Although identification tests of CMC are defined in the Japanese Pharmacopoeia, it is difficult to evaluate its structure using those tests. In the present study, we propose improved methods for evaluating CMC products by analyzing monosaccharides after hydrolysis.

Expression of the clustered NeuAcα2-3Galß O-glycan determines the cell differentiation state of the cells.

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs and evaluated their ability to mark cancer cells, as well as hESCs. MAb7 recognized undifferentiated hESCs and showed a diffuse band with molecular mass of >239 kDa in the lysates of hESCs. Although some amniotic epithelial cells expressed MAb7 antigen, its expression was barely detected in normal human keratinocytes, fibroblasts, or endothelial cells. The expression of MAb7 antigen was observed only in pancreatic and gastric cancer cells, and its levels were elevated in metastatic and poorly differentiated cancer cell lines. Analyses of MAb7 antigen suggested that the clustered NeuAcα2-3Galß O-linked oligosaccharides on DMBT1 (deleted in malignant brain tumors 1) were critical for MAb7 binding in cancer cells. Although features of MAb7 epitope were similar with those of TRA-1-60, distribution of MAb7 antigen in cancer cells was different from that of TRA-1-60 antigen. Exposure of a histone deacetylase inhibitor to differentiated gastric cancer MKN74 cells evoked the expression of MAb7 antigen, whereas DMBT1 expression remained unchanged. Cell sorting followed by DNA microarray analyses identified the down-regulated genes responsible for the biosynthesis of MAb7 antigen in MKN74 cells. In addition, treatment of metastatic pancreatic cancer cells with MAb7 significantly abrogated the adhesion to endothelial cells. These results raised the possibility that MAb7 epitope is a novel marker for undifferentiated cells such as hESCs and cancer stem-like cells and plays a possible role in the undifferentiated cells.

Common glycoproteins expressing polylactosamine-type glycans on matched patient primary and metastatic melanoma cells show different glycan profiles.

Recently, we reported comparative analysis of glycoproteins which express cancer-specific N-glycans on various cancer cells and identified 24 glycoproteins having polylactosamine (polyLacNAc)-type N-glycans that are abundantly present in malignant cells [ Mitsui et al., J. Pharm. Biomed. Anal. 2012 , 70 , 718 - 726 ]. In the present study, we applied the technique to comparative studies on common glycoproteins present in the matched patient primary and metastatic melanoma cell lines. Metastatic melanoma cells (WM266-4) contained a large amount of polyLacNAc-type N-glycans in comparison with primary melanoma cells (WM115). To identify the glycoproteins expressing these N-glycans, glycopeptides having polyLacNAc-type N-glycans were captured by a Datura stramonium agglutinin (DSA)-immobilized agarose column. The captured glycopeptides were analyzed by LC/MS after removing N-glycans, and some glycoproteins such as basigin, lysosome-associated membrane protein-1 (LAMP-1), and chondroitin sulfate proteoglycan 4 (CSPG4) were identified in both WM115 and WM266-4 cells. The expression level of polyLacNAc of CSPG4 in WM266-4 cells was significantly higher than that in WM115 cells. In addition, sulfation patterns of chondroitin sulfate (CS) chains in CSPG4 showed dramatic changes between these cell lines. These data show that characteristic glycans attached to common proteins observed in different stages of cancer cells will be useful markers for determining degree of malignancies of tumor cells.

Analysis of O-glycans as 9-fluorenylmethyl derivatives and its application to the studies on glycan array.

A method is proposed for the analysis of O-glycans as 9-fluorenylmethyl (Fmoc) derivatives. After releasing the O-glycans from the protein backbone in the presence of ammonia-based media, the glycosylamines thus formed are conveniently labeled with Fmoc-Cl and analyzed by HPLC and MALDI-TOF MS after easy purification. Fmoc labeled O-glycans showed 3.5 times higher sensitivities than those labeled with 2-aminobenzoic acid in fluorescent detection. Various types of O-glycans having sialic acids, fucose, and/or sulfate residues were successfully labeled with Fmoc and analyzed by HPLC and MALDI-TOF MS. The method was applied to the comprehensive analysis of O-glycans expressed on MKN45 cells (human gastric adenocarcinoma). In addition, Fmoc-derivatized O-glycans were easily converted to free hemiacetal or glycosylamine-form glycans that are available for fabrication of glycan array and neoglycoproteins. To demonstrate the availability of our methods, we fabricate the glycan array with Fmoc labeled glycans derived from mucin samples and cancer cells. The model studies using the glycan array showed clear interactions between immobilized glycans and some lectins.

Specific detection of N-glycolylneuraminic acid and Galα1-3Gal epitopes of therapeutic antibodies by partial-filling capillary electrophoresis.

The oligosaccharide structure is very important in biopharmaceuticals because of its effects on protein function, including efficacy and half-life. N-glycolylneuraminic acid (Neu5Gc) and Galα1-3Gal (α-Gal) residues are known to show immunogenicity in humans. It is now understood that murine cell lines, such as NS0 or SP2, which are typically used for biopharmaceutical manufacture, produce proteins containing Neu5Gc and α-Gal residues. The expression of these specific residues is affected by the cell line and culture conditions. Therefore, monitoring and controlling the levels of these epitopes are important for the quality control of biopharmaceuticals. To detect the two epitopes on a therapeutic antibody produced by NS0 cells, we applied partial-filling capillary electrophoresis using anti-Neu5Gc antibody and α-galactosidase. In the anti-Neu5Gc antibody filling method, one minor glycan peak with Neu5Gc residues at the nonreducing end disappeared specifically from the electropherogram. In the α-galactosidase filling method, some minor peaks with α1,3-linked Gal residues disappeared. However, in a therapeutic antibody from Chinese hamster ovary cells, no peaks disappeared with the two methods. These results show this method can be used to specifically detect and quantify the two epitopes on biopharmaceuticals with high sensitivity.

Comparative studies on glycoproteins expressing polylactosamine-type N-glycans in cancer cells.

In the series of our previous reports, we showed that some cancer cell lines specifically express polylactosamine-type N-glycans and such glycans were often modified with fucose and sulfate residues. To confirm the proteins expressing these glycans, glycopeptide mixture obtained by digestion of whole protein fractions with trypsin was captured by a polylactosamine-specific lectin, Datura strasmonium agglutinin (DSA). And the peptides and glycans of the captured glycopeptides after digestion with N-glycoamidase F were extensively analyzed by HPLC and MS techniques. We found that some glycoproteins such as CD107a and CD107b commonly contained polylactosamine-type glycans in all the examined cancer cells. But integrin-α5 (CD49e) and carcinoembryonic antigen-related cell adhesion molecule 5 (CD66e) having these glycans were specifically found in U937 (human T-lymphoma) and MKN45 (human gastric cancer) cells, respectively. These data clearly indicate that specific glycans attached to specific proteins will be promising markers for specific tumors with high accuracy.

Analysis of nonhuman N-glycans as the minor constituents in recombinant monoclonal antibody pharmaceuticals.

Minor N-linked glycans containing N-glycolylneuraminic acid residues and/or α-Gal epitopes (i.e., galactose-α1,3-galactose residues) have been reported to be present in recombinant monoclonal antibody (mAb) therapeutics. These contaminations are due to their production processes using nonhuman mammalian cell lines in culture media containing animal-derived materials. In case of the treatment of tumors, we inevitably use such mAbs by careful risk-benefit considerations to prolong patients' lives. However, expanding their clinical applications such as for rheumatism, asthma, and analgesia demands more careful evaluation of the product characteristics. The present work for detailed evaluations of N-glycans demonstrates the methods using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and a combination of high-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry. The CE-LIF method provides excellent separation of both major and minor N-glycans from six commercial mAb pharmaceuticals within 30 min and clearly indicates that a possible trigger of immunogenicity in humans due to the presence of nonhuman N-glycans is present. We strongly believe that the proposed method will be a powerful tool for the analysis of N-glycans of recombinant mAb products in various development stages, such as clone selection, process control, and routine release testing to ensure safety and efficacy of the products.

One-pot characterization of cancer cells by the analysis of mucin-type glycans and glycosaminoglycans.

We developed an automated apparatus for rapid releasing of O-glycans from mucin-type glycoproteins [Anal. Biochem. 371 (2007) 52-61; Anal. Chem. 82 (2010) 7436-7443] and applied the device to analyze them in some cancer cell lines [J. Proteome Res. 8 (2009) 521-537]. We also found that the device is useful to release glycosaminoglycans from proteoglycans [Anal. Biochem. 362 (2007) 245-251]. Based on these studies, we developed a method for one-pot analysis of mucin-type glycans and glycosaminoglycans after releasing them from total protein pool obtained from some cancer cell lines. Mucin-type glycans were analyzed by a combination of high-performance liquid chromatography and mass spectrometry techniques, and glycosaminoglycans were analyzed by capillary electrophoresis as fluorescent-labeled unsaturated disaccharides after digestion with specific eliminases followed by fluorescent labeling. Ten cancer cell lines, including blood cancer cells as well as epithelial cancer cells, were used to assess the method. The results clearly revealed that both mucin-type glycans and glycosaminoglycans showed quite interesting profiles. Thus, the current technique will be a powerful tool for discovery of glycan markers of diseases.

Comparative studies of HPLC-fluorometry and LC/MS method for the determination of N-acetylneuraminic acid as a marker of deteriorated ophthalmic solutions.

Methods for determining the deterioration of ophthalmic solutions using both high-performance liquid chromatography (HPLC) with fluorescence detection and liquid chromatography coupled with selected ion monitoring mass spectrometry (LC/MS) are described. The methods are based on the determination of N-acetylneuraminic acid (NeuAc) released by the hydrolysis of foreign bodies that contaminate eye drops during use. The released NeuAc was either labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) for fluorometric detection or detected without derivatization by mass spectrometry. The calibration curves for NeuAc showed good linearity between 1.2 ng/mL and 39 ng/mL for fluorometric HPLC and 5.0 ng/mL and 100 ng/mL for LC/MS, respectively. Detection limits for fluorometric HPLC and LC/MS were 0.20 ng/mL and 0.88 ng/mL, respectively. The NeuAc content of some model glycoproteins determined by LC/MS method were 62-78% of those determined by fluorometry. The differences are attributed to matrix effects but the LC/MS method afforded sufficiently high sensitivity that NeuAc in the foreign bodies could be determined in eight of nine test samples.

Recent advances in the analysis of carbohydrates for biomedical use.

Glycans are widely distributed in biological systems in free state as well as conjugated forms as parts of glycoproteins, glycolipids, and proteoglycans. Because glycans are not synthesized directly by the corresponding genes but a combination of the related enzymes and substrates, the structures of glycans are quite diverse and sensitive with the changes of physiological conditions. Due to the extremely complex heterogeneities of glycans, it has been a big challenging target to analyze comprehensive glycan profiles (i.e. glycome) and determine characteristic glycan(s) for clinical use. Recent advances in separation sciences such as capillary/microchip electrophoresis and mass spectrometry have made it possible to analyze glycans for various practical uses. New emerging technologies of microarray and bioinformatics have also been applied to glycome/glycomics studies. In this review, recent topics on glycan analysis in clinical use are described with their historical background. Some results obtained by our studies are also shown.

Yokukansan inhibits neuronal death during ER stress by regulating the unfolded protein response.

BACKGROUND: Recently, several studies have reported Yokukansan (Tsumura TJ-54), a traditional Japanese medicine, as a potential new drug for the treatment of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress is known to play an important role in the pathogenesis of AD, particularly in neuronal death. Therefore, we examined the effect of Yokukansan on ER stress-induced neurotoxicity and on familial AD-linked presenilin-1 mutation-associated cell death. METHODS: We employed the WST-1 assay and monitored morphological changes to evaluate cell viability following Yokukansan treatment or treatment with its components. Western blotting and PCR were used to observe the expression levels of GRP78/BiP, caspase-4 and C/EBP homologous protein. RESULTS: Yokukansan inhibited neuronal death during ER stress, with Cnidii Rhizoma (Senkyu), a component of Yokukansan, being particularly effective. We also showed that Yokukansan and Senkyu affect the unfolded protein response following ER stress and that these drugs inhibit the activation of caspase-4, resulting in the inhibition of ER stress-induced neuronal death. Furthermore, we found that the protective effect of Yokukansan and Senkyu against ER stress could be attributed to the ferulic acid content of these two drugs. CONCLUSIONS: Our results indicate that Yokukansan, Senkyu and ferulic acid are protective against ER stress-induced neuronal cell death and may provide a possible new treatment for AD.

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.

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.

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.

Comparison of methods for profiling O-glycosylation: Human Proteome Organisation 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 Initiative's 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.

[Survey of relationship between measurement of abdominal circumference and metabolic syndrome on new health check in university].

In April 2008, a system of special health checks and health guidance was initiated with the aim of identifying people with metabolic syndrome (visceral fat syndrome) and pre-metabolic syndrome. In this study, we investigated the relationship between health check results and abdominal circumference in 632 university faculty members. The standard value for abdominal circumference in women (> or =90) is set higher than that for men (> or =85). When this standard value is used, only 7.4% of women may be able to receive special health guidance. In the future, a follow-up survey of female subjects focusing on the difference in the standard value and an evaluation of its relationship with lifestyle-related diseases will probably be necessary. In the present study, significant positive correlations were seen between abdominal circumference and GPT and gamma-GTP, which are related to hepatic function, and between abdominal circumference and uric acid levels in males. These results indicate the possibility that abdominal circumference is related to alcohol intake. In addition, white blood cell count, red blood cell count, hemoglobin level, and hematocrit level were significantly higher in subjects with large abdominal circumference, and there were differences in hematopoietic function. There have been virtually no studies on the relationship between visceral fat and hepatic and hematopoietic functions, and a follow-up study of the present subjects in the future may shed new light on risk factors for various diseases with abdominal circumference as an indicator.

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.

Capillary electrophoresis-electrospray ionization mass spectrometry for rapid and sensitive N-glycan analysis of glycoproteins as 9-fluorenylmethyl derivatives.

It is well known that most protein therapeutics such as monoclonal antibody pharmaceuticals and other biopharmaceuticals including cancer biomarkers are glycoproteins, and thus the development of high-throughput and sensitive analytical methods for glycans is essential in terms of their determination and quality control. We previously reported a novel alternative labeling method for glycans involving 9-fluorenylmethyl chloroformate (Fmoc-Cl) instead of the conventional reductive amination procedure. The derivatives were analyzed by high-performance liquid chromatography (HPLC) (Kamoda S, Nakano M, Ishikawa R, Suzuki S, Kakehi K. 2005. Rapid and sensitive screening of N-glycans as 9-fluorenylmethyl derivatives by high-performance liquid chromatography: A method which can recover free oligosaccharides after analysis. J Proteome Res. 4:146-152). This method was rapid and simple; however, it was time-consuming in terms of analysis by HPLC and did not provide so much information such as the detailed structures and mass numbers of glycans. Here we have developed a high-throughput and highly sensitive method. It comprises three steps, i.e., release of glycans, derivatization with Fmoc, and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI MS) analysis. We analyzed several glycoproteins such as fetuin, alpha1 acid glycoprotein, IgG, and transferrin in order to validate this method. We were able to analyze the above glycoproteins with the three-step procedure within only 5 h, which provided detailed N-glycan patterns. Moreover, the MS/MS analysis allowed identification of the N-glycan structures. As novel applications, the method was employed for the analysis of N-glycans derived from monoclonal antibody pharmaceuticals and also from alpha-fetoprotein; the latter is known as one of the tumor markers of hepatocellular carcinomas. We were able to easily and rapidly determine the detailed structures of the N-glycans. The present method is very useful for the analysis of large numbers of samples such as a routine analysis.

Accumulation of free complex-type N-glycans in MKN7 and MKN45 stomach cancer cells.

During the N-glycosylation reaction, it has been shown that 'free' N-glycans are generated either from lipid-linked oligosaccharides or from misfolded glycoproteins. In both cases, occurrence of high mannose-type free glycans is well-documented, and the molecular mechanism for their catabolism in the cytosol has been studied. On the other hand, little, if anything, is known with regard to the accumulation of more processed, complex-type free oligosaccharides in the cytosol of mammalian cells. During the course of comprehensive analysis of N-glycans in cancer cell membrane fractions [Naka et al. (2006) J. Proteome Res. 5, 88-97], we found that a significant amount of unusual, complex-type free N-glycans were accumulated in the stomach cancer-derived cell lines, MKN7 and MKN45. The most abundant and characteristic glycan found in these cells was determined to be NeuAcalpha2-6Galbeta1-4GlcNAcbeta1-2Manalpha1-3Manbeta1-4GlcNAc. Biochemical analyses indicated that those glycans found were cytosolic glycans derived from lysosomes due to low integrity of the lysosomal membrane. Since the accumulation of these free N-glycans was specific to only two cell lines among the various cancer cell lines examined, these cytosolic N-glycans may serve as a specific biomarker for diagnosis of specific tumours. A cytosolic sialidase, Neu2, was shown to be involved in the degradation of these sialoglycans, indicating that the cytosol of mammalian cells might be equipped for metabolism of complex-type glycans.