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.

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.

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.

[Capillary electrophoresis analysis of contaminants in heparin sodium for the Japanese pharmacopoeia purity test].

Heparin is widely used as an anticoagulant for the treatment and prevention of thrombotic disorders. Recently, hundreds of cases of anaphylactic reaction as adverse effects were reported by the presence of contaminating oversulfated chondroitin sulfate (OSCS) in some heparin preparations. In addition, these heparin preparations often contaminated dermatan sulfate (DS). Unfortunately, the Japanese Pharmacopoeia (JP) does not include appropriate purity tests. In the present paper, we show that capillary electrophoresis (CE) is a powerful tool for the analysis of OSCS and DS in heparin preparations. CE method shows high resolution and good quantification of OSCS in heparin preparations. This method (OSCS method) was evaluated for accuracy (93.7 %), repeatability (R.S.D.=2.11), linearity (R(2)=0.9996), detection limit (0.1% OSCS) and specificity. In contrast, DS was not able to be detected in high sensitivity by OSCS method. However, a modified CE method (DS method) using the buffer at lower pHs showed good parameters for accuracy (88.1%), repeatability (R.S.D.=1.99), linearity (R(2)=0.9998), detection limit (0.25% DS) and specificity. In conclusion, CE will be an alternative to the NMR method which is being adopted for purification test of heparin sodium in the present version of JP.

Electrophoresis studies on the contaminating glycosaminoglycans in commercially available hyaluronic acid products.

Hyaluronic acid (HA) samples showing inhibition effect on digestion with testicular hyaluronidase (HAase) were found from 16 commercially available HA products, which were supplied from 11 different manufacturers. Most of these HA samples (six samples) were derived from the rooster comb, and one sample was derived from the human umbilical cord. HA oligosaccharides produced by exhaustive digestion of these HA samples with testicular HAases were monitored by capillary electrophoresis, and we found that a few HA samples gave no oligosaccharide products. Detailed analysis of HA samples by cellulose acetate membrane electrophoresis revealed that the HA samples were not digested with HAase because of the presence of a small amount of dermatan sulfate (DS). Analysis of disaccharide units of these HA samples produced by digestion with chondroitinase ABC supported the observations. And the content of DS in the sample was estimated to be ca. 8%. In contrast, these HA samples were easily digested with bacterial hyaluronate lyases from Streptomyces hyalurolyticus and Streptococcus dysgalactiae and gave endproducts of unsaturated disaccharide or unsaturated tetra- or hexasaccharides. The results suggested that the inhibitory effect of DS on HAase is specific to endo-type hydrolase (i.e. testicular HAase). In addition, pharmaceutical preparations of HA derived from rooster comb were easily digested with testicular HAase. These findings will be useful information for clinical or cosmetic use of HA preparations in terms of their half-life.