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Objectives Mucins are heavily glycosylated large glycoproteins produced in the salivary glands that contribute to salivary viscosity. This study aimed to characterize age-related changes in mucin production in mouse submandibular salivary gland (SMG). Methods The paraffin sections of the SMGs of the young and aged mice were stained with HE or Alcian blue (AB). SMGs mucins derived from the young and aged mice were separated using supported molecular matrix electrophoresis (SMME). After SMME, the membranes were stained with AB and subsequent glycan analysis or subjected to immunoblotting. The expression of 18 mucin genes and 4 sialyltransferase genes in the young and aged SMGs were determined by qPCR. The neuraminidase activity in the SMG homogenates was determined using Neuraminidase Assay Kit. Results The mouse SMG is more strongly stained by AB with increasing age. On SMME, a characteristic band not found in the young SMG is detected in aged SMG. Based on migration position and the MALDI MS, the band that appeared specifically with aging was determined to be acidic mucin. Additionally, most glycans of this acidic mucin were sialo-oligosaccharides. Furthermore, there was an increase in the expression of sialyltransferase genes ST6GalNAc I and ST6GalNAc II, but not a decrease in neuraminidase activity, in the SMG of aged mice. Conclusion A sialomucin or sialylated mucin-like molecule not found in the SMGs of young WT mice is expressed in aged WT mice. The increase in the sialo-oligosaccharide content in this aging-associated molecule may be attributed to the increased expression of the sialyltransferase genes.

Mucins are difficult to separate using conventional gel electrophoresis methods such as SDS-PAGE and agarose gel electrophoresis, owing to their large size and heterogeneity. On the other hand, cellulose acetate membrane electrophoresis can separate these molecules, but is not compatible with glycan analysis. Here, we describe a novel membrane electrophoresis technique, termed "supported molecular matrix electrophoresis" (SMME), in which a porous polyvinylidene difluoride (PVDF) membrane filter is used to achieve separation. This description includes the separation, visualization, and glycan analysis of mucins with the SMME technique.

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.

グライコミクスにおける方法論の確立や，糖タンパク質ならびに疾患関連糖鎖バイオマーカーの探索では，タンパク質の糖鎖修飾を迅速かつ高感度に解析できる技術が要求される。我々は，還元末端を保持した O-結合型糖鎖をコアタンパク質から高速に遊離させる装置，AutoGlycoCutter (AGC) の開発を行った（Matsuno et al., Anal. Biochem., 362, 2007, 245-257. Yamada et al., Anal. Biochem., 371, 2007, 52-61）。本総説では，AGC を用いるムチン型糖鎖やプロテオグリカン型糖鎖の解析例を紹介しながら，本法の性能と有用性を紹介する。さらに AGC を応用した最近の研究成果として，培養癌細胞株中のムチン型糖鎖プロファイルの比較解析や，AGC と MS スポッターの連結による分析の高スループット化についても報告する。

Rapid and sensitive analysis of glycans is essential for glycomics. We previously reported an apparatus, the AutoGlycoCutter (AGC), for rapid release of O-linked glycans under alkaline conditions and its application to rapid analysis of glycans in proteoglycans. We now report an application of the AGC to obtain mucin-type glycans with reducing end (i.e., hemiacetal group) within only 3 min. The released oligosaccharides could be labeled with fluorescent 2-aminobenzoic acid for analysis by normal-phase high-performance liquid chromatography (NP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We could detect O-glycans from as low as 5 pmol of bovine caseino glycomacropeptide (CGMP) by the proposed procedures. The validity of the current method was shown by the analyses of the released O-glycans from some standard glycoproteins: bovine submaxillary mucin, bovine fetuin, porcine stomach mucin, and human colostrum immunoglobulin A. The advantage of the current method was also demonstrated in comparative analysis of mucin-type glycans in CGMP derived from three different animal species.

An apparatus, AutoGlycoCutter (AGC), was developed as a tool for rapid release of O-linked-type glycans under alkaline conditions. This system allowed rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in proteoglycans (PGs). After digestion of PGs with chondroitinase ABC, the oligosaccharides at the linkage region were successfully released from the protein core by AGC within 3 min. The reducing ends of the released oligosaccharides were labeled with 2-aminobenzoic acid and analyzed by a combination of capillary electrophoresis (CE) and matrix-assisted laser desorption time-of-flight mass spectrometry. In addition, the unsaturated disaccharides produced by chondroitinase ABC derived from the outer parts of the glycans were labeled with 2-aminoacridone and analyzed by CE to determine the disaccharide compositions. We evaluated AGC as a method for structural analysis of glycosaminoglycans in some chondroitin-sulfate-type PGs (urinary trypsin inhibitor, bovine nasal cartilage PG, bovine aggrecan, bovine decorin, and bovine biglycan). Recoveries of the released oligosaccharides were 57-73% for all PGs tested in the present study. In particular, we emphasize that the use of AGC achieved ca. 1000-fold rapid release of O-glycans compared with the conventional method.

Alcian blue staining has been widely used to visualize acidic mucins and mucopolysaccharides in supported molecular matrix electrophoresis (SMME) and on membrane transferred from electrophoresis gels. Mucins with low acidic glycan content, however, cannot be stained with Alcian blue, which is one of the major drawbacks of this staining method. On the other hand, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, regardless of the acidic residue content; however, periodic acid-Schiff staining decomposes glycans. Here, we introduce succinylation-Alcian blue staining as an alternative staining method to visualize mucins, regardless of the acidic residue content, and without glycan decomposition.

Mucins are difficult to separate using conventional gel electrophoresis methods such as SDS-PAGE and agarose gel electrophoresis, owing to their large size and heterogeneity. On the other hand, cellulose acetate membrane electrophoresis can separate these molecules, but is not compatible with glycan analysis. Here, we describe a novel membrane electrophoresis technique, termed "supported molecular matrix electrophoresis" (SMME), in which a porous polyvinylidene difluoride (PVDF) membrane filter is used to achieve separation. This description includes the separation, visualization, and glycan analysis of mucins with the SMME technique.

Alcian blue staining has been widely used to visualize acidic mucins and mucopolysaccharides in supported molecular matrix electrophoresis (SMME) and on membrane transferred from electrophoresis gels. Mucins with low acidic glycan content, however, cannot be stained with Alcian blue, which is one of the major drawbacks of this staining method. On the other hand, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, regardless of the acidic residue content; however, periodic acid-Schiff staining decomposes glycans. Here, we introduce succinylation-Alcian blue staining as an alternative staining method to visualize mucins, regardless of the acidic residue content, and without glycan decomposition.

Protein blotting is often used for identification and characterization of proteins on a membrane to which proteins separated by gel electrophoresis are transferred. The transferring process is sometimes problematic, in particular, for mucins and proteoglycans. Here, we describe a novel membrane electrophoresis technique, termed supported molecular matrix electrophoresis (SMME), in which a porous polyvinylidene difluoride (PVDF) membrane filter is used as the separation support. Proteins separated by this method can be immunoblotted without any transferring procedures.

Protein blotting is often used for identification and characterization of proteins on a membrane to which proteins separated by gel electrophoresis are transferred. The transferring process is sometimes problematic, in particular, for mucins and proteoglycans. Here, we describe a novel membrane electrophoresis technique, termed supported molecular matrix electrophoresis (SMME), in which a porous polyvinylidene difluoride (PVDF) membrane filter is used as the separation support. Proteins separated by this method can be immunoblotted without any transferring procedures.

Supported molecular matrix electrophoresis (SMME), in which a hydrophilic polymer such as PVA serves as a support within a porous PVDF membrane, was recently developed. This method is similar to cellulose acetate membrane electrophoresis but differs in the compatibility to glycan analysis of the separated bands. In this report, we describe the first instance of the application of SMME to human serum fractionation, and demonstrate the differences with serum fractionation by cellulose acetate membrane electrophoresis. The SMME membrane exhibited almost no EOF during electrophoresis, unlike the cellulose acetate membrane, but afforded comparative results for serum fractionation. The visualization of each fraction was achieved by conventional staining with dye such as Direct Blue-71, and objective quantification was obtained by densitometry after inducing membrane transparency with 1-nonene. Immunostaining was also achieved. Moreover, mass spectrometric analysis of both N-linked and O-linked glycans from the separated bands was demonstrated. Serum fractionation and glycan profiling of each fraction using SMME will enable novel insights into the relationships between various glycosylation profiles and disease states.

The characterization of mucins is critically important for gaining insights into the molecular pathology of diseases, including cancers, as well as for the discovery of biomarkers for disease diagnosis. However, no practical method has yet been reported for identifying mucin proteins. Here, we report a technique for immunological identification of mucins separated by supported molecular matrix electrophoresis (SMME), a recently developed membrane electrophoresis method. The technique involves on-membrane deglycosylation of mucins by using mild periodate oxidation/base-catalyzed elimination, followed by immunostaining with an antibody that specifically recognizes the mucin tandem repeat (TR) peptide. We demonstrated the method's feasibility by using MUC1 derived from 2 cancer cell lines, T47D and HPAF-II. The present method shows potential as an alternative approach for the identification of mucins separated by SMME or blotted from conventional gel electrophoresis, on a PVDF membrane.

The isolation and characterization of mucins are critically important for obtaining insight into the molecular pathology of various diseases, including cancers and cystic fibrosis. Recently, we developed a novel membrane electrophoretic method, supported molecular matrix electrophoresis (SMME), which separates mucins on a polyvinylidene difluoride (PVDF) membrane impregnated with a hydrophilic polymer. Alcian blue staining is widely used to visualize mucopolysaccharides and acidic mucins on both blotted membranes and SMME membranes; however, this method cannot be used to stain mucins with a low acidic glycan content. Meanwhile, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, but is incompatible with glycan analysis, which is indispensable for mucin characterizations. Here we describe a novel staining method, designated succinylation-Alcian blue staining, for visualizing mucins on a PVDF membrane. This method can visualize mucins regardless of the acidic residue content and shows a sensitivity 2-fold higher than that of Pro-Q Emerald 488, a fluorescent periodate Schiff-base stain. Furthermore, we demonstrate the compatibility of this novel staining procedure with glycan analysis using porcine gastric mucin as a model mucin.

Identification of core proteins carrying the CA19-9 (carbohydrate antigen, sialyl Lewis a) epitope from various tissues will improve the diagnosis of pancreatic cancer in terms of specificity and sensitivity. In this study, we attempted to identify sialyl Lewis a-carrier proteins specifically expressed in pancreatic cancer. Pancreatic cancer is difficult to detect in the early stages of the disease, resulting in a high level of mortality. Therefore, in order to determine the correct course of treatment, it is vital to distinguish cancer from obstruction of the bile duct or other diseases. Our strategy to identify the carrier proteins was as follows: glycoproteins carrying sialyl Lewis a antigen were enriched from pancreatic cancer cell lines using anti-sialyl Lewis a antibody and then subjected to Peptide Mass Fingerprinting analysis. Based on these studies we identified nine glycoproteins carrying the sialyl Lewis a epitope. We evaluated candidate molecules by biochemical analyses of culture supernatants and human sera. In particular, we focused on one candidate molecule carrying a sialyl Lewis a epitope, Galectin-3BP/MAC2BP; M2BP, which was analyzed in detail. These results verified that our candidate molecule is a core protein carrying the sialyl Lewis a epitope. Furthermore, we demonstrated sandwich ELISA, which showed that the glycoprotein was able to detect CA19-9 antigen in culture supernatants. Our approach facilitated the identification of the core protein carrying the sialyl Lewis a epitope. We believe our approach will enable future developments in cancer glycobiomarker identification.

Mucins are a family of heavily glycosylated high molecular mass proteins that have great potential as novel clinical biomarkers for the diagnosis of various malignant tumors. Supported molecular matrix electrophoresis (SMME) is a new type of membrane electrophoresis that can be used to characterize mucins. In SMME, mucins migrate in a molecular matrix supported by membrane materials. Here, we have developed an immunostaining method for the identification of SMME-separated mucins. The novel method involves stably fixing the mucins onto the SMME membrane and optimizing the molecular matrix for the fixation process. We applied this technique for the detection of MUC1 produced from three cancer cell lines (T47D, HPAF-II and BxPC3) and also analyzed their O-linked glycans by mass spectrometry. Our results revealed that properties of the MUC1 molecules from the three cell lines are different in terms of migrating position in SMME and glycan profile. The present method allows simple and rapid characterization of mucins in terms of both glycans and core proteins. The method will be a useful tool for the exploration of mucin alterations associated with various diseases such as cancer.

Glycosaminoglycans (GAGs), linear negatively charged polysaccharides, possess highly structural heterogeneity caused by modifications of amino and hydroxyl groups. Heterogeneity of molecular sizes is also an important feature. These characteristics participate in many cellular events and physiological and pathological processes. Capillary electrophoresis (CE) is one of the most powerful technologies for characterization of such heterogeneous macromolecules based on its high resolving performance. Glycosaminoglycans are generally analyzed by CE both as the intact form and as their enzymatic digested products. This chapter outlines the CE analysis of GAGs. Furthermore, microchip-based capillary electrophoresis for the analysis of GAGs is also introduced, which affords rapid analysis on a time scale of second. This technology has great potential as a tool for routine assessment of pharmaceutical preparations and for clinical diagnosis.

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. Copyright © 2010 John Wiley & Sons, Ltd.

A new concept of separation technology, supported molecular matrix electrophoresis (SMME), is described. In SMME, analytes migrate in a molecular matrix supported by backbone materials. Here we introduce a novel strategy for the separation and characterization of mucins using SMME. Mucin, a highly tumor-associated glycoprotein, has great potential as clinical biomarker for diagnosis of various malignant tumors. However, due to their large size, polymeric nature, and heterogeneous glycosylation, analysis of mucins has been left behind by modern techniques. For mucin analysis, we employed a poly(vinylidene difluoride) (PVDF) membrane and poly(vinyl alcohol) (PVA) as the backbone material and the matrix molecule, respectively. Combining SMME with mass spectrometry and capillary electrophoresis, we demonstrate that a crude porcine stomach mucin consists of a neutral and a sulfated mucin and is contaminated by chondroitin sulfate-containing proteoglycan and hyaluronic acid. Furthermore, to demonstrate the feasibility of the strategy for biomarker discovery, we analyzed mucins in human pancreatic juice, which is an important source for clinical biomarkers of pancreatic tumors. This work revealed the presence of three types of mucin with distinct glycan profiles in human pancreatic juice.

Recently, glycans have been recognized as valuable biomarkers for various disease states. In particular, sialoglycans, which have sialic acids at their terminal end, are likely to have relevance to diseases such as cancer and inflammation. Mass spectrometry (MS) has become an indispensable tool for biomarker discovery. However, matrix-assisted laser desorption ionization (MALDI) MS of sialoglycans normally causes loss of sialic acid. Methylesterification or amidation of carboxyl functionality in sialic acid has been reported to suppress the loss of sialic acids. We found that the modifications of alpha2,3-linked sialic acids proceed less efficiently than those at alpha2,6-linkages. Furthermore, the modifications of the alpha2,3-linked sialic acids are incomplete. This variability in the extent of derivatization presents a major problem in terms of glycan biomarker discovery using MALDI MS. In this study, we developed a novel amidation using acetohydrazide which can completely modify both types of linkages of sialoglycans. With the use of this method, we demonstrate MS profiling of N-linked glycans released from a bovine fetuin which is rich in alpha2,3-linked sialic acids.

Urinary trypsin inhibitor (ulinastatin) is a characteristic protein pharmaceutical which contains both glycosaminoglycans and N-linked glycans in its molecule and has been used for treatment of acute pancreatitis. The comparability of ulinastatin preparations of different lots or from different companies was studied by using conventional analytical approaches such as SDS-PAGE, cellulose acetate membrane electrophoresis, and HP size-exclusion chromatography (SEC) and also by using newly developed techniques such as CE and MALDI-TOF MS. The methods using SEC and SDS-PAGE according to The Japanese Pharmacopoeia showed similar molecular masses for two different preparations, and the estimated molecular masses were significantly different from those observed with MALDI-TOF MS. We also showed that the electrophoretic methods using cellulose acetate membrane electrophoresis and CE can be used for comparability assessments of ulinastatin preparations. In addition, we analyzed the unsaturated disaccharides derived from glycosaminoglycan (chondroitin 4-sulfate chain) and N-linked oligosaccharides attached to ulinastatin by CE after releasing them by enzymatic digestion followed by fluorescent labeling with 2-aminoacridone and 2-aminobenzoic acid, respectively. The results indicated that carbohydrate chains are important as markers for comparability assessments of ulinastatin pharmaceutical preparations.

We analyzed some glycosaminoglycans and natural and artificial acidic polysaccharides using microchip electrophoresis in the buffer containing ethidium bromide, and found that they were successfully separated and detected within 150 s with comparable sensitivity with that of conventional electrophoresis using cellulose acetate membrane. We applied the technique to the analysis of glycosaminoglycans in pharmaceutical preparations and also in cultured cancer cells. Rapidness and easy operation of the proposed technique are quite useful for routine analysis of glycosaminoglycans.

Microchip electrophoresis is a powerful tool for fast analysis of nucleic acids and has expanded its applicability to the analysis of various biological materials including proteins and carbohydrates. Glycosaminoglycans have intrinsic negative charges, and are good targets for electrophoretic analysis. In the present paper, we developed a method to analyze oligosaccharides and unsaturated disaccharides derived from some glycosaminoglycans after digestion with specific enzymes followed by derivatization with 2-aminoacrydone (AMAC) by reductive amination. The method described here allowed rapid analysis of oligosaccharides derived from glycosaminoglycans within 150 s with high sensitivity. We show an application of the present technique to the glycosaminoglycan analysis in cultured HeLa cells.