A Simple Quality Evaluation Method for Proteoglycan after Addition to Beverages.

Over the past 10 years, many products utilizing the functionality of salmon cartilage proteoglycan have come on the market, and consumer awareness of proteoglycan has increased. During this period, the biggest issue has been how to evaluate the amount and quality of proteoglycan in the cartilage extract blended in the products. In this study, we propose an immunological method that can easily evaluate the amount and quality of proteoglycan in the proteoglycan-containing compositions. By the present method, it is possible to evaluate not only the retention of the functional domains of the core protein of proteoglycan, but also that of chondroitin sulfate chains linked to the core protein. Furthermore, the binding activity of proteoglycan to hyaluronan can be evaluated if hyaluronan is used as a probe instead of an antibody. This method is expected to be useful for proteoglycan quality evaluation during the manufacturing process and product storage.

Machine learning diagnosis by immunoglobulin N-glycan signatures for precision diagnosis of urological diseases.

Early diagnosis of urological diseases is often difficult due to the lack of specific biomarkers. More powerful and less invasive biomarkers that can be used simultaneously to identify urological diseases could improve patient outcomes. The aim of this study was to evaluate a urological disease-specific scoring system established with a machine learning (ML) approach using Ig N-glycan signatures. Immunoglobulin N-glycan signatures were analyzed by capillary electrophoresis from 1312 serum subjects with hormone-sensitive prostate cancer (n = 234), castration-resistant prostate cancer (n = 94), renal cell carcinoma (n = 100), upper urinary tract urothelial cancer (n = 105), bladder cancer (n = 176), germ cell tumors (n = 73), benign prostatic hyperplasia (n = 95), urosepsis (n = 145), and urinary tract infection (n = 21) as well as healthy volunteers (n = 269). Immunoglobulin N-glycan signature data were used in a supervised-ML model to establish a scoring system that gave the probability of the presence of a urological disease. Diagnostic performance was evaluated using the area under the receiver operating characteristic curve (AUC). The supervised-ML urologic disease-specific scores clearly discriminated the urological diseases (AUC 0.78-1.00) and found a distinct N-glycan pattern that contributed to detect each disease. Limitations included the retrospective and limited pathological information regarding urological diseases. The supervised-ML urological disease-specific scoring system based on Ig N-glycan signatures showed excellent diagnostic ability for nine urological diseases using a one-time serum collection and could be a promising approach for the diagnosis of urological diseases.

Effect of glycosaminoglycan structure on all-trans-retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells.

Glycosaminoglycan polysaccharides are components of animal extracellular matrices and regulate cell functions based on their various sulfation and epimerization pattern structures. The present study aimed to find glycosaminoglycan structures to promote neural differentiation. We investigated the effect of exogenous glycosaminoglycans with well-defined structures on the all-trans-retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells, which is an ideal model culture system for studying neural differentiation. We found that chondroitin sulfate E and heparin, but not any other glycosaminoglycans, upregulated the expressions of neural specific markers but not a grail specific marker. Chondroitin sulfate E was suggested to function during spheroid formation, however, equimolar concentration of its oligosaccharide did not show promotive effect on the neural differentiation. Another finding was that hyaluronan oligosaccharide mixture markedly downregulated the expressions of a myelin specific marker. These findings suggested that the specific sulfation pattern and/or chain length of exogenous added glycosaminoglycan is important to regulate neural differentiation and myelination.

N-glycan signature of serum immunoglobulins as a diagnostic biomarker of urothelial carcinomas.

Discriminating between urothelial carcinoma (UC), including bladder cancer (BCa) and upper urinary tract UC (UTUC), is often challenging. Thus, the current study evaluated the diagnostic performance of N-glycosylation signatures of immunoglobulins (Igs) for detecting UC, including BCa and UTUC. N-glycosylation signatures of Igs from serum samples of the training cohort, including 104 BCa, 68 UTUC, 10 urinary tract infection, and 5 cystitis cases, as well as 62 healthy volunteers, were measured retrospectively using automated capillary-electrophoresis-based N-glycomics. UTUC or BCa scores were then established through discriminant analysis using N-glycan signatures of Igs. Diagnostic performance was evaluated using the area under receiver operating characteristics curve (AUC) and decision curve analyses (DCA). Our result showed that BCa and UTUC scores for discriminating BCa (AUC: 0.977) and UTUC (AUC: 0.867), respectively, provided significantly better clinical performance compared to urine cytology, gross hematuria, or clinical T1 cases. DCA revealed that adding BCa and UTUC scores to gross hematuria status was the best combination for detecting UC and avoiding the need for more intervention without overlooking UC (risk threshold: 13%-93%). The UC nomogram based on the combination of gross hematuria, UTUC score, and BCa score could detect UC with an AUC of 0.891, indicating significantly better performance compared to gross hematuria status in the validation cohort (251 patients). The limitations of this study include its small sample size and retrospective nature. The UC nomogram based on gross hematuria and N-glycosylation signatures of Igs can be a promising approach for the diagnosis of UC.

Essential hyaluronan structure for binding with hyaluronan-binding protein (HABP) determined by glycotechnological approach.

Hyaluronan specifically binds to aggrecan globular domain 1, which is often referred to as just hyaluronan binding protein (HABP), however, the hyaluronan carbohydrate structure recognized by HABP had not been studied in detail. The aim of the present study was to investigate the important structure of hyaluronan for binding to HABP. We prepared hybrid oligosaccharides from hyaluronan and chondroitin, with or without modification of the reducing or non-reducing terminus, as tools to determine the preferred structure of hyaluronan for binding to the HABP by a competitive ELISA-like method. The non-reducing terminal structure was critical, especially, the glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) of the hyaluronan-unit are essential for complete HABP binding activity, and for any HABP binding activity, respectively. It is possible to replace GlcUAß-1-3GlcNAc of the internal disaccharide units with GlcUAß-1-3N-acetylgalactosamine (GalNAc), if the chain length is decasaccharide or larger.

A Chondroitin Sulfate Chain of Urinary Trypsin Inhibitor Enhances Protease Inhibitory Activity of the Core Protein.

Human urinary trypsin inhibitor (UTI) is a proteoglycan composed of one core protein covalently linked to one glycosaminoglycan, which is a low sulfated chondroitin 4-sulfate. It is used as an anti-inflammatory medicine based on the protease inhibitory activity of the core protein. However, functions of the chondroitin sulfate have not been clarified. Recently, we succeeded in remodeling the UTI chondroitin sulfate to hyaluronan to create hyaluronan hybrid UTI, without changing the core protein. Here, we investigated the effect of the remodeled chondroitin sulfate on the activities of serine proteases. Native UTI showed stronger protease inhibitory activity than hyaluronan hybrid UTI or hydrolyzed glycosaminoglycan UTI. Chondroitin 4-sulfate chains with a small peptide derived from the native UTI did not show any protease inhibitory activity. These results suggest that the chondroitin sulfate chain linked covalently to core protein enhances protease inhibitor activity of UTI although the chondroitin sulfate chain itself does not.

Dermatan sulfate oligosaccharides having reducing end 2, 5-anhydro-d-talose inhibit bovine testicular hyaluronidase activity.

Dermatan sulfate oligosaccharides having reducing end 2,5-anhydro-d-talose were prepared by partial N-deacetylation of dermatan sulfate polysaccharide with hydrazine followed by deamination with nitrous acid, and the effect of these oligosaccharides on the activity of bovine testicular hyaluronidase was investigated. Hydrolysis activity and transglycosylation activity of this enzyme were assessed in an independent reaction system by analyzing the products by HPLC. Dermatan sulfate oligosaccharides inhibited hyaluronan hydrolysis by bovine testicular hyaluronidase. Kinetic analysis of the hydrolysis reaction revealed that the inhibition mode by dermatan sulfate oligosaccharides was as competitive as that previously shown by chondroitin sulfate oligosaccharides. Transglycosylation of hyaluronan by bovine testicular hyaluronidase, as a reverse reaction of hydrolysis of hyaluronan, was also inhibited. These inhibitory effects were dependent on the dose and sulfation degree of dermatan sulfate.

Proteoglycan-substrate gel zymography for the detection of chondroitin sulfate-degrading enzymes.

Chondroitin sulfate (CS), a linear polysaccharide, is a major component of the cartilage matrix. Although CS plays various roles in several biological and pathological processes, most details regarding its metabolism are still poorly understood. Some CS-degrading enzymes have been identified in mammals, but their expression patterns and localizations remain unclear. Here we present a simple zymography procedure to detect CS-degrading enzymes using salmon nasal cartilage proteoglycans as substrates. This method should be useful to explore CS-degrading enzymes.

4-Methylumbelliferone Decreases the Hyaluronan-rich Extracellular Matrix and Increases the Effectiveness of 5-Fluorouracil.

BACKGROUND/AIM: Pancreatic cancer responds poorly to most chemotherapeutic agents. Several studies have reported that hyaluronan (HA)-rich extracellular matrix (ECM) is a biological barrier against chemotherapeutic agents. 4-methylumbelliforone (MU) led to inhibition of HA synthesis and its preservation in ECM, which may enhance 5-fluorouracil (5-FU) cytotoxicity. Thus, new therapy with MU and 5-FU may be developed for pancreatic cancer. MATERIALS AND METHODS: A 5-fluorouracil (5-FU) concentration and 4-methylumbelliferone (MU) dosage was analyzed by high-performance liquid chromatography (HPLC). Change in antitumor efficacy of 5-FU in combination with MU was also examined in vivo and in vitro. RESULTS: Combined 5-FU and MU treatment inhibited cell proliferation better than 5-FU alone; 0.01 mM 5-FU alone decreased cell proliferation by 37.7 %, while 0.01 mM 5-FU with 0.5 mM MU decreased cell proliferation by 57.4%. MU enhanced the intracellular concentration of 5-FU by 47.3% compared to control. Mice tumors treated with 5-FU and MU decreased in size and animal survival was prolonged. Moreover, MU decreased cohesiveness of the intercellular space. CONCLUSION: Combination therapy of 5-FU with MU was effective. A novel therapy can be designed for pancreatic cancer by using ECM modulation.

Chondroitin sulfate proteoglycans from salmon nasal cartilage inhibit angiogenesis.

Because cartilage lacks nerves, blood vessels, and lymphatic vessels, it is thought to contain factors that inhibit the growth and development of those tissues. Chondroitin sulfate proteoglycans (CSPGs) are a major extracellular component in cartilage. CSPGs contribute to joint flexibility and regulate extracellular signaling via their attached glycosaminoglycan, chondroitin sulfate (CS). CS and CSPG inhibit axonal regeneration; however, their role in blood vessel formation is largely unknown. To clarify the function of CSPG in blood vessel formation, we tested salmon nasal cartilage proteoglycan (PG), a member of the aggrecan family of CSPG, for endothelial capillary-like tube formation. Treatment with salmon PG inhibited endothelial cell adhesion and in vitro tube formation. The anti-angiogenic activity was derived from CS in the salmon PG but not the core protein. Salmon PG also reduced matrix metalloproteinase expression and inhibited angiogenesis in the chick chorioallantoic membrane. All of these data support an anti-angiogenic role for CSPG in cartilage.

Characterization of Proteoglycan and Hyaluronan in Hot Water Extract from Salmon Cartilage.

Salmon cartilage proteoglycan fractions have recently gained favor as ingredients of functional food and cosmetics. An optimal hot water method to extract proteoglycan from salmon cartilage has recently been developed. The extracted cartilage includes hyaluronan and collagen in addition to proteoglycan as counterparts that interact with each other. In this study, biochemical analyses and atomic force microscopical analysis revealed global molecular images of proteoglycan in the hot water extract. More than seventy percent of proteoglycans in this extract maintained their whole native structures. Hyaluronan purified from the hot water extract showed a distribution with high molecular weight similar to hyaluronan considered to be native hyaluronan in cartilage. The current data is evidence of the quality of this hot water cartilage extract.

A mechanism for evasion of CTL immunity by altered O-glycosylation of HLA class I.

Anti-tumour immunity by cytotoxic T-lymphocytes (CTLs) is essential to suppress tumour progression. Cancer cells that evade CTL immunity proliferate in the host, promoting metastasis, but mechanisms underlying this capacity remain unknown. Here we report that bladder cancer cells metastasized to lymph nodes evade CTL immunity by a new mechanism via altered glycosylation. CTLs normally recognize and kill cancer cells presenting antigenic peptides on human leukocyte antigen (HLA) class I. We show bladder cancer cells expressing the O-glycan processing enzyme, core2 ß-1,6-N-acetylglucosaminyltransferase (C2GnT) exhibit HLA class I O-glycan modified with poly-N-acetyllactosamine and are highly susceptible to CTL. In those cells, poly-N-acetyllactosamine on HLA class I O-glycan binds galectin-3 to form a cell-surface molecular lattice, enabling efficient cell-surface retention of HLA class I. In contrast, bladder cancer cells in which C2GnT is downregulated show decreased levels of poly-N-acetyllactosamine on HLA class I O-glycans, attenuating lattice formation and reducing the cell-surface half-life of HLA class I. These tumour cells present antigenic peptides less efficiently, thereby evading CTL lysis and facilitating metastasis.

4-Methylumbelliferone Suppresses Hyaluronan Synthesis and Tumor Progression in SCID Mice Intra-abdominally Inoculated With Pancreatic Cancer Cells.

OBJECTIVES: Pancreatic ductal adenocarcinoma contains large amounts of the glycosaminoglycan hyaluronan (HA), which is involved in various physiological processes. Here, we aimed to clarify the anticancer mechanisms of 4-methylumbelliferone (MU), a well-known HA synthesis inhibitor. METHODS: MIA PaCa-2 human pancreatic cancer cells were used. We evaluated cellular proliferation, migration, and invasion in the presence of MU, exogenous HA, and an anti-CD44 antibody. We also analyzed apoptosis, CD44 expression, and HA-binding ability using flow cytometry. The HA content in tumor tissue was quantified and histopathologically investigated in mice who had been inoculated with cancer cells. RESULTS: In vitro, MU inhibited pericellular HA matrix formation; however, HAS3 mRNA was up-regulated. Treatment with 0.5 mM MU suppressed cellular proliferation by 26.4%, migration by 14.7%, and invasion by 22.7%. Moreover, MU also significantly increased apoptosis. CD44 expression and HA-binding ability were not altered by MU. In vivo, MU suppressed HA accumulation in pancreatic tumors and improved survival times in tumor-bearing mice. CONCLUSIONS: 4-Methylumbelliferone indirectly caused apoptosis in pancreatic cancer cells by inhibiting HA production. 4-Methylumbelliferone may be a promising agent in the treatment of pancreatic cancer.

Hyaluronan Production Regulates Metabolic and Cancer Stem-like Properties of Breast Cancer Cells via Hexosamine Biosynthetic Pathway-coupled HIF-1 Signaling.

Cancer stem cells (CSCs) represent a small subpopulation of self-renewing oncogenic cells. As in many other stem cells, metabolic reprogramming has been implicated to be a key characteristic of CSCs. However, little is known about how the metabolic features of cancer cells are controlled to orchestrate their CSC-like properties. We recently demonstrated that hyaluronan (HA) overproduction allowed plastic cancer cells to revert to stem cell states. Here, we adopted stable isotope-assisted tracing and mass spectrometry profiling to elucidate the metabolic features of HA-overproducing breast cancer cells. These integrated approaches disclosed an acceleration of metabolic flux in the hexosamine biosynthetic pathway (HBP). A metabolic shift toward glycolysis was also evident by quantitative targeted metabolomics, which was validated by the expression profiles of key glycolytic enzymes. Forced expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), an HBP rate-limiting enzyme, resembled the results of HA overproduction with regard to HIF-1α accumulation and glycolytic program, whereas GFAT1 inhibition significantly decreased HIF-1α protein level in HA-overproducing cancer cells. Moreover, inhibition of the HBP-HIF-1 axis abrogated HA-driven glycolytic enhancement and reduced the CSC-like subpopulation. Taken together, our results provide compelling evidence that HA production regulates the metabolic and CSC-like properties of breast cancer cells via HBP-coupled HIF-1 signaling.

Antitumor effects of the hyaluronan inhibitor 4-methylumbelliferone on pancreatic cancer.

Hyaluronan (HA) is a major component of the extracellular matrix (ECM), and influences tumor invasion and metastasis. In a previous study, the present authors reported for the first time that 4-methylumbelliferone (MU) inhibited HA synthesis and suppressed tumor growth. However, the localization of HA and the changes in ECM morphology caused by MU in pancreatic cancer remain to be examined in detail. In the present study, the cytotoxicity of MU and its effect on cellular proliferation was evaluated in the human pancreatic cancer cell line MIA PaCa-2. The amount of HA synthesized and the retention of HA around the cells were quantitatively and immunohistochemically analyzed in vitro and in vivo. Structural changes in the ECM in the tumor tissue were investigated using an electron microscope. MU treatment led to a decrease in extracellular HA retention, as evidenced by a particle exclusion assay and immunohistochemical staining. Cell proliferation was suppressed by MU in a dose-dependent manner. The release of lactate dehydrogenase into the culture medium due to damage to the cellular membrane did not increase following MU administration. In tumor-inoculated mice, MU suppressed any increase in tumor volume and decreased the quantity of HA. Electron microscopy revealed that MU attenuated the intercellular space and caused it to be less cohesive. These data indicate that MU inhibits HA synthesis and reduces the amount of HA in the ECM while exhibiting no obvious cytotoxic effect. These findings suggest that MU has potential as a novel therapeutic agent for pancreatic cancer.

Effects of C-xylopyranoside derivative on epithelial regeneration in an in vitro 3D oral mucosa model.

Identifying substandard tissue-engineered oral mucosa grafts with a poor epithelium before clinical use is critical to ensure quality assurance/control in regenerative medicine, leading to success of grafting. This study investigated the effects of one of the C-xylopyranoside derivatives, ß-D-xylopyranoside-n-propane-2-one (XPP), on oral epithelial regeneration. Using a three-dimensional oral mucosa model, we analyzed changes of the epithelial structure, glycosaminoglycan (GAG) synthesis, the expression levels of basement membrane zone markers, and substrates of Akt/mTOR signaling. Compared with the control, 2 mM XPP treatment increased the mean and minimal epithelial thickness, and reduced the variation of epithelial thickness. It also stimulated expressions of decorin and syndecan-1 with change of GAG amount and/or composition, and enhanced the expressions of integrin α6, CD44, and Akt/mTOR signaling substrates. These findings suggest that XPP supplementation contributes to consistent epithelial regeneration. Moreover, upregulation of those markers may play a role in increasing the quality of the oral mucosal epithelium.

Enzymatic synthesis of hyaluronan hybrid urinary trypsin inhibitor.

Human urinary trypsin inhibitor is a proteoglycan that has a single low-sulfated chondroitin 4-sulfate chain at the seryl residue in position 10 of the core protein as a glycosaminoglycan moiety, and is used as an anti-inflammatory medicine based on the protease inhibitory activity of the core protein. However, the functions of the glycosaminoglycan moiety have not yet been elucidated in detail. In the present study, the glycosaminoglycan chains of a native urinary trypsin inhibitor were remodeled to hyaluronan chains, with no changes to the core protein, using transglycosylation as a reverse reaction of the hydrolysis of bovine testicular hyaluronidase, and the properties of the hybrid urinary trypsin inhibitor were then analyzed. The trypsin inhibitory activitiy of the hyaluronan hybrid urinary trypsin inhibitor was similar to that of the native type; however, its inhibitory effect on the hydrolysis of hyaluronidase were not as strong as that of the native type. This result demonstrated that the native urinary trypsin inhibitor possessed hyaluronidase inhibitory activity on its chondroitin sulfate chain. The hyaluronan hybrid urinary trypsin inhibitors obtained affinity to a hyaluronan-binding protein not exhibited by the native type. The interactions between the hyaluronan hybrid urinary trypsin inhibitors and phosphatidylcholine (abundant in the outer layer of plasma membrane) were stronger than that of the native type. Hyaluronan hybrid urinary trypsin inhibitors may be useful for investigating the functions of the glycosaminoglycan chains of urinary trypsin inhibitors and hyaluronan, and our hybrid synthesizing method may be used widely in research for future medical applications.

Inhibitory effect of chondroitin sulfate oligosaccharides on bovine testicular hyaluronidase.

Hyaluronan and chondroitin sulfates are prominent components of the extracellular matrices of animal tissues; however, their functions in relation to their oligosaccharide structures have not yet been fully elucidated. The oligosaccharides of hyaluronan and chondroitin sulfate were prepared and used to investigate their effects on the hydrolysis and transglycosylation reactions of bovine testicular hyaluronidase when hyaluronan was used as a substrate. Hydrolysis and transglycosylation activities were assessed in independent reaction systems by analyzing the products by HPLC. The hydrolysis and transglycosylation reactions of bovine testicular hyaluronidase were dose-dependently inhibited by chondroitin sulfate oligosaccharides, but not by hyaluronan or chondroitin oligosaccharides. A kinetic analysis of the hydrolysis reaction using hyaluronan octasaccharide revealed that the inhibition mode by chondroitin sulfate oligosaccharides was competitive.

Chondroitin sulfate cluster of epiphycan from salmon nasal cartilage defines binding specificity to collagens.

Epiphycan (EPY) from salmon nasal cartilage has a glycosaminoglycan (GAG) domain that is heavily modified by chondroitin 4-sulfate and chondroitin 6-sulfate. The functional role of the GAG domain has not been investigated. The interaction of EPY with collagen was examined in vitro using surface plasmon resonance analysis. EPY was found to bind to type I collagen via clustered chondroitin sulfate (CS), while a single chain of CS was unable to bind. Types I, III, VII, VIII and X collagen showed high binding affinity with EPY, whereas types II, IV, V, VI and IX showed low binding affinities. Chemical modification of lysine residues in collagen decreased the affinity with the clustered CS. These results suggest that lysine residues of collagen are involved in the interaction with the clustered CS, and the difference in lysine modification defines the binding affinity to EPY. The clustered CS was also involved in an inter-saccharide interaction, and formed self-associated EPY. CS of EPY promoted fibril formation of type I collagen.

Biochemical and atomic force microscopic characterization of salmon nasal cartilage proteoglycan.

Biological activities of salmon nasal cartilage proteoglycan fractions are known, however, structural information is lacking. Recently, the major proteoglycan of this cartilage was identified as aggrecan. In this study, global molecular images and glycosaminoglycan structure of salmon nasal cartilage aggrecan purified from 4M guanidine hydrochloride extract were analyzed using HPLCs and atomic force microscopy with bovine tracheal cartilage aggrecan as a control. The estimated numbers of sulfates per disaccharide unit of chondroitin sulfate chains of salmon and bovine aggrecans were similar (approximately 0.85). However, the disaccharide composition showed a higher proportion of chondroitin 6-sulfate units in salmon aggrecan, 60%, compared to 40% in bovine. Gel filtration HPLC and monosaccharide analysis showed the salmon aggrecan had a lower number (approximately one-third), but 1.5-3.3 times longer chondroitin sulfate chains than the bovine aggrecan. Atomic force microscopic molecular images of aggrecan supported the images predicted by biochemical analyses.