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.

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.

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.

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.

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.

Effect of a cholesterol-rich lipid environment on the enzymatic activity of reconstituted hyaluronan synthase.

Hyaluronan synthase (HAS) is a unique membrane-associated glycosyltransferase and its activity is lipid dependent. The dependence however is not well understood, especially in vertebrate systems. Here we investigated the functional association of hyaluronan synthesis in a cholesterol-rich membrane-environment. The culture of human dermal fibroblasts in lipoprotein-depleted medium attenuated the synthesis of hyaluronan. The sequestration of cellular cholesterol by methyl-ß-cyclodextrin also decreased the hyaluronan production of fibroblasts, as well as the HAS activity. To directly evaluate the effects of cholesterol on HAS activity, a recombinant human HAS2 protein with a histidine-tag was expressed as a membrane protein by using a baculovirus system, then successfully solubilized, and isolated by affinity chromatography. When the recombinant HAS2 proteins were reconstituted into liposomes composed of both saturated phosphatidylcholine and cholesterol, this provided a higher enzyme activity as compared with the liposomes formed by phosphatidylcholine alone. Cholesterol regulates HAS2 activity in a biphasic manner, depending on the molar ratio of phosphatidylcholine to cholesterol. Furthermore, the activation profiles of different lipid compositions were determined in the presence or absence of cholesterol. Cholesterol had the opposite effect on the HAS2 activity in liposomes composed of phosphatidylethanolamine or phosphatidylserine. Taken together, the present data suggests a clear functional association between HAS activity and cholesterol-dependent alterations in the physical and chemical properties of cell membranes.

One set system for the synthesis and purification of glycosaminoglycan oligosaccharides reconstructed using a hyaluronidase-immobilized column.

Using the transglycosylation reaction as a reverse reaction for the hydrolysis of hyaluronidase, new artificial oligosaccharides may be synthesized by reconstructing natural glycosaminoglycans (GAGs) according to preliminary planned arrangements. However, as some problems have been associated with the method, including the low yields of reaction products and complicated processes of separation and purification, improvements in this method were investigated. Transglycosylation reactions were carried out using bovine testicular hyaluronidase-immobilized resin packed in a column. For the transglycosylation reaction, pyridylaminated (PA) GAG hexasaccharides, which were the minimum size for hydrolysis sensitivity and the transglycosylation reaction, were used as acceptors, whereas large size GAGs were used as donors. The reaction mixture was pooled after incubation in the hyaluronidase-immobilized resin column and was then introduced into continuously joined HPLC columns constructed from three steps: the first step of ion-exchange HPLC for concentrating newly synthesized GAG oligosaccharides as reaction products, the second step of reverse phase HPLC for separating PA oligosaccharides from non-PA oligosaccharides, and the third step of size fractionation HPLC for fractionating newly synthesized oligosaccharides. Newly synthesized oligosaccharides were obtained by one complete cycle of the transglycosylation reaction and separation.

Epiphycan from salmon nasal cartilage is a novel type of large leucine-rich proteoglycan.

Chum salmon (Oncorhynchus keta) nasal cartilage was examined by next-generation DNA sequencing and mass spectrometric analyses, and 14 types of proteoglycans including epiphycan (EPY) were found. A cDNA encoding EPY was cloned and sequenced. The cDNA encoded 589 amino acids comprised a glycosaminoglycan (GAG) domain containing 55 potential GAG-modified sites (Ser-Gly and/or Gly-Ser), a cysteine cluster and 6 leucine-rich repeats. EPY was purified from salmon nasal cartilage and the structure of the GAG was characterized. As a result of unsaturated disaccharide analysis, GAG was found to be composed of chondroitin 6-sulfate (58.0%), chondroitin 4-sulfate (26.5%) and non-sulfated chondroitin (15.3%). The average molecular weight of GAG was estimated to be 3.0 × 10(4). Ser-100 and Ser-103 were identified as serine residues substituted by GAG chains by chemical modification and mass spectrometric analysis. More than 50 serine residues were assumed to be substituted by GAG chains. EPY is heavily substituted by chondroitin sulfate, giving an overall molecular weight of just under 2 × 10(6). EPY from salmon nasal cartilage is a novel type of large leucine-rich proteoglycan.

Hyaluronan-chondroitin hybrid oligosaccharides as new life science research tools.

Hyaluronan and chondroitin are glycosaminoglycans well-known as components of pharmaceutical agents and health foods. From these attractive molecules, using transglycosylation reaction of testicular hyaluronidase, we synthesized hybrid neo-oligosaccharides not found in nature. We also found a new site between the chondroitin disaccharide unit and hyaluronan disaccharide unit recognized by a hyaluronan lyase specific to hyaluronan using these hybrid oligosaccharides as substrates. We hope that these hybrid oligosaccharides will help to elucidate the involvement of hyaluronan, chondroitin, and chondroitin sulfates in the mechanisms of cell functions and diseases, based on the structures of these glycosaminoglycans.

Synthesis of neoproteoglycans using the transglycosylation reaction as a reverse reaction of endo-glycosidases.

A method for the synthesis of carbohydrate chains (glycosaminoglycans) and their coupling to peptides was investigated using proteoglycans. Glycosidases generally catalyze a hydrolytic reaction, but can also mediate the reverse reaction, which in this case is a transglycosylation. In the transglycosylation reaction of bovine testicular hyaluronidase, which is an endoglycosidase, glycosaminoglycans (hyaluronan and chondroitin sulfates) release disaccharide (uronic acid-N-acetylhexosamine) moieties from non-reducing terminal sites, and then the liberated disaccharides are transferred immediately to the non-reducing termini of other glycosaminoglycan chains. Using such continuous reactions, it is possible to synthesize glycosaminoglycan chains according to a specific design. It then becomes possible to transfer glycosaminoglycan chains synthesized on a peptide to other peptides using the transglycosylation reaction of endo-ß-xylosidase acting on the linkage region between a peptide and glycosaminoglycan chains of proteoglycans. We believe this approach will open a new field for the synthesis of homogeneous proteoglycans or their corresponding analogues.

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.

Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates.

Glycosaminoglycans were prepared as salts of different divalent cations and tested as donors in bovine testicular hyaluronidase catalyzed transglycosylation reactions. All of the metal cations examined had similar binding efficiency of divalent cations to hyaluronan. However, cations bound with different efficiencies to chondroitin sulfate species and the differences were marked in the case of chondroitin 6-sulfate; the numbers of cations bound per disaccharide unit were estimated to be 0.075 for Mn, 1.231 for Ba, 0.144 for Zn, and 0.395 for Cu. While barium salt of chondroitin sulfates enhanced transglycosylation, the zinc salt of chondroitin sulfates inhibited transglycosylation. Therefore, by selecting the proper divalent cation salt of chondroitin sulfates as a donor in the transglycosylation reaction it is possible to improve the yields of the products.

Identification of proteoglycan from salmon nasal cartilage.

There has been no structural information about the core protein of salmon nasal cartilage proteoglycan although its physiological activities have been investigated. Internal amino acid sequencing using nano-LC/MS/MS revealed that the salmon proteoglycan was aggrecan. Primer walk sequencing based on the amino acid information determined that the salmon aggrecan cDNA is comprised of 4207bp nucleotides predicted to encode 1324 amino acids with a molecular mass of 143,276. It exhibited significant similarities to predicted pufferfish aggrecan, zebrafish similar to aggrecan, zebrafish aggrecan, bovine aggrecan and human aggrecan isoform 2 precursor; whose amino acid identities were 56%, 55%, 49%, 31% and 30%, respectively. Salmon cartilage aggrecan had globular domains G1, G2 and G3 as in mammalian aggrecans. Neither the putative keratan sulfate attachment domain enriched with serine, glutamic acid and proline, nor the putative chondroitin sulfate attachment domain with repeating amino acid sequence containing serine-glycine, found in mammalian aggrecans were observed in salmon, however, random serine-glycine (or glycine-serine) sequences predicted to the sugar chain attachment sites were observed. Based on cDNA analysis and amino acid analysis after ß-elimination, the ratio of serine attached to sugar chains was calculated to be approximately 37.7% of total serine, that is, 46 of 123 serine residues.

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.

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.

Novel proteoglycan glycotechnology: chemoenzymatic synthesis of chondroitin sulfate-containing molecules and its application.

Proteoglycans consist of a protein core, with one or more glycosaminoglycan chains (i.e., chondroitin sulfate, dermatan sulfate and heparin sulfate) bound covalently to it. The glycosaminoglycan chains account for many of the functions and properties of proteoglycans. The development of proteoglycan glycotechnology to exploit the functionality of glycosaminoglycan chains is an extremely important aspect of glycobiology. Here we describe an efficient and widely applicable method for chemoenzymatic synthesis of conjugate compounds comprising intact long chondroitin sulfate (ChS) chains. An alkyne containing ChS was prepared by an enzymatic transfer reaction and linked with a chemically synthesized core compound containing an azido group using click chemistry. This method enabled highly efficient introduction of ChS into target materials. Furthermore, the ChS-introduced compounds had marked stability against proteolysis, and the chemically linked ChS chain contributed to the stability of these core compounds. We believe the present method will contribute to the development of proteoglycan glycobiology and technology.

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.

Novel products in hyaluronan digested by bovine testicular hyaluronidase.

When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid chromatography (HPLC), minor peaks were detected just before the main even-numbered oligosaccharide peaks. The amount of each minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer, and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH.

Stimulation of small proteoglycan synthesis by the hyaluronan synthesis inhibitor 4-methylumbelliferone in human skin fibroblasts.

Human skin fibroblasts cultured with 4-methylumbelliferone (MU), a hyaluronan synthesis inhibitor, produce a hyaluronan-deficient extracellular matrix (See [9]). Our present study investigated the effects of MU on proteoglycan, which is the other main component of the extracellular matrix, and interacts with hyaluronan. Proteoglycans isolated from culture medium in the presence or absence of MU were characterized by gel-filtration chromatography, ion-exchange HPLC, electrophoresis, and immunoblotting. We found that MU had only a negligible effect on the synthesis of large proteoglycan but increased the production of small proteoglycan in comparison with cultures lacking MU. This small proteoglycan was identified by immunoblotting as decorin. The structures of decorin synthesized in the presence and absence of MU were compared by gel-filtration chromatography, and the data indicated that cells incubated with MU produced a larger decorin molecule than cells incubated without MU. Furthermore, the two decorins had galactosaminoglycan chains of different sizes. These results suggest that MU inhibits the synthesis of hyaluronan and accelerates production of the larger decorin in the extracellular matrix.