Detection Of Glycosaminoglycans Research Articles

Detection of Glycosaminoglycans in Biological Specimens.

Proteoglycans (PGs) are macromolecules formed by a protein backbone to which one or more glycosaminoglycan (GAG) side chains are covalently attached. Most PGs are present in connective tissues, cell surfaces, and intracellular compartments. The major biological function of PGs derives from the GAG component of the molecule, which is involved in cell growth and proliferation, embryogenesis, maintenance of tissue hydration, and interactions of the cells via receptors. PGs are categorized into four groups based on their cellular and subcellular localization, including cell surfaces and extracellular, intracellular, and pericellular locations. GAGs are a crucial component of PGs involved in various physiological and pathological processes. GAGs also serve as biomarkers of metabolic diseases such as mucopolysaccharidoses and mucolipidoses. Detection of specific GAGs in various biological fluids helps manage various genetic metabolic disorders before it causes irreversible damage to the patient (Amendum et al., Diagnostics (Basel) 11(9):1563, 2021). There are several methods for detecting GAGs; this chapter focuses on measuring GAGs using enzyme-linked immunosorbent assay, liquid chromatographic tandem mass spectrometry, and automated high-throughput mass spectrometry.

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Cell Biological Assays for Measuring Chondrogenic Activities of CCN2 Protein.

Growth-plate chondrocytes undergo proliferation, maturation, hypertrophic differentiation, and calcification; and these processes can be reproduced in vitro in a chondrocyte culture system. Using this system, we have shown that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes all stages of proliferation, maturation, hypertrophic differentiation, and calcification, thus suggesting that CCN2 is a multifunctional growth factor for chondrocytes and plays important roles in chondrocyte proliferation and differentiation. In this chapter, we describe how to evaluate CCN2 functions in these processes occurring in cultured chondrocytes. Evaluation strategies for cell proliferation include measuring DNA synthesis by [3H]-thymidine incorporation, cellular metabolic activity, and cell number with a hemocytometer. Next, evaluation strategies to assess maturation are analysis of the gene expression of markers of mature chondrocytes, and examination of proteoglycan and collagen synthesis by using radioactive compounds. In addition, cytohistochemical detection of glycosaminoglycans (GAGs), such as chondroitin sulfate, by use of alcian blue and toluidine blue staining is useful to evaluate chondrocyte maturation. These methods can be also used for evaluation of physiological functions of CCN2 in permanent chondrocytes such as articular and auricular chondrocytes, which do not calcify under physiological conditions. Next, evaluation of hypertrophic differentiation is performed by detecting type X collagen, which is specific marker of hypertrophic chondrocytes, and by measuring alkaline phosphatase (ALP) activity. Finally, evaluation of calcification is performed by detecting matrix calcification by use of alizarin red staining and by examining the incorporation of 45Ca into cartilaginous matrix. These methods would be useful for the evaluation not only of CCN2 but also of its derivatives and other CCN proteins.

Integrative Self-assembly of Graphene Quantum Dot and Biopolymer into a Versatile Biosensing Toolkit.

Hybrid self-assembly has become a reliable approach to synthesize soft materials with multiple levels of structural complexity and synergistic functionality. In this work, photoluminescent graphene quantum dots (GQDs, 2-5 nm) are used for the first time as molecule-like building blocks to construct self-assembled hybrid materials for label-free biosensors. Ionic self-assembly of disc-shaped GQDs and charged biopolymers is found to generate a series of hierarchical structures that exhibit aggregation-induced fluorescence quenching of the GQDs and change the protein/polypeptide secondary structure. The integration of GQDs and biopolymers via self-assembly offers a flexible toolkit for the design of label-free biosensors in which the GQDs serve as a fluorescent probe and the biopolymers provide biological function. The versatility of this approach is demonstrated in the detection of glycosaminoglycans (GAGs), pH, and proteases using three strategies: 1) competitive binding of GAGs to biopolymers, 2) pH-responsive structural changes of polypeptides, and 3) enzymatic hydrolysis of the protein backbone, respectively. It is anticipated that the integrative self-assembly of biomolecules and GQDs will open up new avenues for the design of multifunctional biomaterials with combined optoelectronic properties and biological applications.

Histomorphometry and detection of glycosaminoglycans in the endocervical epithelium of pregnant rats after local administration of hyaluronidase

The aim of the study was to detect the presence of glycosaminoglycans and to investigate histomorphometric aspects of the endocervical epithelium in pregnant rats after local administration of hyaluronidase. Ten pregnant rats were randomly distributed into two groups. On day 18 of pregnancy 1 mL of distilled water and 0.02 mL of hyaluronidase were administered to the control group (CG) and the study group (SG), respectively On day 20 the rats were sacrificed, followed by dissection and removal of the uterine cervix, which was prepared for histomorphometry (endocervical epithelium thickness and leucocyte infiltration) and for immunohistochemistry with alcian blue reaction and its respective blockers. The paired Student t test was used to compare the groups. The SG was characterized by reduced epithelial thickness (mean: 291.01 +/- 71.1 vs. 764.30 +/- 50.94; p < 0.0001) and a larger number of eosinophils (mean: 3.72 +/- 1.60 vs. 0.54 +/- 0.70; p < 0.0001). Alcian blue staining (pH 0.5) indicated a very strong reaction (3+) for the CG. With pH 2.5, the staining was also very intense (4+) in the CG. With methylation, both groups showed negative reactions after alcian blue staining (pH 2.5). With the methylation reaction followed by saponification and with enzymatic digestion of the lamina, staining showed a weak reaction (1+) in both groups. The SG presented with significant alterations related to the reduction of epithelial thickness and an increase in leucocyte infiltration. Furthermore, the use of hyaluronidase resulted in a significant decrease of the sulfated glycosaminoglycans.

Immunohistochemical and charge-specific localization of anionic constituents in pseudoexfoliation deposits on the central anterior lens capsule from individuals with pseudoexfoliation syndrome.

Pseudoexfoliation (PSX) syndrome is a degenerative systemic disorder that is characterized primarily by deposits of distinct fibrillar material on the surface lining the anterior and posterior chambers of the eye and is often associated with cataract and glaucoma. Although some components of the PSX material have been identified, the precise composition is obscure. High-resolution scanning electron microscopy in conjunction with colloidal cationic gold labeling was used to localize anionic constituents at the surface of PSX aggregates. Transmission electron microscopy was applied for the immunocytochemical detection of glycosaminoglycans, and to monitor the charge-specific distribution of colloidal thorium dioxide and ferritin in PSX material. The specific binding of antibodies was confirmed by immunohistological staining of paraffin-embedded specimens. Paraffin-embedded tissue sections revealed immunoreactivity for keratan sulfate and dermatan sulfate proteoglycan within PSX material deposited on the surface of the anterior lens capsule. Post-embedding immunogold labeling of keratan sulfate demonstrated an intense label of PSX aggregates primarily associated with mature PSX fibrils, whereas dermatan sulfate proteoglycon appeared to be present in low quantities. Additionally, keratan sulfate was found at the humoral periphery of the lens capsules. To further investigate the distribution of anionic sites in PSX material, we used cationic colloidal tracers of different size, such as gold, thorium dioxide and ferritin. PSX aggregates exhibited a strong negative charge, resulting very likely from glycosaminoglycan chains of proteoglycans. The density of anionic sites was higher at the interfibrillar matrix. Lens capsules associated with PSX material revealed a diminished accumulation of cationic ferritin at the humoral surfaces. Increased amounts of different glycosaminoglycans identified in PSX material suggest an important role of proteoglycans for the pathogenic pathway in PSX.

Detection of glycosaminoglycans as a copper(II) complex in high-performance liquid chromatography

Glycosaminoglycans including heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and hyaluronan were analyzed by high-performance gel filtration chromatography. Detection was achieved at 240 nm based on the formation of copper(II) complex in copper sulfate solution at low pH. Detection of the copper(II)-heparin complex is sensitive, permitting the analysis of as little as 10 −7 g. This method was also successfully applied to the analysis of the chemically derivatized glycosaminoglycans (de-N-acetylated and/or oversulfated chondroitin sulfate) that cannot be depolymerized by enzymes such as heparin and chondroitin lyases.

Detection of glycosaminoglycans as a copper (II) complex in capillary electrophoresis.

Glycosaminoglycans including heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and hyaluronic acid were analyzed by reversed polarity capillary electrophoresis. Detection was achieved at 240 nm based on the formation of a copper (II) complex in copper sulfate solution at low pH. Glycosaminoglycans having a high ratio of iduronic acid to glucuronic acid, as well as ones having lower molecular weight, gave the highest detection sensitivity using this method. Detection of the copper (II)-heparin complex is extremely sensitive, permitting the analysis of as little as 10(-9) g. This method was also successfully applied to the analysis of heparin oligosaccharides that lacked a chromophore prepared from heparin using controlled, low pH nitrous acid depolymerization.

Poly-l-lysine-gold complexes used at different pH are probes for differential detection of glycosaminoglycans and phosphoproteins in the predentine and dentine of rat incisor

At neutral pH, poly-L-lysine-gold complexes labelled the predentine extensively, whereas in dentine the number of gold complexes was reduced by half. Hyaluronidase pretreatment of the section at pH 6.8, prior to labelling, suppressed most of the staining in predentine and did not affect dentine. In contrast, alkaline phosphatase pretreatment at pH 9 enhanced the gold complex labelling in predentine and removed most of the labelling in dentine. This proves that at pH 7.2, the polyanions which are stained include a heterogeneous population of glycosaminoglycans, located in predentine, and phosphoproteins, visualized in dentine. At acidic pH levels (2.9 and 1.1), the number of scored gold complexes decreased, but the ratio between predentine and dentine labelling remained constant. Hyaluronidase pretreatment removed or firmly reduced the gold complex labelling both in predentine and dentine, whereas alkaline phosphatase pretreatment of the sections at pH 9 prior to labelling did not induce any change. This argues in favour of an increased specificity of polylysine-gold complex staining for glycosaminoglycans, stained at low pH in both predentine and dentine. Differential staining of glycosaminoglycans and phosphoproteins according to the pH provides a useful tool for studying the role played respectively by the two matrix components in dentine mineralization.

Detection of glycosaminoglycans on the surface of human umbilical vein endothelial cells using gold-conjugated poly-l-lysine with silver enhancement

Endothelial glycosaminoglycans are important in a diverse range of vascular functions. In the course of a biochemical and histological study exploring the role of glycosaminoglycans in inflammation, we have investigated the use of gold-conjugated poly-L-lysine with silver enhancement to establish the nature and physical location of glycosaminoglycans on the surface of cultured human umbilical vein endothelial cells. Cationic gold was effective in locating anionic sites in both cultured endothelial cells and in paraffin-embedded renal tissue. By manipulating pH, and by using enzymes specific for degrading glycosaminoglycans, it was found that, at pH 1.2, staining was directed primarily at glycosaminoglycans. The surface of human umbilical vein endothelial cells was found to be extensively covered in heparan sulphate, the histological appearance of which was dependent upon the fixation procedure employed. Heparan sulphate was also seen to co-distribute with the extracellular matrix protein, fibronectin, when endothelial cultures were simultaneously stained with cationic gold and an antibody to cellular fibronectin.

Detection of glycosaminoglycans at the one-nanogram level by 125I-cytochrome c

The basic protein cytochrome c forms stable ionic complexes with all known glycosaminoglycans. When labeled with 125I, cytochrome c is capable of detecting exceptionally small quantities of glycosaminoglycans. Subsequent to electrophoresis on cellulose acetate strips using pyridine formate buffer at pH 3, followed by ethanol fixation, and treatment with 125I-cytochrome c, all the known glycosaminoglycans are detected at minimum levels of 1 ng/0.25-μl application. The method can be used for quantification of glycosaminoglycans in other electrophoretic buffer systems also.

Detection of glycosaminoglycans in human gliomas by histochemical methods.

Sulphatized mucopolysaccharides or glycosaminoglycans (GAG) were examined by histochemical methods in biopsy specimens of 69 human cerebral gliomas. The intensity of cell-surface associated GAGs, particularly of the -SO3-groups, was highest in isomorphic oligodendrogliomas and astrocytomas except for pilocytic forms. It showed progressive decrease with increasing degree of anaplasia of these tumors. Anaplastic gliomas only rarely show a very weak reaction for -SO3-groups which was completely absent in glioblastomas, where GAGs are only found in blood vessel walls and mesenchymal stroma. In accordance with observations in experimental gliomas, the rapid loss of membrane-associated GAGs in anaplastic gliomas is considered to indicate a progressive cellular dedifferentiation of glial cells due to hitherto unknown metabolic changes.