Selection of Glycosaminoglycan-Deficient Mutants
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Mutant cell lines provide an excellent model for studying the structure, assembly and function of proteoglycans under the controlled conditions of tissue culture. Numerous proteoglycan-deficient strains have been isolated, mostly in Chinese hamster ovary cells, and in many cases the defects have been characterized both genetically and biochemically (see Table 1). Biochemical analysis of the mutants has confirmed that various enzyme activities detected in cell-free extracts using synthetic substrates actually play a role in proteoglycan assembly in vivo. The cell lines have allowed investigators to study how altering the composition of proteoglycans affects fundamental properties of cells, such as adhesion and signaling. Moreover, animal cell mutants provide the background for predicting the phenotype of organismal mutants defective in proteoglycan assembly. Table 1 Cell Mutants with Defined Defects in Glycosaminoglycan Biosynthesis ComplementationGroup Biochemical Defect Phenotype pgsA (CHO) (28) Xylosyltransferase Glycosaminoglycan-deficient pgsB (CHO) (29) Galactosyltransferase I Glycosaminoglycan-deficient pgsG (CHO) (20) Glucuronosyltransferase I Glycosaminoglycan-deficient pgsD (CHO) (30) N-acetylglucosaminyl/glucuronosyltransferase (EXT-1) Heparan sulfate-deficient Gro2C (mouse L-cells) (3,31) N-acetylglucosaminyl/glucuronosyltransferase (EXT-1) Heparan sulfate-deficient ldlD (CHO) (32,33) UDP-glucose/galactose (GlcNAc/GalNAc) 4-epimerase Chondroitin sulfate-deficient when starved for GalNAc; GAG-deficient when starved for galactose pgsC (CHO) (34) Sulfate transporter Normal glycosaminoglycans; deficient labeling with 35SO4 pgsE (CHO) (35) N-deacetylase/N-sulfotransferase 1 (NDST-1) Undersulfated heparan sulfate CM-15 (COS cells) (36) N-deacetylase/N-sulfotransferase (undefined locus) Undersulfated heparan sulfate pgsF (CHO) (26) 2-O-sulfotransferase Deficient 2-O-sulfation of heparan sulfateKeywords:
Perlecan
Keratan sulfate
Sulfotransferase
Dermatan sulfate
Glycosaminoglycans (GAGs) are a family of complex polyanionic polysaccharides best known for their hexosamine-containing disaccharide repeating units. Based on structural features, GAGs can be classified into four major groups: hyaluronan or hyaluronic acid (HA), chondroitin sulfate (CS) and dermatan sulfate (DS), heparin and heparan sulfate (HS), and keratan sulfate (KS). Structure–activity relationship (SAR) evaluations are needed to understand the extent of binding and the specific nature of the binding sites within the GAG chain. The utmost precision of chemical synthesis in defining the length and functionalization patterns ensures a prominent role in GAG acquisition for SAR studies. GAG syntheses remain formidable, but recent advances are instrumental in providing the necessary compounds with increasing complexity and relative ease. This chapter describes the chemical strategies that are currently utilized for the acquisition of GAG-based oligosaccharides and their mimics.
Dermatan sulfate
Keratan sulfate
Chondroitin
Disaccharide
Iduronic acid
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Glycosaminoglycan isolated from the urine of a patient with the Hunter syndrome was composed of heparan sulfate (59.9%), dermatan sulfate (30.6%) and chondroitin sulfate (9.5%), and was heterogeneous in molecular weight (1, 500-10, 000) and in sulfate content (0.35-2.05 moles/mole of hexosamine). About 60% of dermatan sulfate and 10% of heparan sulfate had molecular weight of 7, 000 to 10, 000, while about 10% of the former and 60% of the latter had those of 1, 500 to 3, 500. Sulfate contents of dermatan sulfate and heparan sulfate were inversely related to their molecular weights. Higher total and N-sulfate contents were measured in smaller molecularweight heparan sulfate, and higher acetyl content was in larger molecular-weight heparan sulfate. On the basis of the chemical properties of dermatan sulfate and heparan sulfate isolated in this experiment, their catabolic processes in the Hunter syndrome were discussed.
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Perlecan
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Perlecan is primarily a heparan sulfate containing proteoglycan found in all basement membranes. Rotary shadowed images of perlecan show it to contain three glycosaminoglycan (GAG) side chains extending from one end of its core protein. Domain I is at the N terminus of perlecan and contains three closely spaced Ser-Gly-Asp sequences that may serve in GAG attachment. We evaluated the serines in these three sequences for GAG attachment by preparing a cDNA construct encoding for the N-terminal half (domains I, II, and III) of perlecan and then a series of constructs containing deletions and mutations within domain I of the domain I/II/III construct, expressing these constructs in COS-7 cells, and then analyzing the recombinant product for GAG side chains and GAG type. The results showed that all three serine residues in the Ser-Gly-Asp sequences in domain I can accept both chondroitin and heparan sulfate side chains but that a cluster of acidic residues N-terminal to these sequences is the primary determinant responsible for targeting these sites for heparan sulfate. Furthermore, there are two elements that can enhance heparan sulfate synthesis at a targeted site: 1) the presence of a the SEA module in the C-terminal region of domain I and 2) the presence of multiple acceptors in close proximity. These results indicate that the proportion of heparan and chondroitin sulfate at any one site in domain I of perlecan is regulated by multiple factors. Perlecan is primarily a heparan sulfate containing proteoglycan found in all basement membranes. Rotary shadowed images of perlecan show it to contain three glycosaminoglycan (GAG) side chains extending from one end of its core protein. Domain I is at the N terminus of perlecan and contains three closely spaced Ser-Gly-Asp sequences that may serve in GAG attachment. We evaluated the serines in these three sequences for GAG attachment by preparing a cDNA construct encoding for the N-terminal half (domains I, II, and III) of perlecan and then a series of constructs containing deletions and mutations within domain I of the domain I/II/III construct, expressing these constructs in COS-7 cells, and then analyzing the recombinant product for GAG side chains and GAG type. The results showed that all three serine residues in the Ser-Gly-Asp sequences in domain I can accept both chondroitin and heparan sulfate side chains but that a cluster of acidic residues N-terminal to these sequences is the primary determinant responsible for targeting these sites for heparan sulfate. Furthermore, there are two elements that can enhance heparan sulfate synthesis at a targeted site: 1) the presence of a the SEA module in the C-terminal region of domain I and 2) the presence of multiple acceptors in close proximity. These results indicate that the proportion of heparan and chondroitin sulfate at any one site in domain I of perlecan is regulated by multiple factors.
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Growth factors bind to extracellular matrices and onto cell surfaces, and this binding is mainly mediated by proteoglycans. Proteoglycans are proteins that carry an unusual carbohydrate, a glycosaminoglycan. The glycosaminoglycans are large carbohydrates that are composed of repeating disaccharide units and exist in four main forms: heparan sulfate and heparin, chondroitin sulfate and dermatan sulfate, keratan sulfate, and hyaluronic acid. The first three are protein-bound glycosaminoglycans in their natural form, and they all contain sulfate; hyaluronic acid is made as a free glycosaminoglycan and lacks sulfate (see Ruoslahti 1989; Kolset and Gallagher 1990). The glycosaminoglycan substitution depends on the ability of the protein to serve as an acceptor for xylosyltransferase, the enzyme that begins the synthesis of most types of glycosaminoglycans.
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Keratan sulfate
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Disaccharide
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Although interleukin‐2 (IL‐2) is typically considered a soluble cytokine, our laboratory has shown that the availability of IL‐2 in lymphoid tissues is regulated, in part, by an association with heparan sulfate glycosaminoglycan. Heparan sulfate is usually found in proteoglycan form, in which the heparan sulfate chains are covalently linked to a specific core protein. We now show that perlecan is one of the major IL‐2‐binding heparan sulfate proteoglycans in murine spleen. IL‐2 binds perlecan via heparan sulfate chains, as enzymatic removal of heparan sulfate from splenic perlecan abolishes its ability to bind IL‐2. Furthermore, we demonstrate that perlecan‐bound IL‐2 supports the proliferation of an IL‐2‐dependent cell line. Identification of perlecan as a major heparan sulfate proteoglycan that binds IL‐2 has implications for both the localization and regulation of IL‐2 in vivo .
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Syndecan 1
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Abstract The glycosaminoglycan (GAG) family of polysaccharides includes the unsulfated hyaluronan and the sulfated heparin, heparan sulfate, keratan sulfate, and chondroitin/dermatan sulfate. GAGs are biosynthesized by a series of enzymes, the activities of which are controlled by complex factors. Animal cells alter their responses to different growth conditions by changing the structures of GAGs expressed on their cell surfaces and in extracellular matrices. Because this variation is a means whereby the functions of the limited number of protein gene products in animal genomes is elaborated, the phenotypic and functional assessment of GAG structures expressed spatially and temporally is an important goal in glycomics. On‐line mass spectrometric separations are essential for successful determination of expression patterns for the GAG compound classes due to their inherent complexity and heterogeneity. Options include size exclusion, anion exchange, reversed phase, reversed phase ion pairing, hydrophilic interaction, and graphitized carbon chromatographic modes and capillary electrophoresis. This review summarizes the application of these approaches to on‐line MS analysis of the GAG classes. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 28:254–272, 2009
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Chondroitin
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G (GAGs) are large complexes of negatively charged heteropolysaccharide chains composed of a repeating disaccharide unit [acidic sugar and amino sugar]. The amino sugar is either D-glucosamine or D-galactosamine, the acidic sugar is either D-glucuronic acid or L-iduronic acid. GAGs are located primarily on the surface of cells or in the extracellular matrix (ECM). The specific GAGs of physiological significance are hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparin, heparan sulfate, and keratan sulfate. Hyaluronic acid may be important in permitting tumor cells to migrate through the ECM. Chondroitin sulfate most abundant GAG. Heparan sulfate, extracellular GAG contains higher acetylated glucosamine than heparin and less sulphated groups. Some tumor cells have less heparan sulfate at their surfaces. Heparin is an intracellular GAG, component of intracellular granules of mast cells. Heparin is an important anticoagulant. Its most important interaction is with plasma anti-thrombin III. Dermatan sulfate is a glycosaminoglycan found mostly in skin. Keratan sulfate originally the designations KSI and KSII were based on differences between KS from cornea and that of cartilage. GAGs such as heparin, heparan sulfate (HS) and dermatan sulfate (DS) serve as key biological response modifiers by acting as co-receptors for growth factors, cytokines and chemokines; regulators of enzyme activity; signaling molecules in response to infection, wounding and and targets for viral, bacterial and parasitic virulence factors for attachment and immune system evasion.
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Glycosaminoglycans (GAGs) are complex linear polysaccharides, which are covalently attached to core proteins (except for hyaluronan) to form proteoglycans. They play key roles in the organization of the extracellular matrix, and at the cell surface where they contribute to the regulation of cell signaling and of cell adhesion. To explore the mechanisms and pathways underlying their functions, we have generated an expanded dataset of 4,290 interactions corresponding to 3,464 unique GAG-binding proteins, four times more than the first version of the GAG interactome (Vallet, Clerc, and Ricard-Blum. J Histochem Cytochem 69: 93–104, 2021). The increased size of the GAG network is mostly due to the addition of GAG-binding proteins captured from cell lysates and biological fluids by affinity chromatography and identified by mass spectrometry. We review here the interaction repertoire of natural GAGs and of synthetic sulfated hyaluronan, the specificity and molecular functions of GAG-binding proteins, and the biological processes and pathways they are involved in. This dataset is also used to investigate the differences between proteins binding to iduronic acid-containing GAGs (dermatan sulfate and heparin/heparan sulfate) and those interacting with GAGs lacking iduronic acid (chondroitin sulfate, hyaluronan, and keratan sulfate).
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Keratan sulfate
Chondroitin
Iduronic acid
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