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Life Science

BioFiles Volume 3, Number 10

Glycobiology Glycosaminoglycans and Polysaccharides

Glycosaminoglycan Sulfation and Signaling Dextrans and Related Polysaccharides Chondroitin sulfate proteoglycans modulate secreted chemotactic proteins that regulate axon guidance in neural cell development. These guidance proteins have distinct binding specificities for sulfate groups (sulfur shown in red) within these glycosaminoglycan chains.

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BioFiles Volume 3, Number 10

Table of Contents Introduction..........................................................3

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Glycobiology Webinar The GlycoProfile™ Metabolic Labeling Reagent Technology Webinar explains the GlycoProfile kits and reagents abilities to incorporate, express and detect unnatural glycoproteins within cells. Applications covered in the webinar: • Selectively targeting labeled sugars • Monitoring differential presentation of important glycans • Visualizing internally and externally labeled glycoproteins • Purifying glycoproteins View the webinar at sigma-aldrich.com/glycoprofile

Glycosaminoglycan Sulfation and Signaling.......................................................4 Biosynthesis of Heparan/Heparin and Chondroitin/Dermatan............................... 4 Heparan Sulfate Signaling................................... 6 Chondroitin Sulfate/Dermatan Sulfate Signaling............................................... 7 Research Techniques........................................... 8 Glycosaminoglycans and Proteoglycans............ 10 Glycosaminoglycan Degrading Enzymes........... 13 Antibodies......................................................... 14 Prestige Antibodies™................................... 14 Antibodies to PG Core Proteins.................... 15 Antibodies to GAG Synthesis/Modification Enzymes................... 15 Additional Antibodies.................................. 15 Glycosaminoglycan Disaccharides..................... 16 Monosaccharide Sulfates.................................. 16 Dextran and Related Polysaccharides................17 Dextrans............................................................ 19 Dextran....................................................... 19 Dextran Sulfate............................................ 22 Immobilized Dextran.................................... 22 Dextrins and Pullulans...................................... 23 Additional Gel Permeation Chromatography (GPC) Standards and Sets......................................... 23 GlycoProfile™ β-Elimination Kit.........................24 Related Kits for Deglycosylation....................... 26

Enzymes for Carbohydrate Analysis The carbohydrate analysis resource from the Enzyme Explorer allows you to select from either the enzyme or carbohydrate to find detailed enzyme specificity diagrams and information for complex carbohydrates. • Information on 25 enzymes and over 20 complex carbohydrates • Quick links to products and references • Complete list of enzymes for PTM analysis and carbohydrate analysis Start using the carbohydrate analysis resource at sigma-aldrich.com/carboanalysis 2

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Introduction Vicki Caligur Product Manager, Specialty Biochemistry vicki.caligur@sial.com

Even within the specific class of glycosaminoglycans, the diversity of function is so extensive that a comprehensive review is not possible. Glycosaminoglycans are primarily found in animal tissue, and their function was initially thought to be solely that of space-occupying components of the extracellular matrix. We now know that glycosaminoglycans contribute to the functionality of proteoglycans and participate in cellular signaling, neuron development, lipoprotein metabolism, inflammation, and bacterial infection. The structural complexity of glycosaminoglycans is necessary for functionality but also makes structure confirmation extremely difficult, as demonstrated by the heparin contamination crisis in early 2008. In the early 20th century, heparin was isolated and found to act as an anticoagulant, and it has been used medically for over 70 years. Low molecular weight heparin modified by various techniques has been developed for use as an anticoagulant to reduce heparin-induced thrombocytopenia. In 1983, the pentasaccharide sequence within heparin that possesses the ability to block factor Xa and antithrombin activity was identified.1 The pentasaccharide fondaparinux is now commercially synthesized and has been approved for medical use in the United States and Europe as an anticoagulant under the trade name ARIXTRA®. Although these alternatives are available, heparin from natural sources continues to be widely used clinically. In the spring of 2008, at least 20 patient deaths were associated with contaminated heparin that was not detected by routine analysis. An international team of researchers identified the impurity as a variant of chondroitin sulfate, a different glycosaminoglycan found in cartilage, which had been modified with sulfate groups in an unnatural pattern. The conclusive identification required multiple techniques, including multidimensional NMR spectroscopy supported by enzymatic digestion and HPLC analysis and was validated by chemical synthesis of an oversulfated chondroitin that matched the unknown contaminant.2 As a result of this exhaustive analysis,

Introduction

Polysaccharides are structural components of plant, animal, and bacterial cells. As a group, polysaccharides, such as starch, glycogen, and cellulose, are ubiquitous and common. However, when specific classes of polysaccharides are examined, it is clear they have great variation based on origin, structure, and function. In this Glycobiology issue of BioFiles, we have focused on two specific types of polysaccharides: glycosaminoglycans and dextran.

capillary electrophoresis and NMR spectroscopy are now required by the United States FDA to screen heparin for similar contamination. Dextran, a glucose polymer isolated from the bacteria Leuconostoc mesenteroides was first applied to medical use as a plasma volume expander and an antithrombotic during World War II. The structural features and applications of dextran, dextran sulfate, and pullulan are reviewed in this issue. Finally, the GlycoProfile™ β-Elimination Kit is Sigma®’s newest product for removing glycans from glycoproteins. Unlike other chemical deglycosylation techniques, β-elimination allows researchers to recover both the glycan pool and the protein core for downstream analysis and application. The GlycoProfile β-Elimination Kit allows for complete glycoproteomic analysis of O-linked glycoproteins as never before possible. References: 1. Petitou, M., Casu, B., and Lindahl, U., 1976-1983, a critical period in the history of heparin: the discovery of the antithrombin binding site. Biochimie, 85, 83-89 (2003). 2. Guerrini, M., Beccati, D., Shriver, Z., Naggi, A., Viswanathan, K., Bisio, A., Capila, I., Lansing, J.C., Guglieri, S., Fraser, B., Al-Hakim, A., Gunay, N.S., Zhang, Z., Robinson, L., Buhse, L., Nasr, M., Woodcock, J., Langer, R., Venkataraman, G., Linhardt, R.J., Casu, B., Torri, G., and Sasisekharan, R. Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events. Nat. Biotechnol., 26, 669-75 (2008).

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Glycosaminoglycan Sulfation and Signaling

Glycosaminoglycan Sulfation and Signaling

Glycosaminoglycans are large linear polysaccharides constructed of repeating disaccharide units. The disaccharide units that are the basis of all glycosaminoglycans (except keratan) are a combination of an uronic acid (either glucuronic acid or iduronic acid) and an amino sugar (either N-acetyl-D-glucosamine or N-acetyl-D-galactosamine). There are four classifications of glycosaminoglycans: • Hyaluronan • Chondroitin sulfate/dermatan sulfate • Heparan sulfate/heparin, and • Keratan sulfate. A given glycosaminoglycan will contain only one type of amino sugar, but may contain both uronic acid forms, as iduronic acid is produced by enzyme-induced epimerization of glucuronic acid. The core disaccharide unit of keratan is N-acetyl-D-lactosamine ([1→3]β-D-galactose-β[1→4]-N-acetyl-D-glucosamine; β-D-Gal-[1→4]GlclNAc-[1→3]) and the galactose residue is not available for epimerization. Glycosaminoglycan structures have a high degree of heterogeneity (termed “fine structure”) with respect to molecular weight, disaccharide construction, and sulfation. This variability can be attributed to the fact that glycosaminoglycan synthesis is dynamic. While protein and nucleic acid syntheses are template-based processes, glycosaminoglycan synthesis has no template but is modulated by the processing enzymes present. In addition, glycosaminoglycans, with the exception of hyaluronan, may have sulfate groups added to specific locations of the disaccharide subunit. However, enzymatic sulfation and epimerization reactions do not go to completion, resulting in structural heterogeneity within the polysaccharide molecule. This heterogeneity and dynamic processing make investigation of the structure/function relationships of glycosaminoglycans challenging. Proteoglycans are proteins that have one or more glycosaminoglycan polymers attached to the protein core via an O-linkage through a serine residue. Glycosaminoglycans, whether separately or as a component of a proteoglycan, can bind to and interact with proteins. These glycosaminoglycan-protein interactions have essential roles in cellular processes including proliferation, cellular differentiation and development, and there have been efforts to relate specific sulfation patterns with resultant processes. Hyaluronan is the only glycosaminoglycan that is not sulfated and does not attach to proteins to form proteoglycans. It has been associated with CD44 binding and signaling. The key function of keratan sulfate is as a structural component of cornea proteoglycans and not signaling effects. The glycosaminoglycans primarily involved in cellular signaling are heparan sulfate (HS)/heparin and chondroitin sulfate (CS)/dermatan sulfate (DS). This discussion will focus on heparan/heparin and chondroitin/dermatan and how sulfation impacts protein binding interactions and signaling effects by those glycosaminoglycans.

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Biosynthesis of Heparan/Heparin and Chondroitin/Dermatan The synthesis of heparan and chondroitin chains on a proteoglycan begins in the same way via an O-linked tetrasaccharide. The GAG chain synthesis is initiated by the attachment of xylose (Xyl) by xylosyltransferase (XylT) to a serine residue of the protein core. Two galactose (Gal) sugars are attached to the xylose sequentially by the enzymes galactosyltransferase I (GalT-I) and galactosyltransferase II (GalT-II). Glucuronyltransferase I (GlcAT-I) attaches the fourth sugar, glucuronic acid (GlcA) to the initial chain. At this point, the synthesis diverges (see Figure 1). A heparan polysaccharide continues with the attachment of an N-acetyl-D-glucosamine (GlcNAc) residue by N-acetylglucosaminyltransferase I. Alternatively, a chondroitin polysaccharide continues with the attachment of an N-acetyl-D-galactosamine (GalNAc) residue by N-acetyl­galactos­ aminyl­transferase I.1,2 Ser

Ser

Ser

Ser

Ser

Ser

Figure 1. Biosynthesis of heparan and chondroitin chains. After the core tetrasaccharide (Xyl-Gal-Gal-GlcA-) is synthesized, the polymer can become a heparan chain by the addition of GlcNAc (shown left) or a chondroitin chain by the addition of GalNAc (shown right).

For heparan, the glycosaminoglycan is polymerized by the alternating addition of glucuronic acid and N-acetyl-D-glucosamine residues, to a final length of up to 200 disaccharides. The critical glycosyltransferases for heparan polymerization are exostosin 1 (EXT1) and exostosin 2 (EXT2). Modification of the polysaccharide is necessary for development of the heterogeneous fine structure. Heparan is modified by the N-deacetylase/N-sulfotransferase enzymes NDST1 and NDST2, which replace the N-acetyl groups (GlcNAc) with N-sulfate groups (GlcNS) on a glucosamine residue. The glucuronic acid units of the heparan chain are also susceptible to modification. Glucuronic acid (GlcA) may be converted to its epimer iduronic acid by the enzyme glucuronyl C5-epimerase. The iduronic acid moiety (IdoA) may be further modified by sulfation at the 2-O-position (IdoA-2S) through the activity of the enzyme heparan sulfate 2-O-sulfotransferase (HS2ST).2,3

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These disaccharides may also be sulfated at the C-6 position of glucosamine (see Figure 2). Three variants of the heparan sulfate 6-O-sulfotransferase gene (HS6ST) have been identified.4 The uncommon form of sulfation is at the 3-O-position of the glucosamine ring and is mediated by the enzyme heparan sulfate 3-O-sulfotransferase (HS3ST). There are six isoforms of the HS3ST enzyme (1, 2, 3A, 3B, 4, and 5). Both the HS6ST and HS3ST enzymes have specific substrate preferences.

Heparin is a specific member of the heparan sulfate family of glycosaminoglycans. Heparin is expressed exclusively by mast cells, including mucosa mast cells, and heparin isolated from porcine intestinal cells has been approved by medical regulatory agencies for use as an anticoagulant. This anticoagulant activity of heparin has been shown to be due to the specific pentasaccharide (five-sugar) structure (see Figure 4). 6S

Ser

NS

NS

NS

Glucuronyl C5-epimerase Ser NS

NS

NS

NS

HS2ST Ser NS 2S NS

NS

NS

HS6ST (1,2,3) 6S

6S

6S 6S

NS 2S NS

Ser NS

NS

HS3ST (1,2,3A,3B,4,5) 6S

6S

3SNS 2S NS Figure 4. The pentasaccharide antithrombin binding site of heparin. Sites of sulfation are indicated.

Ser NS

6S

This sequence contains a rare 3-O-sulfated glucosamine and is a specific ligand for the protease inhibitor antithrombin. Heparin binding of antithrombin blocks the activation of factors of the coagulation cascade mechanism. While heparin is frequently referred to in conjunction with heparan sulfate, it should be considered as a unique member of the heparan sulfate family and not interchangeable with heparan sulfate.2,7 For chondroitin synthesis, the glycosaminoglycan is polymerized by the alternating addition of glucuronic acid and N-acetyl-D-galactosamine residues. The completed polymer typically has 40 to over 100 disaccharide units. Modification of the chondroitin structure is also dynamic, generating fine structures that have type classifications that have not been applied to heparan. Chondroitins have been classified into 4 major types, based on location of sulfation on the disaccharide:

Ser 3SNS 2S NS

NS

• Chondroitin sulfate A (CS-A) GlcA-GalNAc-4-SO4 (ΔDi-4S; A)

NS

• Chondroitin sulfate C (CS-C) GlcA-GalNAc-6-SO4 (ΔDi-6S; C) •C  hondroitin sulfate D (CS-D) GlcA-2-SO4-GalNAc-6-SO4 (ΔDi-diSD; D)

Heparin Antithrombin III Pentasaccharide Figure 2. The modification of the heparan core is caused by a cascade of enzymes. The resultant heparan sulfate contains IdoA residues and domains with a high degree of sulfation. The pentasaccharide antithrombin II sequence associated with heparin is shown in the final image. See text for further explanation.

• Chondroitin sulfate E (CS-E) GlcA-GalNAc-4,6-diSO4 (ΔDi-diSE; E) Less common chondroitin disaccharides B, H, and K have also been identified.

Sulfation is concentrated in specific regions of the heparan polysaccharide. As a result, heparan sulfate (HS) has regions of low sulfation interspersed with regions of high sulfation, with transitional regions separating the two. Highly sulfated domains, termed “composite sulfated regions” or S-domains, contain between 2 and 7 or 8 sequential GlcNS-IdoA-2S disaccharides. Other regions of the heparan polymer are largely unmodified and remain acetylated, constructed of GlcNAc-GlcA disaccharide units. Transition zones connect the two domains (see Figure 3). The fine structure complexity of HS is caused by both the variation in the disaccharide units and the overall variability created by the domain structure.5,6

S-Domain

6S

Glycosaminoglycan Sulfation and Signaling

NDST1, NDST2

6S

S-Domain

Key to Monosaccharide Symbols

β-D-Glucose (Glc)

β-D-Xylose (Xyl)

β-D-Mannose (Man)

α-N-Acetylneuraminic acid; Sialic acid (NeuNAc)

β-D-Galactose (Gal)

β-D-Glucuronic acid (GlcA)

β-D-N-Acetylglucosamine (GlcNAc)

α-L-Iduronic acid (IdoA)

β-D-N-Acetylgalactosamine (GalNAc)

α-L-Fucose (Fuc)

S-Domain

N-Acetylated Domains Figure 3. Heparan sulfate has regions of high sulfation (S-domains) interspersed with less modified regions (N-acetylated domains). A transition zone connects the two regions.

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Glycosaminoglycan Sulfation and Signaling

Dermatan, also known as chondroitin sulfate B, undergoes significant epimerization of glucuronic acid (GlcA) to iduronic acid (IdoA). The enzyme 2-sulfotransferase preferentially sulfates IdoA adjacent to GalNAc-4-SO4. As a result of this enzymatic specificity, the primary disaccharide unit of dermatan sulfate is IdoA-2-SO4-GalNAc-4-SO4 (iB). Other rare dermatan sulfates have been recognized, and are named similarly to their chondroitin sulfate counterparts (iA, iC, iD, and iE), using the letter “i� to indicate the iduronic acid residue. As the sulfation and epimerization reactions occur to varying degrees, there is no absolute structure of any chondroitin sulfate/dermatan sulfate, and chondroitin/dermatan contains many fine structures much like heparan sulfate.1

Heparan Sulfate Signaling Heparan sulfate proteoglycans (HSPGs) have been extensively studied for their role in extracellular signaling. HSPGs interact with and regulate growth factors, chemokines, and cytokines. Signaling pathways for apoptosis, cellular development, and adhesion are regulated by proteoglycans. Genetic defects in heparan synthesis and modifying enzymes may affect growth factor signaling and produce morphological defects.8 Drosophila mutants and gene knockout mouse models have been used to eliminate critical heparan synthesis and modification enzymes, and subsequently identify the developmental processes disrupted. Experiments that use Drosophila containing mutations of the protein core of proteoglycans have been performed and have shown that the protein core also affects development. It has been theorized that both the HS and the protein core have roles in the mediation of growth factor binding and cell signaling.6 HS and HSPGs participate in fibroblast growth factor-2 (FGF-2) activation in response to injury and inflammation. After injury, HSs break free from the cell membrane or their proteoglycan serine linkage and become soluble. Heparanase that is released by cells involved in inflammation cleaves the released heparan sulfate into smaller heparin-like oligosaccharides. While intact HS does not activate FGF-2, these smaller oligosaccharides are able to activate FGF-2. This processing of free HS by heparanase in response to inflammation has been theorized to be a necessary step to wound healing.2,9 The binding of HSPGs to FGF-2 has been studied as well for a possible association with malignant transformation of breast carcinoma cells by FGF-2 in vitro. Fibroblast growth factors require the presence of heparan sulfate or heparin to stabilize binding with tyrosine kinase signaling receptors (FGFR-1 through FGFR-4). Mundhenke found that HSPGs isolated from the MCF-7 breast-carcinoma cell line had an elevated ability to promote formation of the FGF-2/HSPG/FGFR-1 complex as compared to normal epithelial cells. HSPGs have also been shown to affect signaling by attachment to other growth factors including FGF-7.10 Using glycosaminoglycan microarrays, Shipp and Hsieh-Wilson showed that individual members of the FGF family require specific sulfation patterns for HS binding. In addition to confirming sulfation specificities previously reported for FGF-1, FGF-2, FGF-4, they were able to characterize the heparan sulfation requirements

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for FGF-16 and FGF-17. Using the microarrays, chemotactic proteins, including slit2, that regulate axon extension were found to have preferential binding to specific sulfation patterns. This finding was validated by co-culturing embryonic olfactory bulb cells with HEK293 cells expressing slit2. The cultures were supplemented with heparan sulfate variants containing different sulfation patterns. After staining with anti-Ď„ antibody for axon visualization, the cultures showed that highly sulfated heparan sulfate reduced slit2-mediated axon repulsion, while desulfated heparin or N-acetylated heparin had no effect on slit2 repulsion.11 The genes for the heparan polymerases exostosin 1 (EXT1) and exostosin 2 (EXT2) were initially thought to be tumor suppressor genes. Mutations of EXT-type genes in Drosophila reduced the level of heparan synthesis and inhibit hedgehog (Hh), Wingless (Wg, which correlates to Wnt), and Dpp signaling pathways, indicating that HS participates in the signaling function of these pathways.12,13 The protein core of the proteoglycan has been shown to contribute to HSPG signaling effects as well. Wnt signaling was modulated by the processed protein core of the HSPG glypican 3, with the implication that proper signaling requires contributions by both the HS and the core protein components.14 A similar protein-glycosaminoglycan synergy toward binding affinity was identified for the association of interleukin-8 (IL-8) with the HSPG syndecan 2 on human umbilical vein endothelial cells. IL-8 is a chemokine that is secreted at sites of inflammation by cytokine activated endothelial cells and which is retained on the cell surface by interactions with HSPGs. While glycosylated syndecan 2 showed strong affinity for IL-8, the nonglycosylated protein core also demonstrated weak IL-8 binding.15 CD44 (Pgp-1, ECM-III, H-CAM) functions as a homing receptor and is most commonly the cellular receptor for hyaluronic acid. Jones, et al., found that some CD44 isoforms exist as heparan sulfate proteoglycans, and that CD44/HSPG was able to bind FGF-2, vascular endothelial growth factor (VEGF), and heparinbinding epidermal growth factor. CD44/HSPG isoforms were overexpressed by macrophages within inflamed rheumatoid arthritis tissue but minimally expressed in noninflamed osteoarthritis tissue. Based on the experimental data, it was theorized that CD44/HSPG isoforms are involved in the regulation of growth factor activity in inflammatory arthritis.16 You, et al., reported that the tumor necrosis factor receptor family member Decoy receptor 3 (DcR3) could induce apoptosis in dendritic cells (DC) through binding between DcR3 and DC surface heparan sulfate proteoglycans This was based on the observation that HBD.Fc, the recombinant protein containing both the heparan sulfate-binding domain (HBD) of DcR3 and the Fc portion of human IgG1, functions in the same manner as DcR3.Fc to induce apoptosis in dendritic cells.17 In addition to sulfate modifications present on the heparan chain, cell surface sulfatases are critical to growth factor signaling. The human sulfatases Sulf1 and Sulf2 are 6-O-endosulfatases and are able to remove 6-O-sulfate from the highly sulfated regions of the polysaccharide.6 The association between 6-O-sulfation of HS and FGF signaling has been studied from many aspects. In a review by Nakato and Kimata, the authors discussed how the fine structures

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The vascular endothelial growth factor VEGF165 contains a heparinbinding domain that is only weakly activated by 2-O-desulfated heparin or 6-O-desulfated heparin.20 6-O-sulfation has also been shown to affect the signaling pathways of Wnt and bone morphogenic protein (BMP). It has been suggested that there is a dynamic interaction between the Sulf enzymes and HS biosynthetic enzymes, rather than independent processes.6

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Chondroitin Sulfate/Dermatan Sulfate Signaling While HS has been of primary interest in glycosaminoglycan signaling effects, chondroitin sulfate (CS) and dermatan sulfate (DS) have also been recognized as affecting signaling pathways,21 and that these interactions are dependent on the polysaccharide fine structure. Both the glycosaminoglycan components and the proteoglycan have been investigated for contributions to biological function. Because of the fine structure and heterogenetity, both CS and DS units may exist as hybrids.22 Research has primarily focused on the contribution by the specific chondroitin suflate proteoglycans (CSPGs) and dermatan sulfate proteoglycans (DSPGs) aggrecan, decorin, biglycan, versican and the syndecans. Additional CSPGs/DSPGs of interest are neurocan, brevican, bamacan, betaglycan, serglycin, endocan, and appican. In much the same manner as HS, CS/DS and CSPGs /DSPGs participate in fibroblast growth factor activation in response to injury and inflammation.2,22 After injury, the glycosaminoglycan chains are cleaved at their serine linkage, or the proteoglycans separate from the cell membrane and become soluble. Soluble dermatan sulfate is able to activate FGF-2 23 and FGF-7, initiating cellular proliferation via FGF-7 through the mitogen-activated protein-kinase (MAPK) pathway.24 The minimum dermatan sulfate structure required by FGF-2 is an octasaccharide and the requirement by FGF-7 is a decasaccharide. 4-O-Sulfation is also required for FGF-dependent cell proliferation, while additional 2-O-sulfation or 6-O-sulfation did not increase the disaccharide activity.25

Glycosaminoglycan Sulfation and Signaling

of HS, especially those containing 6-O-sulfation, modulate the growth factor signaling functions of HSPGs.8 Increased Sulf-1 expression by the breast cancer cell line MDA-MB-468 inhibited angiogenesis and cancer cell proliferation in vivo. The inhibition was attributed in part to the reduced ability of the vascular HS to form a stable FGF-2/HSPG/FGFR-1 complex. The inference is that Sulf-1 overexpression reduces the degree of 6-O-sulfation in HS fragments, and that 6-O-sulfation contributes to the stabilization of the FGF-2/HSPG/FGFR-1 complex.18 More recently, experiments with murine embryonic fibroblasts (MEF) HS-6-O-transferase deficient mice (HS6ST-1-KO, HS6ST-2-KO, and double knock-out) showed that FGF-2 and FGF-4-dependent signaling in MEF was significantly reduced compared to that of wild-type fibroblasts.19

The hepatic growth factor/scatter factor (HGF/SF) is known to participate in morphogenesis and cellular differentiation, organogenesis, and angiogenesis. Endocan, a DSPG that is regulated by proinflammatory cytokines, requires the presence of the glycosaminoglycan moiety in order to promote HGF/SF-induced proliferation of human embryonic kidney cells.26 HGF/SF binding demonstrated preferential binding by 2,6-O-disulfated DS and highly sulfated HS, but not CS, indicating a selective requirement for either iduronic acid residues or a high degree of sulfation, although highly sulfated polysaccharides were less bound than the iduronic acid-containing glycosaminoglycans.27 While the fine structure of CS/DS affects binding and signaling effects, it turns out that growth factors can modulate the expression and structure of the CS/DS glycosaminoglycan. Cultured human lung fibroblasts were treated with transforming growth factor-β1 (TGF-β1) alone, or in combination with epidermal growth factor and platelet-derived growth factor. Cells treated with TGF-β1 or the combination of growth factors increased their expression of CS/DS proteoglycans. In addition, the sulfation and epimerization profiles of the chondroitin/dermatan chains were significantly modified compared to untreated fibroblasts.28

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Glycosaminoglycan Sulfation and Signaling

The highly sulfated CS-E type has been shown to bind to heparin binding growth factors midkine (MK), pleiotropin (PTN), heparin-binding epidermal growth factor-like growth factor (HB-EGF), FGF-16, and FGF-18. As many of these growth factors are expressed in the mammalian brain, it was proposed that CS-E and CSPGs are critical to the development of the brain and central nervous system.29 Subsequent work using an antibody specific for both the CS-E disaccharide and the oversulfated DS-E disaccharide (IdoA-GalNAc-4,6-diSO4; iE) revealed the presence of these glycosaminoglycan sequences in the mouse developing brain. The strong expression of the gene for GalNAc4S-6ST was associated with brain development, and demonstrated that E/iE-containing CS/DS chains are critical in brain development with the implication that E/iE-containing CS/DS chains participate in neurogenesis, axon guidance, and/or neuronal survival.30 While the glycosaminoglycan sequence and sulfation pattern regulate signaling, the protein core of the glycosaminoglycan also contributes to the signaling ability. The ligand binding of the CS/DS proteoglycans decorin and biglycan to transforming growth factor-β (TGF-β), tumor necrosis factor-β (TNF-β), and Wnt-induced secreted protein-1 (WISP1) is mediated by both the DS component and the protein cores. A recent review by Seidler and Dreier analyzed both the galactosaminoglycan component and the protein core of decorin as critical to the ability of decorin to function as a ligand for other signaling molecules.31 The enzymes N-acetylgalactosamine-4-sulfatase (arylsulfatase B; ASB) and galactose-6-sulfatase (GALNS) hydrolyze sulfate groups of CS/DS. MCS-7 cells that overexpressed ASB or GALNS produced increased expression of CS/DS proteoglycans syndecan-1 and decorin. Alernatively, when siRNA was used to silence ASB or GALNS expression in MCS-7 cells, the level of CS content increased and syndecan-1 expression was inhibited. The results demonstrate a role for these sulfatases in CS development and the regulation of proteoglycan expression.32

Genes of Heparan and Chondroitin Biosynthesis Enzymes Gene

Enzyme Name

ARSB

Arylsulfatase B (N-Acetylgalactosamine-4-sulfatase )

C4ST1 (CHST11)

Chondroitin 4-O-sulfotransferase 1

C4ST2 (CHST12)

Chondroitin 4-O-sulfotransferase 2

C4ST3 (CHST13)

Chondroitin 4-O-sulfotransferase 3

ChGn

Chondroitin β1,4 N-acetylgalactosaminyltransferase

CHST1

Carbohydrate (chondroitin 6/keratan) sulfotransferase 1

CHST2

Carbohydrate (chondroitin 6/keratan) sulfotransferase 2

CHST3

Carbohydrate (chondroitin 6) sulfotransferase 3

CHST7 (C6ST-2)

Chondroitin 6-sulfotransferase 2

CHSY1

Chondroitin synthase (Carbohydrate synthase 1)

D4ST1 (CHST14)

Dermatan 4-sulfotransferase 1

DSE

Dermatan sulfate epimerase

EXT1

Exostosin 1 (N-Acetylglucosaminyl-proteoglycan 4-β-glucuronosyltransferase)

EXT2

Exostosin 2 (N-Acetylglucosaminyl-proteoglycan 4-β-glucuronosyltransferase)

GALNS

Galactosamine (N-acetyl)-6-sulfate sulfatase (N-Acetylgalactosamine-6-sulfatase)

GLCE

Glucuronic acid epimerase (Heparin/heparan sulfate glucuronic acid C5 epimerase)

HS2ST1

Heparan sulfate 2-O-sulfotransferase I

HS3ST1

Heparan sulfate 3-O-sulfotransferase I

HS6ST1

Heparan sulfate 6-O-sulfotransferase I

NDST1

N-Deacetylase/N-sulfotransferase 1

NDST2

N-Deacetylase/N-sulfotransferase 2

NDST3

N-Deacetylase/N-sulfotransferase 3

SULF1

Sulfatase 1

SULF2

Sulfatase 2

Table 1. Genes of heparan and chondroitin biosynthesis enzymes. MISSION® siRNA and/or shRNA are available for all genes listed. Genes of Proteoglycan Core Proteins

Research Techniques A variety of experimental techniques have been employed to identify the structure of the glycosaminoglycan chain, the degree and location of sulfation, and the relationship between polysaccharide component and protein core. Knock out mice that lack a designated heparan or chondroitin/dermatan synthesis enzyme have been used to help identify the pathways affected by the lack of glycosaminoglycan modification. Table 1 lists several of the enzymes involved in glycosaminoglycan synthesis and modification and their genes. Table 2 lists the genes for proteoglycan core protein expression. RNA interference has been applied in vitro as another means to eliminate synthesis and modification enzymes to study downstream cellular processes. ELISA analysis with proteoglycan core specific antibodies has been used to monitor protein expression, while quantitative RT-PCR has been applied to study gene regulation.

Gene

Protein Name

ACAN

Aggrecan

AGRN

Agrin

APP

Amyloid β (A4) precursor protein (Appican)

BGN

Biglycan

DCN

Decorin

GPC1

Glypican 1

GPC2

Glypican 2

GPC3

Glypican 3

GPC4

Glypican 4

GPC5

Glypican 5

GPC6

Glypican 6

HSPG2 (PLC)

Perlecan

SDC1

Syndecan 1

SDC2

Syndecan 2

SDC3

Syndecan 3

SDC4

Syndecan 4

SRGN

Serglycin

Table 2. Genes of proteoglycan core proteins. MISSION® siRNA and/or shRNA are available for all genes listed.

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Poly-L-lysine coated slides were used to create microarrays of desulfated HS glycosaminoglycans. The microarrays were used to profile the binding of FGF-2 and other FGF factors against different sulfation patterns. The microarray technique provides a high throughput method to study protein-glycosaminoglycan binding.11 High-resolution NMR spectroscopy, X-ray crystallography, and molecular modeling are valuable methods applied to the investigation of glycosaminoglycan-protein structural interactions. Some of the resulting glycan-protein data has been recorded and is publicly available from the Centre de Recherches sur les Macromolécules Végétales (CER-MAV; http://www.cermav.cnrs.fr/glyco3d).34 A glycomics approach has been used to monitor the regulation of glycosaminoglycan biosynthesis during differentiation of mouse embryonic stem cells. Glycosaminoglycan content and composition changed during cellular transition from stem cells to embryoid bodies and extraembryonic endodermal cells, with 4 to 6-fold increases in CS/DS synthesis and 5 to 8-fold increases in heparan sulfate synthesis. mRNA transcription levels for glycosaminoglycan synthesis and modifying enzymes were unchanged and did not appear to correlate to the changes in glycosaminoglycan levels found during differentiation.35

Summary Much has been learned in the last few years about glycosaminoglycans. The synthesis mechanisms have been elucidated in great detail and their contribution to proteoglycan structure recognized. The relationship of fine structure and core protein structure to receptor binding is now known to be necessary for multiple cellular processes. Techniques used for genomics, proteomics, and cell biology have been successfully applied to the investigation of glycosaminoglycan signaling pathways. While some aspects of the structural requirements of glycosaminoglycans and proteoglycans for effective signaling have been identified, the reasons for why they participate in cellular processes and how the signaling pathways are initiated and regulated are still to be determined.

References: 1. Silbert, J.E. and Sugumaran, G. Biosynthesis of chondroitin/dermatan sulfate. IUBMB Life, 54, 177-86 (2002). 2. Taylor, K.R., and Gallo, R.L., Glycosaminoglycans and their proteoglycans: hostassociated molecular patterns for initiation and modulation of inflammation. FASEB J., 20, 9-22 (2006). 3. Habuchi, H., et al., Sulfation pattern in glycosaminoglycan: does it have a code? Glycoconj. J., 21, 47-52 (2004). 4. Habuchi, H., et al., The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine. J. Biol. Chem., 275, 2859-68 (2000). 5. Gallagher, J.T., Multiprotein signalling complexes: regional assembly on heparan sulphate. Biochem. Soc. Trans., 34, 438-41 (2006). 6. Lamanna, W.C., et al., The heparanome - the enigma of encoding and decoding heparan sulfate sulfation. J. Biotech., 129, 290-307 (2007). 7. Petitou, M., et al., 1976-1983, a critical period in the history of heparin: the discovery of the antithrombin binding site. Biochimie, 85, 83-89 (2005). 8. Nakato, H., and Kimata, K. Heparan sulfate fine structure and specificity of proteoglycan functions. Biochim. Biophys. Acta, 1573, 312-8 (2002). 9. Kato, M., et al., Physiological degradation converts the soluble syndecan-1 ectodomain from an inhibitor to a potent activator of FGF-2. Nat. Med., 4, 691-7 (1998). 10. Mundhenke, C., et al., Heparan sulfate proteoglycans as regulators of fibroblast growth factor-2 receptor binding in breast carcinomas. Am. J. Pathol., 160, 185-94 (2002). 11. Shipp, E.L. and Hseih-Wilson, L.C., Profiling the sulfation specificities of glycosaminoglycan interactions with growth factors and chemotactic proteins using microarrays. Chem. Biol., 14, 195-208 (2007). 12. Bournemann, D.J., et al., Abrogation of heparan sulfate synthesis in Drosophila disrupts the Wingless, Hedgehog and Decapentaplegic signaling pathways. Development, 131, 1927-1938 (2004). 13. Han, C., et al., Distinct and collaborative roles of Drosophila EXT family proteins in morphogen signalling and gradient formation. Development, 131, 1563-1575 (2004). 14. De Cat, B., et al., Processing by proprotein convertases is required for glypican-3 modulation of cell survival, Wnt signaling, and gastrulation movements. J. Cell Biol., 163, 625-635 (2003). 15. Halden, Y., et al., Interleukin-8 binds to syndecan-2 on human endothelial cells. Biochem. J., 377, 533-538 (2004). 16. Jones, M. et al., Heparan sulfate proteoglycan isoforms of the CD44 hyaluronan receptor induced in human inflammatory macrophages can function as paracrine regulators of fibroblast growth factor action. J. Biol. Chem., 275, 7964-74 (2000). 17. You, R.I., et al., Apoptosis of dendritic cells induced by decoy receptor 3 (DcR3). Blood, 111, 1480-8 (2008). 18. Narita, K., et al., HSulf-1 inhibits angiogenesis and tumorigenesis in vivo. Cancer Res., 66, 6025-32 (2006). 19. Sugaya, N., et al., 6-O-Sulfation of heparan sulfate differentially regulates various fibroblast growth factor-dependent signalings in culture. J. Biol. Chem., 283, 1036676 (2008). 20. Ashikari-Hada, S., et al., Heparin regulates vascular endothelial growth factor165dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins. J. Biol. Chem., 280, 31508-15 (2005). 21. Nandini, C.D. and Sugahara, K., Role of the sulfation pattern of chondroitin sulfate in its biological activities and in the binding of growth factors. Adv. Pharmacol., 53, 253-79 (2006). 22. Sugahara, K., et al., Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate. Curr. Opin. Struct. Biol., 13, 612-20 (2003). 23. Penc, S.F., et al., Dermatan sulfate released after injury is a potent promoter of fibroblast growth factor-2 function. J. Biol. Chem., 273, 28116-21 (1998). 24. Trowbridge, J.M., et al., Dermatan sulfate binds and potentiates activity of keratinocyte growth factor (FGF-7). J. Biol. Chem., 277, 42815-20 (2002). 25. Taylor, K.R., et al., Structural and sequence motifs in dermatan sulfate for promoting fibroblast growth factor-2 (FGF-2) and FGF-7 activity. J. Biol. Chem., 280, 5300-6 (2005). 26. Béchard, D., et al., Endocan is a novel chondroitin sulfate/dermatan sulfate proteoglycan that promotes hepatocyte growth factor/scatter factor mitogenic activity. J. Biol. Chem., 276, 48341-48349 (2001). 27. Catlow, K.R., et al., Interactions of hepatocyte growth factor/scatter factor with various glycosaminoglycans reveal an important interplay between the presence of iduronate and sulfate density. J. Biol. Chem., 283, 5235-5248 (2008). 28. Tiedemann, K., et al., Regulation of the chondroitin/dermatan fine structure by transforming growth factor-β1 through effects on polymer-modifying enzymes. Glycobiology, 15, 1277-1285 (2005). 29. Deepa, S.S., et al., Specific molecular interactions of oversulfated chondroitin sulfate E with various heparin-binding growth factors. J. Biol. Chem., 277, 43707-43716 (2002). 30. Purushothaman, A., et al., Functions of chondroitin sulfate/dermatan sulfate chains in brain development. Critical roles of E and iE disaccharide units recognized by a single chain antibody GD3G7. J. Biol. Chem., 282, 19442-19452 (2007). 31. Seidler, D.G. and Dreier, R., Decorin and its galactosaminoglycan chain: extracellular regulator of cellular function? IUBMB Life, 60, 729-33 (2008). 32. Bhattacharyya, S., et al., Distinct effects of N-acetylgalactosamine-4-sulfatase and galactose-6-sulfatase expression on chondroitin sulfates. J. Biol. Chem., 283, 95239530 (2008). 33. Hitchcock, A.M., et al., Comparative glycomics of connective tissue glycosaminoglycans. Proteomics, 8, 1384-1397 (2008). 34. Imberty, A., et al., Structural view of glycosaminoglycan-protein interactions. Carbohydr. Res., 342, 430-439 (2007). 35. Nairn, A.V., et al., Glycomics of proteoglycan biosynthesis in murine embryonic stem cell differentiation. J. Proteome Res., 6, 4374-87 (2007). 36. Kirkpatrick, C.A. and Selleck, S. B., Heparan sulfate proteoglycans at a glance. J. Cell Sci., 120, 1829-32 (2007).

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Glycosaminoglycan Sulfation and Signaling

Enzymatic degradation is often used to cleave glycosaminoglycans into disaccharide units for subsequent analysis. Amide-hydrophilic interaction chromatography (HILIC) liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been applied for comparative glycomics of chondroitin sulfate isoforms. As carbohydrates are inherently difficult to detect by HPLC, they are often modified by reductive amination to covalently attach a detection molecule, typically 2-aminobenzoic acid (2-AB), 2-anthranilic acid (2-AA) or 2-aminopyridine (2-AP). Stable isotopically labeled 2-anthranilic acid was used to comparatively profile CS disaccharides from different tissues.33

9


Glycosaminoglycans and Proteoglycans

q Heparin Heparin ammonium salt from porcine intestinal mucosa [60800‑63‑7]

Glycosaminoglycans

 activity: ~140 USP units/mg

Chondroitin sulfate B sodium salt Dermatan sulfate sodium salt; β-Heparin [54328‑33‑5]

25,000 units

H6279-100KU

100,000 units

H6279-250KU

250,000 units

H6279-500KU

 from porcine intestinal mucosa, ≥90%, lyophilized powder

H6279-1MU

store at: 2-8°C

Glycosaminoglycan Sulfation and Signaling

H6279-25KU

C3788-25MG

25 mg

C3788-100MG

100 mg

Heparin lithium salt from porcine intestinal mucosa [9045‑22‑1]  activity: ≥150 USP units/mg

Chondroitin sulfate sodium salt from shark cartilage

H0878-100KU

[9007‑28‑7]

H0878-1MU

Natural polymer of β-glucuronic acid-(1→ 3)N-acetyl-β-galactosamine [β-GlcA-(1→3)-GalNAc-(1→4)]. Sulfation may occur at the 6-position and/or 4-position of the galactosamine moiety, with the potential of any individual disaccharide unit to be 6-sulfated, 4-sulfated, 4,6-disulfated, or unsulfated.

100,000 units 1,000,000 units

Heparin sodium salt [9041‑08‑1]  activity: ≥140 USP units/mg

Ratio of β-GlcA-(1→3)-GalNAc-6-sulfate to β-GlcA-(1→3)-GalNAc-4-sulfate is determined by HPLC after enzymatic degradation. store at: 2-8°C

Low calcium content EDTA....................................................................................... none detected Ca................................................................................................... ≤20 ppm

250 mg

store at: 2-8°C

C4384-1G

1 g

H4784-250MG

C4384-5G

5 g

H4784-1G

C4384-25G

25 g

C4384-250MG

500,000 units 1,000,000 units

Chondroitin sulfate A sodium salt from bovine trachea

250 mg 1 g

Heparin sodium salt from bovine intestinal mucosa [9041‑08‑1]

Alternating Co­poly β-glucu­ronic acid-(1→3)-N-acetyl-β-galacto­sa­ mine4-sulfate-(1→4) [39455‑18‑0]  cell culture tested, lyophilized powder Approx. 70%; balance is chondroitin sulfate C

 activity: ≥140 USP units/mg H0777-25KU

25,000 units

H0777-50KU

50,000 units

H0777-100KU

100,000 units

H0777-250KU

250,000 units

Appears to play a regulatory role for chondrocytes, neural cells, and some tumor cells

H0777-500KU

surface coverage................................................................... 20‑2000 μg/cm2

Heparin sodium salt from porcine intestinal mucosa

store at: 2-8°C C9819-5G

5 g

C9819-25G

25 g

He­para­n sulfate sodium salt from bovine kidney Heparitin sulfate sodium salt [57459‑72‑0] Constituent of membrane-associated proteoglycans suggested to contribute to cell-cell adhesion store at: 2-8°C

H0777-1MU

[9041‑08‑1]  Grade I-A, activity: ~170 USP units/mg H3393-10KU

10,000 units

H3393-25KU

25,000 units

H3393-50KU

50,000 units

H3393-100KU

100,000 units

H3393-250KU

250,000 units

H3393-500KU H3393-1MU

H7640-1MG

1 mg

H7640-5MG

5 mg

H7640-10MG

10 mg

He­para­n sulfate fast-moving fraction sodium salt  from porcine intestinal mucosa

500,000 units 1,000,000 units

500,000 units 1,000,000 units

 activity: ≥140 USP units/mg H9399-25KU

25,000 units

H9399-50KU

50,000 units

H9399-100KU

100,000 units

H9399-250KU

250,000 units

Heparitin sulfate [57459‑72‑0]

H9399-500KU H9399-1MU

1,000,000 units

 ≥90% (electrophoresis)

H9399-5MU

5,000,000 units

store at: 2-8°C

500,000 units

Heparin sodium salt from porcine intestinal mucosa

H9902-1MG

1 mg

H9902-5MG

5 mg

[9041‑08‑1]  activity: ≥160 IU/mg Crude Unbleached

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H5515-25KU

25,000 units

H5515-100KU

100,000 units

H5515-250KU

250,000 units

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Proteoglycans

Heparin sodium salt from porcine intestinal mucosa [9041‑08‑1]

Aggrecan from bovine articular cartilage

 average mol wt ~3,000

 lyophilized powder

Depolymerized by peroxidolysis (free-radical induced cleavage).

Major structural proteoglycan of cartilage extracellular matrix. Large proteoglycan with a molecular weight greater than 2,500 kDa. Approximately 100-150 glycosaminoglycan (GAG) chains are attached to the core protein (210-250 kDa). The majority of the GAG chains are chondroitin/dermatan sulfate with the remainder being keratan sulfate. This structural molecule produces a rigid, reversibly deformable gel that resists compression. It combines with hyaluronic acid to form very large macromolecular complexes. Addition of small amounts (0.1-2% w/w) of hyaluronic acid to an aggrecan solution (2mg/ml) results in the formation of a complex with an increased hydrodynamic volume and in a significant increase (30-40%) in the relative viscosity of the solution. Aggrecan is a critical component for cartilage structure and the function of joints. The synthesis and degradation of aggrecan are being investigated for their roles in cartilage deterioration during joint injury, disease, and aging. Contains three globular domains, G1, G2, and G3, that are involved in aggregation and hyaluronan binding, cell adhesion, and chondrocyte apoptosis.

activity: <60 units/mg H3400-50MG

50 mg

H3400-100MG

100 mg

H3400-250MG

250 mg

H3400-1G

1 g

 mol wt 4,000-6,000 Da H8537-50MG

50 mg

H8537-100MG

100 mg

H8537-250MG

250 mg

H8537-1G

1 g

Heparin p Hyaluronan bio­tin sodium salt

Associated gene(s): AGC1 (280985)

Hyaluronic acid bio­tin

Proteoglycans are extracted from articular cartilage with guanidine hydrochloride. Further purification includes gel filtration and ion exchange chromatography. The product is dialyzed against water and sterile-filtered prior to lyophilization.

From cocks comb or fermentation. Highly sensitive probe for detecting nanogram levels of hyaluronan binding proteins (hyaladherins).

salt..........................................................................................essentially free

mol wt ~700 kDa

store at: −20°C

 >97%, soluble powder

A1960-1MG

store at: 2-8°C

1 mg

Biglycan from bovine articular cartilage

B1557-5MG

5 mg

q Hyaluronic acid Poly(β-glucu­ronic acid-[1→3]-β-N-acetyl­gluco­s­amine-[1→4]), alternating [9067‑32‑7]

Hyaluronic acid sodium salt from bovine vitreous humor

Interacts with collagen type I, as well as with fibronectin and TGF-β.

e  ssentially salt-free, lyophilized powder ( from a sterilefiltered solution) Associated gene(s): BGN (280733) mol wt 200-350 kDa (proteoglycan consisting of a 45 kDa core protein and two chrondroitin/dermatan sulfate glycosaminoglycan chains)

(C14H21NaNO11)n

store at: −20°C

High molecular weight polymer composed of repeating dimeric units of glucuronic acid and N-acetyl glucosamine which forms the core of complex proteoglycan aggregates found in extracellular matrix.

B8041-.5MG

store at: −20°C H7630-10MG

10 mg

H7630-50MG

50 mg

Glycosaminoglycan Sulfation and Signaling

Low molecular weight

0.5 mg

Decorin from bovine articular cartilage  salt-free, lyophilized powder Decorin interacts with collagen type I and II, fibronectin, thrombospondin and TGF-β. Decorin is an approx. 100 kDa proteoglycan consisting of a 40 kDa core protein and one chondroitin or dermatan sulfate glycosaminoglycan chain.

Hyaluronic acid sodium salt from rooster comb store at: −20°C

Associated gene(s): DCN (280760)

H5388-100MG

100 mg

H5388-250MG

250 mg

H5388-1G

1 g

store at: −20°C D8428-.5MG

0.5 mg

Hyaluronic acid sodium salt from Streptococcus equi  BioChemika protein....................................................................................................≤1% store at: −20°C 53747-1G

1 g

53747-10G

10 g

Hyaluronic acid p

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11


Glycosaminoglycan Sulfation and Signaling

Glypican 3

8

q Proteoglycan

Sulfate proteoglycan attached to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Regulates cell survival and inhibits cell proliferation during normal development.1 Potential use as marker for early disease detection.2

Chromatographically purified using 7M urea on DEAE-cellulose by modified procedure by Antonopoulos.1

Lit cited: 1. Song, H.H. et al., The loss of glypican-3 induces alterations in Wnt signaling. J. Biol. Chem. 280, 2116-2125 (2005); 2. Wang, X.Y. et al., Glypican-3 expression in hepatocellular tumors: diagnostic value for preneoplastic lesions and hepatocellular carcinomas. Hum. Pathol. 37, 1435-1441 (2006);

Proteoglycan from bovine nasal septum

Lit cited: 1. Antonopoulos, C.A., et al., Biochim. Biophys. Acta 338, 108-119 (1974);

store at: −20°C

Glypican 3, Recombinant human

P5864-10MG

Under non-reducing conditions in SDS-PAGE, the glycanated glypican 3 appears as a smear with an apparent molecular mass of 60 - 100 kDa.

Proteoglycan from chicken sternal cartilage

Lyophilized from a 0.2 μm filtered solution in PBS containing 50 μg of bovine serum albumin per 1 μg of cytokine.

 solid

recombinant, expressed in mouse NSO cells

P5989-2MG

10 mg

store at: −20°C

store at: −20°C

2 mg

Proteoglycan p

G1921-50UG

50 μg

He­para­n sulfate proteoglycan

Tissue Inhibitor of Met­allo­protein­ase-1 human

HSPG

TIMP-1

Extracellular matrix component that binds to fibroblast growth factors, vascular endothelial growth factor (VEGF) and VEGF receptors through its sugar moiety. Acts as a docking molecule for matrilysin (MMP-7) and other matrix metalloproteinases.

TIMP-1 has greater binding efficiency to MMP-9, MMP-1, and MMP-3 than the other MMPs.

 ≥400 μg/mL glycosaminoglycan Composed of a core protein covalently bound to heparan sulfate chains.

Associated gene(s): TIMP1 (7076)

 r ecombinant, expressed in CHO cells, ~500 μg/mL protein, buffered aqueous solution Supplied in 0.01 M sodium phosphate buffer pH 7.3, 0.15 M NaCl.

For cell culture use.

mol wt ~29 kDa

Isolated from basement membrane of Engelbreth-Holm-Swarm mouse sarcoma.

ship: wet ice store at: −20°C

Solution in 50 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, pH 7.4, containing ≥400 μg protein per ml.

Trans­form­ing Growth Factor-β Soluble Receptor III human

Uronic acid................................................................................. ≥100 μg/mL

TGF-β sRIII

ship: dry ice store at: −20°C H4777-.1MG

0.1 mg

T8947-5UG

5 μg

>  97% (SDS-PAGE), recombinant, expressed in mouse NSO cells, lyophilized powder Lyophilized from a 0.2 μm filtered solution in phosphate buffered saline containing 5 mg bovine serum albumin The receptor-mediated activity is measured by its ability to inhibit the TGF-β2 bioactivity in HT-2 cells. Endotoxin.............................................................................................tested store at: −20°C T4567-.1MG

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0.1 mg


Glycosaminoglycan Degrading Enzymes Chondroitinases Name

Specific Activity

Unit Definition

Cat. No.

Chondroitinase ABC from Proteus vulgaris

0.3‑3 units/mg solid using chondroitin sulfate C as substrate

One unit will liberate 1.0 μmole of 2-acetamido-2-deoxy-3-O-(β-d-gluc4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose from chondroitin sulfate A or 1.0 μmole of 2-acetamido-2-deoxy-3-O-(β-d-gluc-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose from chondroitin sulfate C per min at pH 8.0 at 37 °C.

C2905-2UN C2905-5UN C2905-10UN

50‑250 units/mg protein using chondroitin sulfate C as substrate

One unit will liberate 1.0 μmole of 2-acetamido-2-deoxy-3-O-(β-d-gluc4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose from chondroitin sulfate A or 1.0 μmole of 2-acetamido-2-deoxy-3-O-(β-d-gluc-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose from chondroitin sulfate C per min at pH 8.0 at 37 °C.

C3667-5UN C3667-10UN

0.5‑1.5 units/mg solid using chondroitin sulfate A as substrate, also cleaves chondroitin sulfate C

One unit will cause a ΔA232 of 1.0 per minute due to the release of unsaturated disaccharide from chondroitin sulfate A at pH 7.3 at 37 °C. Reaction volume: 3.1 ml (light path 1 cm).

C2780-5UN

100‑300 units/mg solid

One unit will form 0.1 μmole of unsaturated uronic acid per hr at pH 7.5 at 25°C using chondroitin sulfate B as substrate.

C8058-50UN

≥200 units/mg solid

One unit will form 0.1 μmole of unsaturated uronic acid per hr at pH 8.0 at 25 °C using chondroitin sulfate C as substrate.

C0954-75UN

Name

Specific Activity

Unit Definition

Cat. No.

Heparinase I from Flavobacterium heparinum

200‑600 unit/mg solid

One unit will form 0.1 μmole of unsaturated uronic acid per hr at pH 7.5 at 25 °C. One International Unit (I.U.) is equivalent to approx. 600 Sigma units.

H2519-50UN H2519-100UN H2519-250UN

Heparinase II from Flavobacterium heparinum

100‑300 units/mg solid

One unit will form 0.1 μmole of unsaturated uronic acid per hr at pH 7.0 at 25 °C. One International Unit (I.U.) is equivalent to approx. 600 Sigma units.

H6512-10UN H6512-25UN H6512-100UN

200‑600 unit/mg solid

One unit will form 0.1 μmole of unsaturated uronic acid per hr at pH 7.5 at 25 °C. One International Unit (I.U.) is equivalent to approx. 600 Sigma units.

H8891-5UN H8891-10UN H8891-50UN

Lyophilized powder containing Tris buffer salts Chondroitinase ABC from Proteus vulgaris

Chondroitinase AC from Flavobacterium heparinum

lyophilized powder Chondroitinase B from Flavobacterium heparinum lyophilized powder (with BSA as stabilizer) Chondroitinase C from Flavobacterium heparinum lyophilized powder

Heparinases

Glycosaminoglycan Sulfation and Signaling

lyophilized powder

lyophilized powder Heparinase III from Flavobacterium heparinum

Hyaluronidases Name

Specific Activity

Analysis

Cat. No.

Hyaluronidase from bovine testes, Type I-S

400‑1000 units/mg solid

One unit is based on the change in absorbance at 600 nm (change in turbidity) of a USP reference standard hyaluronidase which is assayed concurrently with each lot of this product.

H3506-100MG H3506-500MG H3506-1G H3506-5G

Hyaluronidase from bovine testes, Type IV-S

750‑3000 units/mg solid

One unit will cause a change in A600 of 0.330 per minute at pH 5.7 at 37°C

H3884-50MG H3884-100MG H3884-500MG H3884-1G

Hyaluronidase from bovine testes, Type VIII

~300 units/mg

One unit will cause a change in A600 nm of 0.330 per minute at pH 5.7 at 37 °C

H3757-100MG

Hyaluronidase from bovine testes, Type VI-S

3,000‑15,000 units/mg solid

One unit is based on the change in absorbance at 600 nm (change in turbidity) of a USP reference standard hyaluronidase which is assayed concurrently with each lot of this product.

H3631-3KU H3631-15KU H3631-30KU

Hyaluronidase from sheep testes, Type V

≥1,500 units/mg solid

One unit will cause a change in A600 nm of 0.330 per minute at pH 5.7 at 37 °C

H6254-500MG H6254-1G

Hyaluronidase from sheep testes, Type II

≥300 units/mg

One unit will cause a change in A600 nm of 0.330 per minute at pH 5.7 at 37 °C

H2126-100MG H2126-500MG H2126-1G H2126-5G

-

-

H1136-1AMP

Lyophilized powder containing lactose Hyaluronidase from Streptomyces hyalurolyticus

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Antibodies Prestige Antibodies™

Glycosaminoglycan Sulfation and Signaling

Prestige Antibodies Powered by Atlas Antibodies are developed and validated by the Human Proteome Resource (HPR) project (proteinatlas.org). Each antibody is tested by immunohistochemistry against hundreds of normal and disease tissues. These images can be viewed on the Human Protein Atlas (HPA) site. For each antibody, the site includes multiple immunohistochemical images and a summary of the protein expression and staining level found using different cell lines and tissues. In certain tumor groups, subtypes have been included and efforts have been made to include high and low grade malignancies where applicable. Tumor heterogenity and inter-individual differences are reflected in diverse expression of proteins resulting in variable immunohistochemical staining patterns. The antibodies are also tested using protein array and Western blotting. For application protocols and other useful information about Prestige Antibodies and the HPA, visit sigma.com/prestige.

Immunohistochemistry

Immunohistochemistry

Immunohistochemistry

Anti-BGN: Cat. No. HPA003157: Immunoperoxidase staining of formalin-fixed, paraffin-embedded human soft tissue showing positive staining of chondrocytes in the cartilage.

Anti-HS3ST1: Cat. No. HPA002237: Immunoperoxidase staining of formalin-fixed, paraffin-embedded human cerebral cortex tissue showing positive staining of neuronal and non-neuronal cells.

Anti-SULF2: Cat. No. HPA002325: Immunoperoxidase staining of formalin-fixed, paraffin-embedded human heart muscle tissue showing strong cytoplasmic staining of myocytes.

Prestige Antibodies

14

Product Name

Host

Form

Gene Symbol

Species Reactivity

Anti-BGN 8

rabbit

affinity isolated antibody

BGN, human

human

IHC (p) PA

HPA003157-100UL

Anti-CHST2 8

rabbit

affinity isolated antibody

CHST2, human

human

IHC (p) PA WB

HPA013313-100UL

Anti-CSPG4 8

rabbit

affinity isolated antibody

CSPG4, human

human

IHC (p) PA

HPA002951-100UL

Anti-DCN 8

rabbit

affinity isolated antibody

DCN, human

human

IHC (p) PA

HPA003315-100UL

Anti-DSE 8

rabbit

affinity isolated antibody

DSE, human

human

IHC (p) PA

HPA014764-100UL

Anti-FGF1 8

rabbit

affinity isolated antibody

FGF1, human

human

IHC (p) PA

HPA003265-100UL

Anti-FGF4 8

rabbit

affinity isolated antibody

FGF4, human

human

IHC (p) PA

HPA011209-100UL

Anti-GPC6 8

rabbit

affinity isolated antibody

GPC6, human

human

IHC (p) PA WB

HPA017671-100UL

Anti-HS3ST1 8

rabbit

affinity isolated antibody

HS3ST1, human

human

IHC (p) PA

HPA002237-100UL

Anti-HSPG2 8

rabbit

affinity isolated antibody

HSPG2, human

human

IHC (p) PA

HPA018892-100UL

Anti-SDC1 8

rabbit

affinity isolated antibody

SDC1, human

human

IHC (p) PA

HPA006185-100UL

Anti-SDC3 8

rabbit

affinity isolated antibody

SDC3, human

human

IHC (p) PA

HPA017087-100UL

Anti-SDC4 8

rabbit

affinity isolated antibody

SDC4, human

human

IHC (p) PA

HPA005716-100UL

Anti-SRGN 8

rabbit

affinity isolated antibody

SRGN, human

human

IHC (p) PA

HPA000759-100UL

Anti-SULF2 8

rabbit

affinity isolated antibody

SULF2, human

human

IHC (p) PA

HPA002325-100UL

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Application

Cat. No.


Antibodies to PG Core Proteins Product Name

Host

Clone

Form

Monoclonal Anti-AGC1

mouse

2A8

purified immunoglobulin

Application ELISA (i) WB

WH0000176M1-100UG

Cat. No.

Monoclonal Anti-DCN

mouse

3H4-1F4

purified immunoglobulin

ELISA (i) IHC (p) WB

WH0001634M1-100UG

Monoclonal Anti-GPC5

mouse

1C9

purified immunoglobulin

ELISA (i)

WH0002262M1-100UG

Antibodies to GAG Synthesis/Modification Enzymes Host

Clone

Form

Monoclonal Anti-ARSB

mouse

1A4

purified immunoglobulin

Application ELISA (i) WB

Cat. No. WH0000411M2-100UG

Monoclonal Anti-EXT1

mouse

5A5

purified immunoglobulin

ELISA (i) WB

WH0002131M1-100UG

Monoclonal Anti-EXT2

mouse

3G6

purified immunoglobulin

ELISA (i) WB

WH0002132M1-100UG

Monoclonal Anti-NDST1

mouse

1G10

purified immunoglobulin

ELISA (i) IHC (p) WB

WH0003340M1-100UG

Monoclonal Anti-NDST3

mouse

5B9

purified immunoglobulin

ELISA (i) WB

WH0009348M1-100UG

Monoclonal Anti-CHST3

mouse

1C4

purified immunoglobulin

ELISA (i) WB

WH0009469M1-100UG

Monoclonal Anti-SART2

mouse

6D4

purified immunoglobulin

ELISA (i) WB

WH0029940M3-100UG

Monoclonal Anti-CHST11

mouse

4F1

purified immunoglobulin

ELISA (i) WB

WH0050515M1-100UG

Glycosaminoglycan Sulfation and Signaling

Product Name

Additional Antibodies Product Name

Host

Clone

Form

Monoclonal Anti-Chondroitin Sulfate

mouse

CS-56

ascites fluid

Application ARR IF (i)

Cat. No. C8035-.2ML C8035-.5ML

Application Abbreviation Table Application

Abbreviation

Application

Abbreviation

ANA-indirect immunofluorescence

IF (ANA)

Immunohistochemistry

IHC

Capture ELISA

ELISA (c)

Immunohistochemistry (formalin-fixed, paraffin-embedded sections)

IHC (p)

Direct ELISA

ELISA (d)

Immunohistochemistry (frozen sections)

IHC (f)

Direct immunofluorescence

IF (d)

Immunoprecipitation

IP

Dot blot

DB

Indirect ELISA

ELISA (i)

Dot immunobinding

DIBA

Indirect immunofluorescence

IF (i)

Electron microscopy

EM

Microarray

ARR

Enzyme immunoassay

EIA

Neutralization

Neutral

Flow cytometry

FACS

Ouchterlony double diffusion

ODD

Immunoblotting

WB

Particle immunofluorescence

PIFA

Immunoblotting (chemiluminescent)

WB CL

Quantitative precipitin assay

QPA

Immunocytochemistry

ICC

Radioimmunoassay

RIA

Immunoelectrophoresis

IEP

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Glycosaminoglycan Disaccharides

Glycosaminoglycan Sulfation and Signaling

In the following listings, ΔUA = 4-deoxy-L-threo-hex-4-enopyranosyluronic acid; GalNAc = N-acetyl-D-galactosamine; GlcN = D-glucosamine; GlcNAc = N-acetyl-D-glucosamine; NS = N-sulfo-; 2S = 2-sulfate; 4S = 4-sulfate; 6S = 6-sulfate. The uronic acid moiety loses its chirality on cleavage from the native polysaccharide.

Name

Synonyms

Formula

Formula Weight

Chondroitin disaccharide Δdi-0S sodium salt

α-ΔUA-[1→3]-GalNAc

Chondroitin disaccharide Δdi-4S sodium salt

Cat. No.

C14H20NNaO11

401.30

C3920-5MG C3920-10MG

α-ΔUA-[1→3]-GalNAc-4S

C14H19NNa2O14S

503.34

C4045-5MG C4045-10MG

Chondroitin disaccharide Δdi-6S sodium salt

α-ΔUA-[1→3]-GalNAc-6S

C14H19NNa2O14S

503.34

C4170-5MG C4170-25MG

Chondroitin disaccharide Δdi-UA-2S sodium salt

α-ΔUA-2S-[1→3]-GalNAc

C14H19NNa2O14S

503.34

C5820-1MG

Heparin disaccharide I-A sodium salt

α-ΔUA-2S-[1→4]-GlcNAc-6S

C14H18NNa3O17S2

605.39

H9517-1MG

Heparin disaccharide I-H sodium salt

α-ΔUA-2S-[1→4]-GlcN-6S

C12H17NNa2O16S2

541.37

H8892-1MG

Heparin disaccharide I-S sodium salt

α-ΔUA-2S-[1→4]-GlcNS-6S

C12H15NNa4O19S3

665.40

H9267-1MG H9267-5MG

Heparin disaccharide II-H sodium salt

α-ΔUA-[1→4]-GlcN-6S

C12H18NNaO13S

439.33

H9017-1MG

Heparin disaccharide II-S sodium salt

α-ΔUA-[1→4]-GlcNS-6S

C12H16NNa3O16S2

563.35

H1020-.5MG

Heparin disaccharide III-H sodium salt

α-ΔUA-2S-[1→4]-GlcN

C12H18NNaO13S

439.33

H9142-1MG

Heparin disaccharide III-S sodium salt

α-ΔUA-2S-[1→4]-GlcNS

C12H16NNa3O16S2

563.35

H9392-1MG

Heparin disaccharide IV-A sodium salt

α-ΔUA-[1→4]-GlcNAc

C14H20NNaO11

401.30

H0895-.5MG

Heparin disaccharide IV-H

α-ΔUA-[1→4]-GlcN

C12H19NO10

337.28

H9276-1MG

Monosaccharide Sulfates Name

Synonyms

Formula

Formula Weight

N-Acetyl-d-galactosamine 6-sulfate sodium salt

GalNAc-6S

C8H14NNaO9S

323.25

51947-1MG

N-Acetyl-d-glucosamine 6-sulfate sodium salt

GlcNAc-6S

C8H14NNaO9S

323.25

44001-25MG

d-Galactosamine

2S-GalN

C6H12NNaO8S

281.22

12662-5MG

C6H11NaO9S

282.20

90572-1MG 05801-1MG

d-Galactose

4-sulfate sodium salt

Gal-4S

d-Galactose

6-sulfate sodium salt

Gal-6S

C6H11NaO9S

282.20

d-Glucosamine

2-sulfate sodium salt

GlcN-2S

C6H12NNaO8S

281.22

G7889-100MG

d-Glucosamine

3-sulfate

GlcN-3S

C6H13NO8S

259.23

11631-25MG

d-Glucosamine

6-sulfate

GlcN-6S

C6H13NO8S

259.23

G8641-25MG

Glc-3S

C6H11NaO9S

282.20

G9909-10MG G9909-50MG

Man-6S

C6H11NaO9S

282.20

14652-50MG

d-Glucose

3-sulfate sodium salt

d-Mannose

16

2-sulfate sodium salt

Cat. No.

6-sulfate sodium salt

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Dextran and Related Polysaccharides Dextran O

Historically, dextrans had been long recognized as contaminants in sugar processing and other food production. The formation of dextran in wine was shown by Pasteur to be due to the activity of microbes.1 The name dextran was created by Scheibler in 1874, who demonstrated dextran was a carbohydrate with the formula (C6H10O6)n and a positive optical rotation.2

The secretion of dextrans provides an opportunity for bacteria to modulate adhesion, e.g. in tooth decay, by having a softer or more rigid bacterial cell surface, depending on the polysaccharide itself and the pH and ionic strength. Low bacterial adhesion occurs at low salt conditions with more rigid polysaccharides and a softer surface, while high bacterial adhesion is obtained with more flexible polysaccharides and a rigid bacterial surface. Polymer elasticity is important for structural integrity. and the pyranose ring is the structural unit controlling the elasticity of the polysaccharide. This elasticity results from a force-induced elongation of the ring structure and a final transition from a chair-like to a boat-like conformation of the glucopyranose ring, which plays an important role in accommodating mechanical stress and modulating ligand binding in biological systems.9 Laboratory experiments have demonstrated that cleavage of the pyranose rings of dextran, amylose, and pullulan convert these different polysaccharide chains into similar structures where all the bonds of the polymer backbone can rotate and align under force. After ring cleavage, single molecules of dextran, amylose, and pullulan display identical elastic behavior as measured by atomic force microscopy.

6

HO HO

O 2

5

1

OH

3

O 4

6

HO HO

5 3

O 2

Dextran and Related Polysaccharides

Dextrans are polysaccharides with molecular weights ≥1,000 Dalton, which have a linear backbone of α-linked d-glucopyranosyl repeating units. Three classes of dextrans can be differentiated by their structural features. The pyranose ring structure contains five carbon atoms and one oxygen atom. Class 1 dextrans contain the α(1→6)-linked d-glucopyranosyl backbone modified with small side chains of d-glucose branches with α(1→2), α(1→3), and α(1→4)-linkage (see Figure 1). The class 1 dextrans vary in their molecular weight, spatial arrangement, type and degree of branching, and length of branch chains,3-5 depending on the microbial producing strains and cultivation conditions.6,7 Isomaltose and isomaltotriose are oligosaccharides with the class 1 dextran backbone structure. Class 2 dextrans (alternans) contain a backbone structure of alternating α(1→3) and α(1→6)-linked d-glucopyranosyl units with α(1→3)-linked branches. Class 3 dextrans (mutans) have a backbone structure of consecutive α(1→3)-linked d-glucopyranosyl units with α(1→6)-linked branches. One and two-dimensional NMR spectroscopy techniques have been utilized for the structural analysis of dextrans.8

4

1

OH

O 6

4

HO a-1,4-linked D-Glucopyranoside side chains

5

HO 3

2

O 1

OH O

6

4

HO

5

HO a-1,3-linked D-Glucopyranoside side chains

3

O 2 1

OH O

HO

6

4

5

HO 3

a-1,2-linked D-Glucopyranoside side chains

O 2 1

OH O 4

6

HO HO

5 3

2

O 1

OH O

Figure 1. General structure of class 1 dextrans consisting of a linear backbone of α(1→6)-linked d-glucopyranosyl repeating units. The dextran may have branches of smaller chains of d-glucose linked to the backbone by α(1→2)- , α(1→3)- or α(1→4)- glycosidic bonds.

Dextrans are found as bacterial extracellular polysaccharides. They are synthesized from sucrose by beneficial lactic acid bacteria, such as Leuconostoc mesenteroides and Lactobacillus brevis, but also by the dental plaque-forming species Streptococcus mutans. Bacteria employ dextran in biofilm formation10 or as protective coatings, e.g., to evade host phagocytes in the case of pathogenic bacteria.11 The physical and chemical properties of purified dextrans vary depending on the microbial strains from which they are produced and by the production method, but all are white and tasteless solids. Dextrans have high water solubility and the solutions behave as Newtonian fluids. Solution viscosity depends on concentration, temperature, and molecular weight, which have a characteristic distribution.

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Dextran and Related Polysaccharides

The long history of the safety of dextrans has allowed them to be used as additives to food and chemicals, and in pharmaceutical and cosmetics manufacturing.12 Dextrans have been investigated for the targeted and sustained delivery of drugs, proteins, enzymes, and imaging agents.13 In medicine, clinical grades of dextrans with a molecular weight range of 75-100 kDa have been used as blood-plasma volume expanders in transfusions.14 Other applications include the use of dextrans with polyethylene glycol as components of aqueous two-phase systems for the extraction of biochemicals. The hydroxyl groups present in dextran offer many sites for derivatization, and these functionalized glycoconjugates represent a largely unexplored class of biocompatible and environmentally safe compounds. Cross-linked dextran beads are widely used for chromatography in biochemical research and industry. The classic application of cross-linked dextrans is as gel filtration media in packed-bed columns for the separation and purification of biomolecules with molecular weights in the range of 0.7-200 kDa.15-17 Ion exchange chromatography utilizes dextran that has been derivatized with positively or negatively charged moieties such as carboxymethyl (CM), diethylaminoethyl (DEAE), diethyl(2-hydroxy­propyl)­amino­ethyl (QAE), and sulfopropyl (SP). Sigma® offers a large variety of dextrans with high polydispersity and dextran molecular weight standards with low polydispersity (Mw/Mn values close to 1.0).

Other Polysaccharides Pullulans are structural polysaccharides primarily produced from starch by the fungus Aureobasidium pullulans.18,19 Pullulans are composed of repeating α(1→6)-linked maltotriose (D-glucopyranosyl-α(1→4)-D-glucopyranosyl-α(1→4)-D-glucose) units with the inclusion of occasional maltotetraose units.20 Diffusion-ordered NMR spectroscopy has been used to achieve a simple estimation of the molecular weight of pullulan.21 The solution properties of pullulan in water have been studied, and it was confirmed that pullulan molecules behave as random coils in aqueous solution.22

References: 1. Pasteur, L., Bull. Soc. Chim. Paris, 30-31 (1861). 2. Scheibler, C., Z. Ver. Dtsch. Zucker-Ind., 24, 309-335 (1874). 3. Robyt, J.F., in: Encyclopedia of Polymer Sci. Eng., J.I.Kroschwitz (ed.), 4, 752-767 (1986), Wiley-VCH. 4. Cheetham, N.W.H., et al., Dextran structural details from high-field proton NMR spectroscopy. Carbohydr. Polym. 14, 149-158 (1990). 5. Naessens, M., et al., Leuconostoc dextransucrase and dextran: production, properties and applications. J. Chem. Technol. Biotechnol., 80, 845-860 (2005). 6. Kim, D., et al., Dextran molecular size and degree of branching as a function of sucrose concentration, pH, and temperature of reaction of Leuconostoc mesenteroides B-512FMCM dextransucrase. Carbohydr. Res., 338, 1183-11889 (2003). 7. Côté, G.L., and Leathers, T.D., A method for surveying and classifying Leuconostoc spp. glucansucrases according to strain-dependent acceptor product patterns. J. Ind. Microbiol. Biotechnol. 32, 53-60 (2005). 8. Maina, N.H., et al., NMR spectroscopic analysis of exopolysaccharides produced by Leuconostoc citreum and Weissella confusa. Carbohydr. Res., 343, 1446-1455 (2008). 9. Marszalek, P.E., et al., Polysaccharide elasticity governed by chair-boat transitions of the glucopyranose ring. Nature, 396, 661-664 (1998). 10. Banas, J.A., and Vickermann, M.M., Glucan-binding proteins of the oral streptococci. Crit. Rev. Oral Biol. Med., 14, 89-99 (2003). 11. Meddens, M.J., et al., Br. J. Exp. Pathol., 65, 257-265 (1984). 12. Kato, I., Fragrance J., 33, 59-64 (2005). 13. Mehvar, R., Dextrans for targeted and sustained delivery of therapeutic and imaging agents. J. Controlled Release, 69, 1-25 (2000). 14. Terg, R., et al., Pharmacokinetics of Dextran-70 in patients with cirrhosis and ascites undergoing therapeutic paracentesis. J. Hepatol., 25, 329-333 (1996). 15. Porsch, B., and Sundelöf, L.-O., Size-exclusion chromatography and dynamic light scattering of dextrans in water: Explanation of ion-exclusion behaviour. J. Chromatogr. A, 669, 21-30 (1994). 16. Neyestani, T.R., et al., Isolation of α-lactalbumin, β-lactoglobulin, and bovine serum albumin from cow’s milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity. Protein Expres. Purif., 29, 202-208 (2003). 17. Penzol, G., et al., Use of dextrans as long and hydrophilic spacer arms to improve the performance of immobilized proteins acting on macromolecules. Biotechnol. Bioeng., 60, 518-523 (1998). 18. Gibson, L.H., and Coughlin, R.W., Optimization of high molecular weight pullulan production by Aureobasidium pullulans in batch fermentations. Biotechnol. Prog., 18, 675-678 (2002). 19. Leathers, T.D., Biotechnological production and applications of pullulan. Appl. Microbiol. Biotechnol., 62, 468-473 (2003). 20. Catley, B.J., Pullulan, a relationship between molecular weight and fine structure. FEBS Lett., 10, 190-193 (1970). 21. Viel, S., et al., Diffusion-ordered NMR spectroscopy: a versatile tool for the molecular weight determination of uncharged polysaccharides. Biomacromolecules, 4, 18431847 (2003). 22. Nishinari, K., et al., Solution properties of pullulan. Macromolecules, 24, 5590-5593 (1991). 23. Wahl, G.M., et al., Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc. Natl. Acad. Sci. USA, 76, 3683-3687 (1979). 24. Warnick, G.R., et al., Dextran sulfate-Mg2+ precipitation procedure for quantitation of high-density-lipoprotein cholesterol. Clin. Chem., 28, 1379-1388 (1982). 25. Mitsuya H., et al., Dextran sulfate suppression of viruses in the HIV family: inhibition of virion binding to CD4+ cells. Science, 240, 646-649 (1988).

Dextrins are composed of D-glucopyranosyl units but have shorter chain lengths than dextrans. They start with a single α(1→6) bond, but continue linearly with α(1→4)-linked D-glucopyranosyl units. Dextrins are usually mixtures derived from the hydrolysis of starch and have found widespread use in the food, paper, textile, and pharmaceutical industries. Dextran sulfates are derived from dextran via sulfation. They have become indispensable components in many molecular biology techniques, including the transfer of large DNA fragments from agarose gels and rapid hybridization,23 precipitation procedures for the quantitation of high-density lipoprotein cholesterol,24 and inhibition of virion binding to CD4+ cells.25

18

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Dextrans Dextran For General Applications Name

Mol Wt

Cat. No.

Mr ~1,500

31394-5G 31394-25G 31394-100G

Mr ~6,000

31388-25G 31388-100G 31388-500G

Mr 15,000‑25,000

31387-25G 31387-100G 31387-500G

Mr ~40,000

31389-25G 31389-100G 31389-500G

Mr ~70,000

31390-25G 31390-100G 31390-500G

Mr ~100,000

09184-10G-F 09184-50G-F 09184-250G-F

Dextran

BioChemika enzymatic synth. Dextran from Leuconostoc spp.

Dextran from Leuconostoc spp.

BioChemika Dextran from Leuconostoc spp.

BioChemika Dextran from Leuconostoc spp. Glycobiology Analysis Manual

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Dextran from Leuconostoc spp.

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Dextran and Related Polysaccharides

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Dextran For General Applications, continued Name

Mol Wt

Cat. No.

Mr ~500,000

31392-10G 31392-50G 31392-250G

Mr ~2,000,000

95771-10G 95771-50G 95771-250G

Dextran from Leuconostoc mesenteroides

average mol wt 9,000‑11,000

D9260-10G D9260-50G D9260-100G D9260-500G

Dextran from Leuconostoc mesenteroides

average mol wt 35,000‑45,000

D1662-10G D1662-50G D1662-100G D1662-500G

Dextran from Leuconostoc mesenteroides

average mol wt 60,000‑90,000

D3759-1KG D3759-2KG

Dextran from Leuconostoc mesenteroides

Mr ~60,000

31397-100G 31397-500G

Dextran from Leuconostoc mesenteroides

average mol wt 64,000‑76,000

D4751-10G D4751-50G D4751-100G D4751-500G D4751-1KG

Dextran from Leuconostoc mesenteroides

average mol wt 100,000‑200,000

D4876-50G D4876-100G D4876-500G D4876-1KG

Dextran from Leuconostoc mesenteroides

Mr ~200,000

31398-25G 31398-100G 31398-500G

Dextran from Leuconostoc mesenteroides

average mol wt 425,000‑575,000

D1037-50G D1037-100G D1037-500G

Dextran from Leuconostoc mesenteroides

average mol wt ~2,000,000

D5376-100G D5376-500G

Dextran from Leuconostoc mesenteroides

average mol wt 5,000,000‑40,000,000

D5501-100G D5501-500G D5501-1KG

Dextran from Leuconostoc spp.

BioChemika

Dextran and Related Polysaccharides

Dextran from Leuconostoc spp.

BioChemika

BioChemika

BioChemika

industrial grade

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For Use as Standards in Gel Permeation Chromatography (GPC) Certified dextran standards have been certified by the Deutsches Institut fĂźr Normung e.V. (DIN, Berlin, Germany). Name Dextran

Description

Mn

MP

MW

standard 1,000 certified according to DIN

~1,010

~1,080

~1,270

Cat. No. 00268-500MG

standard 5,000 certified according to DIN

~3,260

~4,440

~5,220

00269-100MG 00269-500MG

standard 12,000 certified according to DIN

~8,110

~9,890

~11,600

00270-100MG 00270-500MG

standard 25,000 certified according to DIN

~18,300

~21,400

~23,800

00271-100MG

standard 50,000 certified according to DIN

~35,600

~43,500

~48,600

00891-100MG

standard 80,000 certified according to DIN

~55,500

~66,700

~80,900

00892-100MG

standard 150,000 certified according to DIN

~100,300

~123,600

~147,600

00893-100MG 00893-500MG

standard 270,000 certified according to DIN

~164200

~196,300

~273,000

00894-100MG 00894-500MG

standard 410,000 certified according to DIN

~236,300

~276,500

~409,800

00895-100MG 00895-500MG

standard 670,000 certified according to DIN

~332,800

~401,300

~667,800

00896-100MG

standard 1,000

~1,000

~1,100

~1,300

31416-100MG

standard 5,000

~3,300

~4,400

~5,200

31417-100MG-F

standard 12,000

~8,000

~10,000

~12,000

31418-100MG

standard 25,000

~20,000

~20,000

~25,000

31419-100MG-F

standard 50,000

~35,000

~45,000

~50,000

31420-100MG-F

standard 80,000

~55,000

~65,000

~80,000

31421-100MG-F

standard 150,000

~100,000

~125,000

~150,000

31422-100MG-F

standard 270,000

~165,000

~195,000

~270,000

31423-100MG-F

standard 410,000

~235,000

~275,000

~410,000

31424-100MG

standard 670,000

~330,000

~400,000

~670,000

31425-100MG-F

standard 1,400,000

~65,100

~1,223,000

~1,394,000

49297-100MG-F

BioChemika Dextran BioChemika Dextran

Dextran BioChemika Dextran BioChemika Dextran BioChemika Dextran BioChemika Dextran

Dextran and Related Polysaccharides

BioChemika

BioChemika Dextran BioChemika Dextran BioChemika Dextran from Leuconostoc mesenteroides BioChemika Dextran from Leuconostoc mesenteroides BioChemika Dextran from Leuconostoc mesenteroides BioChemika Dextran from Leuconostoc mesenteroides BioChemika Dextran from Leuconostoc mesenteroides BioChemika Dextran from Leuconostoc mesenteroides BioChemika Dextran from Leuconostoc mesenteroides BioChemika Dextran from Leuconostoc mesenteroides BioChemika Dextran from Leuconostoc mesenteroides BioChemika Dextran from Leuconostoc mesenteroides BioChemika Dextran from Leuconostoc mesenteroides BioChemika

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Dextran Sulfate Name

Mol. Wt.

Additives

Dextran sulfate sodium salt from Leuconostoc spp.

Mr 5,000

-

Cat. No. 31404-5G-F 31404-25G-F

Dextran sulfate sodium salt from Leuconostoc spp.

6,500‑10,000

-

D4911-1G D4911-10G D4911-50G D4911-100G

Dextran sulfate sodium salt from Leuconostoc spp.

average mol wt 9,000‑20,000

-

D6924-1G D6924-10G D6924-50G

Dextran sulfate sodium salt from Leuconostoc spp.

Mr 100,000

-

66786-50G-F

Dextran sulfate sodium salt from Leuconostoc spp.

average mol wt >500,000 (dextran starting material)

phosphate buffer, pH 6-8 0.5-2.0%

D6001-1G D6001-10G D6001-50G D6001-100G D6001-500G

Dextran sulfate sodium salt from Leuconostoc spp.

average Mw >500,000 (dextran starting material)

phosphate buffer 0.5-2%

D8906-5G D8906-10G D8906-50G D8906-100G D8906-500G

Dextran and Related Polysaccharides

BioChemika

BioChemika

for molecular biology

Immobilized Dextran Name

Synonyms

Particle Size (μm)

CM-D; Carboxymethyl-dextran

-

86524-10G-F 86524-50G-F 86524-100G-F 86524-250G-F 86524-500G-F

DEAE-Dextran hydrochloride

Diethylaminoethyl-dextran hydrochloride

-

D9885-10G D9885-50G D9885-100G

DEAE-Dextran hydrochloride

Diethylaminoethyl-dextran hydrochloride

-

30461-25G

-

50 - 150

01468-250G-F

-

20 - 80

94504-50G-F 94504-250G-F

-

50 - 150

40359-50G-F 40359-250G-F

-

100 - 300

68263-10G-F 68263-50G-F 68263-250G-F

CM-Dextran sodium salt

Cat. No.

BioChemika

BioChemika for molecular biology Dextran cross-linked G-25 BioChemika Dextran cross-linked G-50 BioChemika Dextran cross-linked G-50 BioChemika Dextran cross-linked G-50

BioChemika

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Dextrins and Pullulans Name

Type

Product Line, Grade

Impurities

Dextrin from corn

Type I

-

Reducing sugar ≤5%

Cat. No. D2006-100G D2006-500G D2006-1KG

This grade is prepared by removing a minimum of 95% of the water-insolubles, and almost all of the alcohol-solubles. Type II

commercial grade

-

D2131-500G D2131-2.5KG

Dextrin from corn

Type III

commercial grade

-

D2256-500G D2256-2.5KG

Dextrin from maize starch

-

BioChemika

reducing matter in dry substance ~10%

31410-100G 31410-500G

Dextrin from maize starch

-

BioChemika

reducing matter in dry substance ~20%

31414-100G 31414-500G

Dextrin from potato starch

Type IV

-

Reducing sugar ≤5%

D4894-500G D4894-1KG

Dextrin from potato starch

-

BioChemika, for microbiology

-

31400-250G 31400-1KG

Pullulan from Aureobasidium pullulans

-

-

-

P4516-1G P4516-5G P4516-25G

Dextran and Related Polysaccharides

Dextrin from corn

substrate for pullulanase suitable

Additional Gel Permeation Chromatography (GPC) Standards and Sets Dextran

Pullulan Standard 340

[9004‑54‑0] [C6H10O5]n

[9057‑02‑7]

 BioChemika, for GPC, standard-Set Mp 1,000-400,000

 for GPC

exact values can be taken from the accompanying certificate of analysis

Mn ~342

set contains 10 different dextran standards with 500 mg with Mp ~1,100 (Fluka 31416), ~4,400 (Fluka 31417), ~10,000 (Fluka 31418), ~20,000 (Fluka 31419), ~45,000 (Fluka 31420),~65,000 (Fluka 31421), ~125,000 (Fluka 31422), ~195,000 (Fluka 31423), ~275,000 (Fluka 31424), ~400,000 (Fluka 31425); The components can also be ordered separately under the Fluka product numbers indicated.

Mp ~342

31430-1EA

1 set

Pullulan Standard 180

Mw ~342 Mw/Mn ~1.00 exact values can be taken from the accompanying certificate of analysis 76651-100MG

100 mg

Pullulan Standard Set [9057‑02‑7]

[9057‑02‑7]

 for GPC, Mp 342-710’000

 for GPC

exact values can be taken from the accompanying certificate of analysis

Mn ~180

Set contains 10 different Pullulan Standards with 0.1 g with Mw ~320, ~1’300, ~6’000, ~12’000, ~22’000, ~50’000, ~110’000, ~200’000, ~400’000, ~800’000

Mp ~180 Mw ~180

96351-1KT

Mw/Mn ~1.00

1 kit

exact values can be taken from the accompanying certificate of analysis 90950-100MG

100 mg

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GlycoProfile™ β-Elimination Kit

GlycoProfile™ β-Elimination Kit

O-Glycan Removal: A Novel β-Elimination Method O-Linked glycans are linked through the hydroxyl of Ser (serine) or Thr (threonine) and do not require a consensus sequence, although a number of core structures have been identified. Despite their complexity and apparent lack of uniformity, O-linked glycans are being studied more frequently in recent research. This is due to the increased awareness of their multiple roles and importance in biological function and disease. In order to further understand O-linked glycans, the glycan is often removed from the protein for analysis.

Sigma’s GlycoProfile β-Elimination Kit is unique in that it does not completely destroy the protein, as seen with other traditional methods of β-elimination. SDS-PAGE of proteins before and after deglycosylation. Gel has been stained with EZBlue™ Gel Staining Reagent (Cat. No. G1041) and destained with water.

1 2 3

5

6

7 8

9 10 11 12

Enzymatic methods of O-linked deglycosylation include sequentially hydrolyzing sugars by use of a series of exoglycosidases, followed by O-glycosidase. Although effective at removing the glycan, this process is tedious and does not result in an intact glycan. There are a number of chemical methods used to remove O-glycans. These include hydrazinolysis and trifluoromethanesulfonic (TFMS) acid, which remove both N- and O-linked glycans, and can be destructive to the glycans, thus preventing accurate analysis. Alkaline β-elimination is a chemical method of deglycosylation commonly used to specifically remove O-glycans. Traditionally, this method uses a combination of sodium hydroxide and sodium borohydride. The O-glycan linkage is easily hydrolyzed using dilute alkaline solution under mild conditions. The presence of a reducing agent can keep the glycan from “peeling” after being released, but the process significantly degrades the protein or peptide. Other methods, such as using sodium hydroxide alone or with borane-ammonia, also easily hydrolyze the linkage, but are not very efficient at keeping both moieties intact.

Lane 1: ColorBurst™ Marker, High Range (Cat. No. C1992) Lanes 2 and 3: Fetuin prior to β-elimination Lanes 4 and 5: Fetuin incubated in 50 mM NaOH overnight Lanes 6 and 7: Fetuin incubated in NaBH4 and NaOH overnight (traditional β−elimination) Lanes 8 and 9: Fetuin incubated in Sigma’s β−elimination reagent at 4-8° C overnight Lanes 10 and 11: Fetuin incubated in Sigma’s β−elimination reagent at room temperature overnight Lane 12: SigmaMarker™, Wide Range (Cat. No. S8445)

A novel non-reductive β-elimination kit has been developed that keeps both protein and glycan intact. The method is much easier to use than the traditional β-elimination methodologies. There is no tedious neutralization of the borohydride or ion exchange chromatography to be performed. This technology allows for complete glycoproteomic analysis of O-linked glycoproteins, as never before possible.

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The GlycoProfile β-Elimination Kit allows for proteomic analysis of O-linked glycoproteins after removal of the O-glycan. The following data is summarized from LC-MS analysis of the tryptic peptides of two glycoproteins (TrypZean™ recombinant trypsin, and glycophorin A) before and after deglycosylation. TrypZean, BEFORE DEGLYCOSYLATION TRY1_BOVIN (100%), 25,424.8 Da Cationic trypsin precursor (EC 3.4.21.4) (Beta-Trypsin) [Contains: Alpha-trypsin chain 2] (Fragment) – Bos taurus (Bovine) 24 unique peptides, 25 unique spectra, 463 total spectra, 134/243 amino acids (55% sequence coverage) FIFLALLGAAVAFPVDDDDKIVGGYTCGANTVPYQVSLNSGYHFCGGSLINSQWVVSAAHCYKSGIQVRLGEDNINVVEGNEQFISASKSIVHPSYNSNTLNNDIMLIKLKSAASLNSRVASISLPTSCASAGT QCLISGWGNTKSSGTSYPDVLKCLKAPILSDSSCKSAYPGQITSNMFCAGYLEGGKDSCQGDSGGPVVCSGKLQGIVSWGSGCAQKNKPGVYTKVCNYVSWIKQTIASN

TRY1_BOVIN (100%), 25,424.8 Da Cationic trypsin precursor (EC 3.4.21.4) (Beta-Trypsin) [Contains: Alpha-trypsin chain 2] (Fragment) – Bos taurus (Bovine) 29 unique peptides, 30 unique spectra, 573 total spectra, 134/243 amino acids (55% sequence coverage) FIFLALLGAAVAFPVDDDDKIVGGYTCGANTVPYQVSLNSGYHFCGGSLINSQWVVSAAHCYKSGIQVRLGEDNINVVEGNEQFISASKSIVHPSYNSNTLNNDIMLIKLKSAASLNSRVASISLPTSCASAGT QCLISGWGNTKSSGTSYPDVLKCLKAPILSDSSCKSAYPGQITSNMFCAGYLEGGKDSCQGDSGGPVVCSGKLQGIVSWGSGCAQKNKPGVYTKVCNYVSWIKQTIASN GLYCOPHORIN A, BEFORE DEGLYCOSYLATION GLPA_HUMAN (100%), 16,273.7 Da Glycophorin A precursor (PAS-2) (Sialoglycoprotein alpha) (MN sialoglycoprotein) (CD235a antigen) – Homo sapiens (Human) 17 unique peptides, 18 unique spectra, 79/150 amino acids (53% coverage)

GlycoProfile™ β-Elimination Kit

TrypZean, AFTER DEGLYCOSYLATION

MYGKIIFVLLLSAIVSISASSTTGVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIE NPETSDQ GLYCOPHORIN A, AFTER DEGLYCOSYLATION GLPA_HUMAN (100%), 16,273.7 Da Glycophorin A precursor (PAS-2) (Sialoglycoprotein alpha) (MN sialoglycoprotein) (CD235a antigen) – Homo sapiens (Human) 22 unique peptides, 25 unique spectra, 89/150 amino acids (59% coverage) MYGKIIFVLLLSAIVSISASSTTGVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIE NPETSDQ

# of Unique Peptides

% Sequence Coverage

TrypZean, before deglycosylation*

24

55

TrypZean, after deglycosylation

29

55

Glycophorin A, before deglycosylation

17

53

Glycophorin A, after deglycosylation

22

59

Sample

Navigate!

* Trypzean is recombinant trypsin, bovine sequence, expressed in corn.

GlycoProfile™ β-Elimination Kit

8

Components β-Elimination Reagent 940 μL Sodium hydroxide (Sigma S8263) 60 μL Protein Enrichment Column 10 each Glycan Enrichment Column 10 each store at: Room temp PP0540-1KT

1 kit

Let Sigma help navigate your proteomics research. ■ Full range of progressive technologies for biomarker discovery ■ Proteomic standards, calibrants, and markers ■ Intuitive design tools for peptide libraries

Order your Proteomics Product Guide today. Visit sigma.com/pepguide

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Related Kits for Deglycosylation GlycoProfile™ IV Chemical Deglycosylation Kit

Glycoprofile™ II, Enzymatic In-Solution N-Deglycosylation Kit

 TFMS Deglycosylation System

GlycoProfile™ II has been optimized to provide a convenient and reproducible method to remove

GlycoProfile™ β-Elimination Kit

The GlycoProfile™ IV Kit utilizes trifluoromethanesulfonic acid (TFMS) in a deglycosylation system that completely removes all N- and O-linked glycans while preserving the protein/polypeptide structure. TFMS hydrolysis of glycoproteins results in minimal protein degradation while the released glycans are destroyed. For high molecular weight or complex, nonmammalian glycoproteins, an optional scavenger species is included. Complete deglycosylation of glycoproteins takes place in as little as 30 minutes.

N-linked glycans from glycoproteins and is compatible with subsequent MALDI-TOF mass spectrometric analysis without interference from any of the reaction components. Contains sufficient reagents for a minimum of 20 reactions when the sample size is between one to two mg of a typical glycoprotein. store at: 2-8°C

store at: 2-8°C PP0510-1KT

PP0201-1KT

1 kit

GlycoProfile™ I, In-Gel Deglycosylation Kit

Contains all enzymes and reagents needed to completely remove all N- and simple O-linked including polysialylated carbohydrates from glycoproteins as well as additional enzymes & reagents needed to remove complex O-linked carbohydrates.

The GlycoProfile™ Enzymatic In-gel N-Deglycosylation Kit is optimized to provide a convenient, reproducible method to N-deglycosylate and digest protein samples from one dimensional (1D) and two dimensional (2D) polyacrylamide gel pieces for subsequent mass spectrometry or HPLC analysis.

ship: wet ice store at: 2-8°C EDEGLY-1KT

This procedure is suitable for Coomassie Brilliant Blue and colloidal Coomassie stained gels. Silver stained gels may also be used if properly destained. ®

1 kit

Native Protein Deglycosylation Kit The Native Protein Deglycosylation is designed for the deglycoslylation of N-linked oligosaccharides from PNGase F-resistant native proteins. Endoglycosidases F1, F2, and F3 are less sensitive to protein conformation than PNGase F and are more suitable for removal of all classes of N-linked oligosaccharides without protein denaturation.

store at: 2-8°C PP0200-1KT

1 kit

Enzymatic Protein Deglycosylation Kit

1 kit

ship: wet ice store at: 2-8°C NDEGLY-1KT

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Our Innovation, Your Research — Shaping the Future of Life Science MISSION is a registered trademark of Sigma-Aldrich Co. and its affiliate Sigma-Aldrich Biotechnology, L.P.

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