Digitonin Permeabilization

At the end of the experimental run, remove medium and rinse cells twice with 2 mL of autofluorescence buffer. 2. Reinitiate data acquisition and add digitonin (20-50 g mL). 3. Monitor until fluorescence intensity falls to a stable plateau. 4. Subtract final image from all acquired images. 1. Photobleaching and dye extrusion can be distinguished by briefly interrupting data acquisition and comparing the fluorescence intensity before and after resuming acquisition. If the fluorescent intensities...

Second and Third Round Plaque Assays see Note

Day 1 Initiate culture of 143B cells. Seed one 6-well plate (35-mm-diameter wells) per plaque, with 143B cells at a density of 5 x 105 cells well in MEM 10 . Use the next day when greater than 90 confluent ( 1 x 106 cells per well). 2. Day 2 Infection of 143B cells. Thaw lysate from first-round plaque assay. Sonicate for 30 s, chill on ice for 2 min, and vortex vigorously for 30 s. Repeat once. 3. Incubate 250 L of lysate with an equal volume of trypsin and incubate at 37 C for 30 min, vortex...

And Glycosaminoglycans from Drosophila

Staatz, Hidenao Toyoda, Akiko Kinoshita-Toyoda, Kimberlly Chhor, and Scott B. Selleck The fruitfly, Drosophila melanogaster, has provided a powerful model organism for the study of development. Analysis of patterning in a variety of species from the nematode, C. elegans, to frogs, chicks, and mice have demonstrated that the fundamental mechanisms of morphogenesis are conserved among diverse species. In recent years genetic studies in Drosophila have shown that proteoglycans (PGs) and...

Melissa Handler and Renato V Iozzo

The use of antisense technology has been applied to a number of diverse organisms, including Drosophila melanogaster, plants, and mammals. The purpose is to take advantage of the base-pair specificity of both RNA RNA and RNA DNA interactions, ultimately to block protein production. This technique requires the production of exogenous antisense mRNA, generated either constitutively or inducibly, from a functional promoter. Classically, a constitutive promoter, such as the cytomegalovirus (CMV)...

Exogenous Acceptor Substrates

Deglycosylated core proteins are obtained by treating the proteoglycans with chondroitin ABC lyase or heparan lyase to remove the chondroitin dermatan sulfate chains or heparan sulfate chains and then treating with polyhydrogen fluoride in pyridine with anisole as scavenger (29) in order to remove the GlcA-Gal-Gal-Xyl linkage region (see Note 6). 2. Partially deglycosylated chondroitin sulfate core protein is prepared by digestion with testicular hyaluronidase to remove variable amounts of...

Evaluation of Specificity of Antibodies Using ELISA

The reactivity of the anti-heparan sulfate antibodies with other molecules can be analyzed by ELISA in two ways (1) by application of antibodies to wells of microtiter plates coated with the test molecule, or (2) by an inhibition assay in which the antibodies are incubated with the test molecule. 1. All incubation steps are carried out at room temperature. 2. Coat wells with test molecules (see step 9, Subheading 2.8.) by incubation with 10 g of test molecules mL solution for 16 h at 4 C. 3....

Sample Preparation and Fluorescence Labeling

For studies of interactions involving carbohydrate components of the extra cellular matrix (ECM), glycosaminoglycans such as chondroitin sulfate and hyaluronan are commercially available (e.g., Sigma, Seikagaku) from several tissue sources (e.g., bacteria, cock's comb, umbilical cord). Commercially available FITC-labeled proteins are sometimes unsuitable for binding studies, as they can be excessively substituted (> 10 mol FITC per mole protein) and their binding and diffusion properties may...

Kazuhiro Mio Antonei B Csoka Susan Stair Nawy and Robert Stern

Hyaluronidases are a group of enzymes that degrade the glycosaminoglycan hyaluronan (HA, hyaluronic acid). Many types of hyaluronidases are reported, from prokaryotes to eukaryotes (1,2). These enzymes have a wide variety of properties, including substrate specificity, inhibitor sensitivity, and a range of pH optima. Strep-tomyces hyaluronidase, and the venom hyaluronidases from bee, snake, and scorpion are active at neutral pH. Hyal-1, the best-characterized somatic hyaluronidase (3-5),...

Jeremy E Turnbull 1 Introduction

The functions of heparan sulfate (HS) are determined by specific saccharide motifs within HS chains. These sequences confer selective protein-binding properties and the ability to modulate protein bioactivities (1,2). HS chains consist of an alternating disaccharide repeat of glucosamine (GlcN N-acetylated or N-sulfated) and uronic acid (glucuronic GlcA or iduronic acid IdoA ). The initial biosynthetic product containing N-acetylglucosamine (GlcNAc) and GlcA is modified by N-sulfation of the...

Edward Conrad

With the exception of hyaluronic acid, all glycosaminoglycans are O-sulfated. Heparins and heparan sulfates are, in addition, N-sulfated on many of their glucosamine residues. Other naturally occurring structures are also sulfated. For example, some proteins contain sulfotyrosine residues (1), and some complex lipids are O-sulfated. In addition, certain N-linked oligosaccharides are sulfated (2). In all of these cases, the enzymes that add sulfate residues to these structures utilize...

Background for Molecular Methods to Study HSPGMediated Endocytosis

The underlying model is that each cell-surface HSPG, like other receptors, is organized into domains that mediate specific functions. Portions of the HS side chains serve as the ligand-binding domains, while regions within the core protein are responsible for recruiting the specific cellular machinery used for internalization of that particular species of HSPG see (1,33,78) . In this light, the distinct pathways for cellular uptake of ligands bound to syndecan HSPGs versus the perlecan HSPG can...

Use of Microsomal Preparations

Details in this section refer to synthesis of chondroitin dermatan sulfate and to a lesser extent to synthesis of heparin heparan sulfate. Similar microsomal reaction conditions with slight modifications have been used by U. Lindahl and his associates in their extensive examination of heparin heparan sulfate biosynthesis (1,7). 1. Typically, reaction mixtures using endogenous acceptors are incubated with labeled or nonlabeled 0.05-0.2 mM UDP-GlcA, UDP-GalNAc, or UDP-GlcNAc with or without...

Hplc

It will be necessary to calculate an approximate lag time between the flow cell and the exit point of tubing where you will be collecting peaks. This will be (60 x (internal volume of the tube coming out of the flow cell + half the volume of the flow cell) flow rate) seconds (see Subheading 2.4.). 1. Prepare solvents (If helium sparging is used to remove dissolved gases, perform this before addition of trifluoroacetic acid. In practice, helium sparging is not generally necessary. 3. If sample...

Introduction

Amino acid sequence analysis of proteoglycans is performed using many of the same methods that are used for conventional proteins and glycoproteins, with some specific modifications that result from the glycosaminoglycans that are attached to the protein core. Amino acid sequence analysis of proteoglycans is more challenging than for conventional proteins for two reasons. First, because proteoglycans are large molecules, they have the same problems that are inherent in sequence analysis of...

Capture of Biotinylated Ligands Surface Regeneration and Storage

Streptavidin, avidin, or neutravidin should be immobilized on the appropriate surface according to the instructions of the instrument manufacturer. The following protocol is suitable for the IAsys instrument. It will have to be adapted for the other instruments. In particular, high flow rates (100 L min) should be maintained throughout the capture reaction in BIAcore and BioTul to ensure efficient mixing and hence an even capture of biotinylated ligand over the sensor surface. The cuvet is...

Proteoglycans Analyzed by Composite Gel Electrophoresis and Immunoblotting

Proteoglycans are the protein products of diverse genes posttranslationally modified with highly negatively charged side chains, commonly known as glycosami-noglycans. The latter consist of repeating disaccharides capable of forming polymers of varying size, which, depending on the specific disaccharide composition, are known as the chondroitin sulfates, the iduronate-containing dermatan sulfates, keratan sulfate, and heparan sulfate. Some of the more complex proteoglycans, such as aggrecan,...

Degradation of Heparan Sulfate with Heparin Lyases Laurie A LeBrun and Robert J Linhardt

Heparinase

Glycosaminoglycan (GAG), heparan sulfate (HS), and heparin are a polydisperse mixture of linear polysaccharides composed of glucosamine residues 4 linked to uronic acid residues. The major repeating unit in heparin is 4)-a-d-N-sulfoglu-cosamine-6-sulfate (1 4)-a-l-iduronic acid-2-sulfate (1 , corresponds to 75-90 of its sequence (1) (see Fig. 1A), whereas heparan sulfate consists of 50-75 4)-a-d-N-acetylglucosamine (1 4)- -glucuronic acid (1 and smaller amounts of...

Enzyme Preparation and Storage

Tris-HCl sodium acetate buffer, 50 mM (see Table 2 for pH). Properties of the Chondroitinase Lyases Chondroitinase ABC from Proteus vulgaris Chondroitinase ACI from Flavobacterium heparinum Chondroitinase ACII from Arthrobacter aurescens Chondroitinase B from Flavobacterium heparinum Chondroitinase C from Flavobacterium heparinum 150,000 Tris-HCl sodium acetate 8.0 37 76,000 Tris-HCl sodium acetate 7.5 37 76,000 Tris-HCl sodium acetate 6.0 37 55,000 Tris-HCl sodium acetate 7.5 25 Tris-HCl...

Disaccharide Composition in Glycosaminoglycans Proteoglycans Analyzed by Capillary Zone Electrophoresis

During the past decade, the use of fully automated equipment for capillary electrophoresis (CE) has made this a routine method for the study of soluble analytes. The separation of such compounds in capillaries (20-200 m id) and in strong electric fields (around 50 kV m) seems to be exceptionally efficient for separating both large and small molecules. The most common type of CE is capillary zone electrophoresis (CZE), which is done with the separation buffer free in an otherwise empty...

David G Fernig 1 Introduction

Networks of interacting molecules, operating from the outside of the cell to the cell nucleus, regulate cell behavior. Optical biosensors provide a means of analyzing these interactions and possess key advantages over other methods posttranslationally modified proteins, secondary gene products such as polysaccharides, chemically synthesized molecules, and nucleic acids are all equally susceptible to analysis a quantitative description of an interaction is obtained the structural rules and the...

Immobilization of Proteoglycans Glycosaminoclycan Chains and Oligosaccharides

Binding Dextran Surface Cells

The immobilization of ligands is accomplished by chemically activating a functional group on the surface, e.g., carboxyl on carboxymethyl dextran or amino on aminosilane. Since excellent protocols for ligand immobilization are supplied by the manufacturers, only points specific to proteoglycans will be considered here. There are major difficulties in the efficient direct coupling of proteoglycans, glycosaminogly-can chains, and oligosaccharides to surfaces, due to the highly anionic character...

Introduction Of Proteoglycan

The general features of the biosynthetic assembly of all proteoglycans (see refs. 1-9 for reviews), except the keratan sulfate portions of cartilage and cornea (see Note 1), consist of sequential (1) synthesis of the core protein (2) xylosylation of specific Ser moieties of the core protein (3) addition of two galactose (Gal) residues to the xylose (Xyl) (4) completion of a common tetrasaccharide linkage region by addition of a glucuronic acid (GlcA) residue (5) addition of an...

Electrophoresis

Immediately before electrophoresis, rinse the resolving gel surface with stacking gel buffer (0.125 M Tris-HC1 buffer, pH 6.8, diluted from the 1 M stock solution). 2. Prepare and degas the stacking gel solution (for 5 ml, mix 0.5 mL of T50 C5 acrylamide stock with 0.6 mL of 1 M Tris pH 6.8 and 3.9 mL of distilled water). Fig. 1. Principles of integral glycan sequencing and an example. (A) Fluorescence detection of different amounts of a 2AA-tagged heparin tetrasaccharide run on a 33 minigel....

Labeling Cell Surface Proteoglycans

Apart from standard equipment, a specialty gas mix of 5 CO2 balanced with air should be placed near the laminar-flow hood. A plastic tube from the low-pressure regulator is connected onto a plugged 5- or 10-mL pipet, which acts as sterile filter. 2. Appropriate sterile medium. L cells use Dulbecco's modified Eagle's medium containing 10 fetal bovine serum, 100 units mL of penicillin G, and 100 g mL of streptomycin sulfate (Life Technologies, Inc.). 3. PBS...

Tung Ling L Chen Peiyin Wang Seung S Gwon Nina W Flay and Barbara M Vertel

Proteoglycans undergo numerous synthetic and processing events as they progress through the exocytic pathway. In cells that express genetically engineered constructs encoding proteoglycans, immunolocalization is a useful approach in identifying specific intracellular compartments involved in their processing and trafficking. 2.1. Expression of Target Proteoglycan (Cell Culture and Transfection) 1. Proteoglycan constructs packaged into vectors suitable for expression in target cells (1,2). In...

Wei Luo Chunxia Guo Jing Zheng and Marvin L Tanzer 1 Introduction

Target proteoglycan gene(s) can be rearranged to fulfill special designs for expression, purification, or characterization purposes. These are achieved through several procedures, including polymerase chain reaction (PCR) amplification of a target gene, cloning of PCR products into cloning vectors, subcloning of a correct gene fragment into an expression vector, purification of target gene DNA via a large preparation, transfection into host cells for expression, purification of candidate...

Purification of Proteoglycans by Preparative DEAE Microchromatography

Proteoglycans are partially purified and concentrated with a DEAE microcolumn, prepared by Subheading 2.3., steps 7-9. All effluents are to be considered as radioactive and disposed of accordingly. The proteoglycan-containing supernatant is combined with 6.5 mL of PLB (see Table 3). A pipetman is used to load 1-mL portions. Apply slowly so that the sample runs down the wall and only minimally disturbs the gel bed. see Note 13. 2. Column washing. Wash with 4 mL of urea acid wash,...

Methods

Upon verification of two criteria, this assay can be tailored to detect the activity of any HS sulfotransferase that creates a high-affinity binding site for a protein ligand. First, the protein ligand of interest must preferentially bind either to a subpopulation of HS or to HS from only a subset of cell types. Second, two specific cell lines must be identified an expressive line that produces high levels of the ligand-binding site and a nonexpressive line that produces little or no...

Preparation of Tissue Sections

The binding and localization procedure can be performed using either frozen or paraffin-embedded tissue sections. When fresh-frozen tissues are being examined, the tissue should be embedded in OCT or equivalent compound (see Note 2). 1. (Frozen sections) 4- m sections are thaw-mounted on charged slides (e.g., Fisher Plus slides) and immediately dipped in 70 ethanol and then fixed in 4 paraformaldehyde in PBS at room temperature. The fixation procedure is crucial for reproducible FR1-AP binding...

Paraffin

Deparaffinize tissue sections in three changes of xylene, 7 min each, on slow shaker. 2. Rinse tissue, 2 x 1 min, in 100 EtOH. 3. Quench endogenous tissue peroxidase by incubating tissue in 0.7 H2O2 in absolute methanol for 20 min.(see Note 41). 4. Rinse tissue 2 x 1 min in 100 EtOH, and rehydrate through graded EtOH, 1 min each. 5. Rinse, 2 x 1 min, in H2O, then 10 min in TBS. 6. Epitope unmasking treatment as necessary with enzyme digestion (see Note 43) Chon-droitin ABC lyase 1 h, 37 C...

Sample Preparation

Preparation for Direct N-Terminal Analysis Direct N-terminal analysis is not a particularly helpful method for identification of a proteoglycan. The hydrated glycosaminoglycan chain(s) generally result in high backgrounds, due to nonspecific peptide bond cleavage. However, it is the only method suitable for defining the N-terminus and has proved to be valuable for identifying, for example, the N-termini of the mature forms of the leucine-rich proteoglycans decorin and biglycan (7) (not...

Plastic Removal

Tissue Fixation and Embedding in Plastic (see Note 46) 1. Dissect tissue to 1-mm3 cubes or smaller and fix for 2 h at room temperature. 2. Wash tissue 2 x 5 min with PBS. 3. Quench free aldehyde groups with 0.1 M glycine PBS 2 x 10 min. 5. Dehydrate with 35 , 50 , 70 EtOH, 10 min each on shaker. 6. En bloc stain with 3 uranyl acetate 70 EtOH 1 h. 7. Continue dehydration through 90 , 95 , and 100 ethanol, 10 min each, on shaker. 8. Prepare epoxy resin and use in 1 1 mixture EPON ethanol....

Detection of Antibodies Expressed by Bacterial Clones Using an Immunoblot Assay

0.45- m nitrocellulose filter (Schleicher & Schuell). 4. PBS containing 3 (w v) Marvel and 1 (v v) Tween-20. 5. PBS containing 2 (w v) Marvel and 0.2 (v v) Tween-20. 6. Anti-cMyc antibody hybridoma culture supernatant (clone 9E10, mouse IgG see Note 4). 7. PBS containing 0.1 (v v) Tween-20. 8. Alkaline phosphatase-conjugated rabbit anti-mouse IgG antibodies (DAKO). 9. PBS containing 1 (w v) Marvel and 0.1 (v v) Tween-20. 10. 1 M diethanolamine containing 0.5 mM MgCl2, pH 9.8. 11. -Nitro blue...

Plaque Amplification into T175 Flasks

Day 1 Seed T-175 flasks with HeLa cells. Note that selection agent (BrdU) is no longer required. A single flask is needed for each individual putative recombinant. 2. Day 2 Thaw lysate from T-25 amplification (see Subheading 5.5.2.). Sonicate for 30 s, chill on ice for 2 min, and vortex vigorously for 30 s. Repeat once. 3. Add 250 L of lysate to 1.75 mL of 2.5 DMEM. 4. Aspirate media from cells and infect with 2.0 mL of diluted lysate per flask. 5. Incubate for 2 h with gentle rocking every 15...

Gel Imaging and Product Quantitation see Figs

After completion of the electrophoresis, one gel cassette at a time is removed from the tank. The outside of the glass plates as well as the loading wells are extensively washed with distilled water to remove running buffer and excess unreacted AMAC reagent (see Note 11). 2. The sample wells and the stacking gel are covered with light impermeable tape, and the gel cassette is placed on the transilluminator light box (see Note 12). 3. The gels are viewed and the images captured and stored in...

Proteoglycan Introduction

Heparan sulfate proteoglycans (HSPGs) bind via their heparan sulfate (HS) gly-cosaminoglycan chains to a large variety of extracellular ligands. These ligands include components of the extracellular matrix, other cell surface receptors, viruses, proteases and their inhibitors, and growth factors. The interaction of growth factors with HS has been proposed to affect growth factor function, if by no means other than limiting growth factor diffusion. For certain growth factors, however, work over...

John D Sandy

The full- or partial-length c-DNA and deduced core protein sequence is now available for at least 38 distinct proteoglycans for review, see (1) . Many of these can be placed into several large family groupings, such as the 10 members of the small leu-cine-rich repeat proteoglycans (decorin, biglycan, fibromodulin, lumican, keratocan, PRELP, epiphycan, mimecan, oculoglycan and osteoadherin), the glypicans (GPC-1, cerebroglycan, OCI-5, K-glypican, GPC-5, and GPC-6), the hyaluronan-binding...

Xiaomei Bai Brett Crawford and Jeffrey D Esko

Mutant cell lines provide an excellent model for studying the structure, assembly and function of proteoglycans under the controlled conditions of tissue culture. Numerous proteoglycan-deficient strains have been isolated, mostly in Chinese hamster ovary cells, and in many cases the defects have been characterized both genetically and biochemically (see Table 1). Biochemical analysis of the mutants has confirmed that various enzyme activities detected in cell-free extracts using synthetic...

Isolation of Bovine Milk LpL

3.6 L of raw, fresh whole cows' milk (it is best to milk the cows on the morning the isolation begins, and the milk must be promptly chilled and kept cold). 2. 160 mL of heparin-agarose beads (Bio-Rad cat. no.153-6173 is suitable and inexpensive). 3. One 4-L vacuum Erlenmyer flask with a large scintered glass funnel. 6. A refrigerated table-top centrifuge with centrifuge bottles, to accommodate about 4 L of liquid (Four Beckman 750-mL centrifuge bottles in a GS-6R centrifuge work well). 7....

Proteoglycan Notes

It is possible to digest samples on blots or in gels. This is described in more detail in another volume of this series (3,4). An earlier version of the blot digestion procedure has also been published (11). These methods work well for globular proteins, but in the case of proteoglycans it can be difficult to obtain a sample density that provides an adequate signal-to-noise ratio. Proteoglycans often do not stain well with Coomassie blue or Ponceau S, so it can be difficult to identify the...

Isolation of Bovine Milk LpL see Note

Place the 3.6 L of cold, raw, fresh milk into a 4-L beaker on a stir plate at 4 C. Add 84.1 g of solid NaCl, to bring the final concentration of added NaCl to 0.4 M. Stirring should be fast enough for the salt to dissolve within about 1 h, but not fast enough to cause foaming. 2. While the salted milk is stirring, place 140 mL of heparin-agarose beads into the sintered glass funnel attached to the 4-L vacuum flask. Wash the beads with 3L of chilled distilled water, then 2 L of chilled...

Alan D Murdoch and Renato V Iozzo

For molecules with such extensive posttranslational modifications, it may not be immediately obvious why one would wish to express the protein cores of proteoglycans in prokaryotic systems, where none of this modification can occur. However, there are several cases where this is not a problem, and some where there is a positive advantage to expressing a core protein with none of the normal eukaryotic modifications. Bacterially expressed proteoglycan core proteins have been used successfully to...

Info

Schematic of cationic nylon filter binding assay for growth factor heparan sulfate proteoglycan interactions. Filtration of various mixtures of growth factor, heparan sulfate proteoglycan (E-HS), and compounds that block either the heparin-binding domain on the growth factor or the protein-binding domain on the heparan sulfate chains, through cationic nylon filters can be used to measure the various binding affinities quantitatively. (1) Cationic nylon filters do not specifically retain...

Expression and Purification of RTKBinding Probe

Soluble FGF RTK is expressed as fusion protein containing the extracellular (ligand-binding) portion of the RTK at the amino terminus and carboxy-terminal human placental alkaline phosphatase, which serves as a tag (e.g., FR-AP). Fusion constructs containing the extracellular portion of the FGFR1c and most other FGF receptors and their splice variants have been designed and generated by D. Ornitz (Washington University, St. Louis, MO) (21). FR1c-AP has been extensively characterized in binding...

Mw Stds KDfl

Analysis of G1 domain-containing proteoglycan species isolated by preparative SDS PAGE of a trypsinized ovine articular cartilage sample by 4-20 SDS gradient PAGE and Western (Mab 1-C-6 anti-G1 domain MAb 5-D-4 anti-KS) and affinity blotting (biotinylated hyaluronan). Lane 1 represents the crude trypsinized cartilage extract, which had been purified by HA affinity chromatography and lanes 2-4 show aggrecan fragments isolated from this by preparative SDS PAGE (see Subheadings 3.2 and...

Complete Chondroitin Lyase Catalyzed Depolymerization of Radiolabeled GAGs

Dissolve GAGs sample containing radiolabeled (35S, 14C, or 3H) CS or DS in 1 mL of distilled water. Exhaustively dialyze sample against water using 1000-MWC0 dialysis membrane. Freeze-dry nondialyzable retentate. Add 50 L of Tris-HCl sodium acetate buffer. Alternatively, the radiolabeled sample can be buffer exchanged using a Centricon (YM3, 3000 MWCO) centrifugal filter unit. 2. Thaw 10 L of chondroitin lyase solution at room temperature and use immediately. 3. Add 30 L of samples containing...

Mathematica Fitting Program for Ligand Proteoglycan Binding

Fadds .05, .05, .05, .05, .05, .05, 0.10, 0.10, 0.10, 0.10, 0.10, 0.10,0.20,0.20, 0.20, 0.20, 0.20, 0.20, 0.20, 0.20, 0.20, 0.20, 0.20, 0.20, 0.50, 0.50, 0.50, 0.50, 0.50, 0.50, 0.50, 0.50, 0.50, 0.75, 0.75, 0.75, 1.0, 1.0, 1.0, 1.0, 1.0, 1.0, 1.0, 1.0, 1.0, 2.0, 2.0, 2.0, 2.0, 2.0, 2.0, 2.0, 2.0, 2.0, 2.5, 2.5, 2.5, 4.0, 4.0, 4.0, 5.0, 5.0, 5.0, 5.0, 5.0, 5.0, 6.0, 6.0, 6.0, 10.0, 10.0, 10.0 Ebound 0025, .0035, .0034, .0013, .0055, .0033, .0067, .0069, .0076, .0028,...

Anna L McBain and David M Mann

Conventional approaches to purifying most connective tissue proteoglycans (PGs) generally involve treatments that consequently alter the binding properties of the purified PG (e.g., exposure to strong detergents or harsh denaturants such as guanidinium). Herein we describe a protocol that we use to isolate human decorin in which the PG remains functional in its binding to various ligands (1,2). This method utilizes a eukaryotic expression system that produces high quantities of recombinant...

Proteoglycan Binding Inhibitors Protamine Sulfate

Inhibitor effectiveness analysis is based on steady-state data acquisition with two independent first-order reactions occurring in solution. where P is E-HS, L is the growth factor (bFGF), C1 is the binding complex of E-HS and bFGF, PS is a proteoglycan binding inhibitor (protamine sulfate), and C3 is the binding complex of PS and bFGF. Assuming conservation of mass, the following equation at the steady state can be solved for the KD value (KD3) (Lq - Q)(Pq - Q) - (Lq -Q) C3 KD1C1 (3) where C3...

Cartilage and Smooth Muscle Cell Proteoglycans Detected by Affinity Blotting Using Biotinylated Hyaluronan

Chondrocytes and smooth muscle cells synthesise the large CS-rich proteoglycans aggrecan (1) and versican (2) respectively. Both proteoglycans are capable of interacting with hyaluronan to form molecular aggregates that have important tissue specific functional roles to play. Aggrecan is a major matrix component of cartilage, the aggrecan aggregates are physically entrapped within the collagenous extracellular matrix of this tissue, and it is the collective interplay between this collagenous...

Screening for Full Length Inserts Using PCR and for VH Gene Diversity Using DNA Fingerprinting

In this procedure, the positive clones will be analyzed for the presence of DNA (about 1 kbp) encoding the scFv antibody. In addition, as an initial screening for the diversity of the clones, restriction enzyme analysis will be performed. (Also see Fig. 2). Fig. 2. Restriction fragment analysis of clones expressing anti-heparan sulfate antibodies. Polymerase chain reaction (PCR) is preformed to amplify the region encoding the scFv antibody (A), using a set of primers flanking the VH and VL...

Cell Aggregation Assay see

Aggregation Cell

Prewarm aggregation buffer to 37 C (see Note 1). 2. Collect 5.0 x 106 cells in an eppendorf tube. 3. Centrifuge the cells at 300g for 5 min and remove the supernatant. 4. Wash the cell pellet in 1 mL of aggregation buffer and centrifuge again. 5. Using a transfer pipet, vigorously resuspend the cells in 1 mL of aggregation buffer. During initial experiments, visually inspect the cells using a phase microscope to ensure that the cells are in a single-cell suspension. 6. Split the sample into two...

Purification of Perlecan from Endothelial Cells

Perlecan Endothelial

Perlecan, the major heparan sulfate (HS) proteoglycan of basement membranes and other connective tissues, is a modular molecule with five structural domains. It has been isolated from various sources including kidney and placenta, but most commonly from the mouse Englebroth Holm Swarm (EHS) tumor (1,2). It has also been purified from cultured cell lines such as endothelial cells (3) or fibroblasts (4). When it has been extracted from tissue or from the extracellular matrix of cultured cells, it...

Binding of FGF and FR1cAP to Tissue Sections

FGF Biotinylated (see Subheading 2.1.) or native growth factor. 2. Anti-biotin antibody, for example, mouse monoclonal (Jackson, Cat. 200-002-096, Jackson ImmunoResearch, West Grove, PA) or goat polyclonal (Vector, cat. no. SP-3000, Vector Laboratories, Burlingame, CA). 3. Anti-placental alkaline phosphatase antibody, for example, mouse monoclonal (Sigma, clone 8B6, cat. no. A2951) or rabbit polyclonal (Biomeda, cat. no. A67, Foster City, CA). 4. Heparitinase enzyme Heparitinase (mixture of...

Zoo

Sulfate labeling experiment and GAG chain characterization. (A) Autoradiogra-phy of sulfate-labeled products of empty vector (lane 1) and aggrecan G3 construct (lane 2) from wild-type CHO cells (1). Note that only chondroitin heparan sulfate GAGs are labeled (not the core protein). (B) Autoradiography of sulfate-labeled products of aggrecan G3 construct in Chondroitinase buffer (lane 1) and G3 digested by Chondroitinase ABC (lane 2). Note that the enzyme digestion almost completely...

Analysis of ADisaccharides Using Ultraviolet Detection

Following digestion of samples containing CS and or DS with both chondroitinases ABC and AC, all constituent nonsulfated and variously sulfated disaccharides of these Fig. 3. Electropherograms showing the resolution of GalAG-derived A-disaccharides following degradation of CS DS with chondroitinases ABC and AC. (A) Direct UV detection at 232 nm and (B) derivatization of the A-disaccharides obtained with fluorescent tag 2-aminoacridone and detection using LIF (Xexc 488 nm). In both cases,...

Complete Heparin Lyase Catalyzed Depolymerization of Radiolabeled HS

Dissolve GAG sample containing radiolabeled HS in 50 L of sodium phosphate buffer. Dialyze sample against sodium phosphate buffer using 1000 MWCO dialysis membrane or a Centricon (YM3, MWCO 3000) centrifugal filter unit (see Note 9). 2. Thaw 10 L of heparin lyase III solution at room temperature, immediately prior to use (see Note 5). 3. Add 30 L of sodium phosphate buffer to the 500- L polypropylene microcentrifuge tube containing the enzyme solution. 4. (Optional) (see Note 15). Add 1.7 L...

Novel ConfocalFRAP Analysis of Carbohydrate Protein Interactions Within the Extracellular Matrix

Heng, and Timothy E. Hardingham 1. Introduction Extracellular matrices (ECMs) contain a mixture of fibrillar and nonfibrillar mac-romolecular components, which interact through a range of covalent and noncovalent associations to form a composite structure (1-3). It is the ECM that defines the architecture, the form, and the biomechanical properties of many tissues. In order to perform their functional roles, many of the important noncollagenous components of...

Sandip Patel and Renato V Iozzo

Ca2+ is a crucial biological messenger involved in a host of diverse cellular processes (1). Many hormones, growth factors, and neurotransmitters raise cytosolic Ca2+ levels through activation of phospholipase C, which catalyzes the production of the second messenger, inositol 1,4,5-trisphosphate (IP3) (2). G-protein-linked receptors and receptor tyrosine kinases couple to distinct isoforms of phospholipase C (3). Once generated, IP3raises cytosolic Ca2+ levels by activating Ca2+ channels...

After

(Autoradiogram Phosphorimage) Fig. 1. Analytical ACE schematic. Top panel ACE gels poured using a casting stand and Teflon combs and strips as shown in Fig. 5 are used to create nine parallel rectangular wells, which are filled with protein-agarose mixtures, each at a different protein concentration. Radiolabeled GAG or PG is loaded into the slot above the protein-containing wells (shown as a dark line), and after electrophoresis of the GAG or PG through the protein-containing lanes, its...

John R Couchman and Pairath Tapanadechopone 1 Introduction

Proteoglycans are quite abundant components of many extracellular matrices, while most cell surfaces also bear these macromolecules. Frequently the profiles are complex. For example, several members of the syndecan and glypican families of cell surface heparan sulfate proteoglycans may be present on a single cell type (1,2). Some extracellular matrices, e.g., from brain, may also contain a variety of proteoglycans including several members of the hyalectans or aggregating proteoglycans such as...

David J McQuillan Neung Seon Seo Anne M Hocking and Camille I McQuillan

Only Mammalian Cells Will Make a Proteoglycan Proteoglycans are molecules that comprise a core protein to which at least one glycosaminoglycan (GAG) chain is attached, and a consideration of recombinant proteoglycan expression must operate under this simple definition. There are numerous systems currently available to express proteoglycans (and other complex glycoconju-gates), but this chapter will focus on a system adopted by us to express proteoglycans and other extracellular matrix...

Binding Assays

A single binding assay is illustrated in Fig. 3, and the schematic (see Fig. 4) shows the changes occurring at the surface in the bulk phase. 3.5.1. Footprinting and Multimolecular Complexes In all footprinting experiments, it is essential that the ligates are used at concentrations > Kd for the ligand, when the ligate will occupy > 50 of the binding sites on the ligand. The main measurement made in footprinting experiments is the extent of ligate bound, which requires the binding reaction...

FACE Gel Electrophoresis of Fluorotagged Lyase Digestion Products

The electrophoresis tank is filled with precooled running buffer and set into a large container on ice to maintain tank and buffer temperatures at 4 C before and during the run. 2. The precast Mono composition gels are removed from the package, the loading wells extensively washed with the Mono gel running buffer, and the gel cassette placed into the electrophoresis tank. 3. A 4- to 6- L portion (representing 10-15 of the total) of the standard mixture and the fluorotagged samples is loaded per...

Andrew P Spicer

Hyaluronan (HA) is synthesized at the plasma membrane as a free linear polymer of composition 01 4GlcAp1 3GlcNAc n (1-3). All models suggest that polymerization occurs at the inner face of the plasma membrane while the polymer is coordi-nately translocated or extruded across the membrane to the extracellular face of the cell for review, see (2-5) . In mammals (6), and all vertebrates (Spicer, unpublished data), HA is synthesized by any one of three HA synthases (HAS). The three HAS proteins are...

High Pressure Liquid Chromatography HPLC

Different GAG-charged matrices were also packed into a 3-mL stainless steel column and equilibrated with 20 mM Tris-HCl, pH 7.5, at 0.5 mL min using a Waters HPLC system (see Figs. 2 and 3). Fig. 1. Elution profile of the apoSAAl.1 CNBr peptides on a heparin-Affigel column testing apoSAAl.l CNBr fragments for heparin-binding activity. ApoSAAl.1 CNBr cleavage fragments are shown above the graph 16mer (residues 1-16), 7mer (residues 17-23), 53mer (residues 24-76), and 27mer (residues 77-103). A...

Selection of Phages Displaying Antibodies Reactive with Heparan Sulfate Using Biopanning

For a schematic outline of the selection procedure, see Fig. 1. 3.1.1. Growth of Antibody Phage Display Library 1. Inoculate 50 mL of 2XTY containing 100 g of ampicillin mL and 1 (w v) glucose with about 5 x 108 bacteria from a glycerol stock of the (semi)-synthetic scFv Library 1 (5). Fig. 1. Schematic representation of the selection of phage display-derived anti-heparan sulfate antibodies by biopanning. Fig. 1. Schematic representation of the selection of phage display-derived anti-heparan...

Susan Stair Nawy Antonei B Csoka Kazuhiro Mio and Robert Stern

Biotin Hydrazide

Hyaluronidase is a term applied to a group of very dissimilar enzymes (1-3) that degrade hyaluronan (HA, hyaluronic acid), a high-molecular-weight glycosaminogly-can of the extracellular matrix. Some of these enzymes have the ability to degrade additional glycosaminoglycans, albeit at a slower rate. Most of the hyaluronidases from eukaryotes have both hydrolytic and transglycosidase activity, while those from bacteria operate by -elimination. HA is prominent whenever rapid cell proliferation...

Quantification of Maximum Invasive Depth

Diagram Collagen Seeding Theory

The depth of cell invasion is determined by measuring the distance from the top of the gel to the leading front of migrating cells using the calibrated micrometer present on the fine-focus dial of an inverted phase microscope (see Fig. 2). The leading front is defined as the point at which two of the leading cells within a given field are in the same focal plane under 200x magnification. 1. Measurements are taken in five fields within each well using the center of the well as a landmark and as...

Potassium Dihydrogen Phosphate Gradient

Improved separation of monosulfated disaccharides can be obtained using a gradient of phosphate (see Figure 2c and Note 8). 1. Using two ProPac PA1 columns in series, equilibrate in H2O, pH 6.0. 2. Inject samples and elute with a linear gradient of 0-1 M KH2PO4 buffer, pH 4.6 over 90 min. 3. Monitor 3H radioactivity as under Subheading 3.2.1. 3.3. Separation of HS Heparin Oligosaccharides In addition to separation of disaccharides, much larger HS and heparin oligosac-charides can also be...

Inverse HASubstrate Gels

3 Triton X-100 solution 3 Triton X-100 in 50 mM HEPES, pH 7.4. 2. pH 7.4 hyaluronidase inhibitor assay buffer Prepare 0.5 rTRU mL bovine testicular hyaluronidase (Sigma) 0.15 M NaCl, 1 mM MgCl2 in 50 mM HEPES, pH 7.4 (see Note 3). 3. 0.1 mg pronase mL in phosphate-buffered saline (PBS), pH 7.4. 4. Alcian blue solution. Dissolve 0.5 g of Alcian blue in 3 acetic acid solution. 5. Destaining solution for Alcian blue staining 7 acetic acid. 6. Destaining solution for Coomassie blue staining 50...

Giancarlo Ghiselli and Renato V Iozzo

Overview of Gene Targeting in Mammalian Cells Gene targeting allows the generation of specifically designed mutations in cells by the mean of homologous recombination between exogenous DNA and endogenous genomic sequences (1-3). This is possible because a fragment of genomic DNA introduced into a cell can locate and recombine with the chromosomal homologous sequences (4). Although the mechanisms of exogenous DNA transfer to the nucleus and of homologous recombination are still poorly...

PAGE Separation of Saccharides

Polyacrylamide gel electrophoresis (PAGE) is a high-resolution technique for the separation of HS and heparin saccharides of variable sulfate content and disposition. It provides a level of resolution for oligosaccharides larger than tetrasaccharides that is superior to gel filtration or anion-exchange HPLC (18,19). It is possible to obtain improved resolution using gradient gels. However, these are more difficult to prepare and use routinely and in most cases adequate resolution can be...

Glycosaminoglycan Lyase Digestion

Chondroitinase ABC (Proteus vulgaris), (Seikagaku America), dissolved in water at 1.0 U mL and stored in 10- L aliquots at -80 C for up to 3 mo (see Notes 4-6). 2. 50 mM ammonium acetate, pH 7.0, freshly prepared. 3. Microcentrifuge tubes (1.5 mL capacity). Fig. 2. Schematic representation of chondroitinase ABC cleavage in the glycosaminoglycan chain and the products generated from the interior and the nonreducing terminus. Fig. 2. Schematic representation of chondroitinase ABC cleavage in the...

Maria Jos Hernaiz and Robert J Linhardt

Chondroitin Svenska

Glycosaminoglycans (GAGs) are a family of complex linear polysaccharides characterized by a repeating core disaccharide structure typically comprised of an -substituted hexosamine and an uronic acid residue. They can be categorized into four main structural groups hyaluronate, chondroitin sulfate (CS) dermatan sulfate (DS) heparan sulfate heparin and keratan sulfate. The biological roles of chondroitin and dermatan sulfate GAGs are poorly understood and their exact chemical structures have not...

Quantitation of Proteoglycans in Biological Fluids Using Alcian Blue

Madeleine Karlsson and Sven Bjornsson 1. Introduction Alcian blue is a tetravalent cation with a hydrophobic core that contains a copper atom, hence its blue color (1). The four charges allow Alcian Blue to bind to gly-cosaminoglycans (GAGs) at high ionic strength, in contrast to other cationic dyes, which are all monovalent. The molecular structure, that is, the plane tetragonal hydro-phobic core with positive charges attached at its corners, may facilitate formation of aggregates of several...

Modified Movats Pentachrome

Alcian blue, 1 Alcian Blue 8GX 1 g in distilled water DW 100 mL , stable for 6 mo. 2. Alkaline alcohol 95 EtOH 180 mL , conc. ammonium hydroxide 20 mL , stable a. Alcoholic hematoxylin, 2 stock Hematoxylin 2 g in 95 EtOH 100 mL , stable for 3 mo. b. Ferric Chloride, 1.48 stock Ferric chloride hexahydrate 2.48 m in DW 99 mL , conc. HCl 5 mL , stable for 6 mo. c. Iodine stock Potassium iodide 4 g in DW 20 mL , iodine 2 g , DW 80 mL stable for 6 mo. d. Working solution Mix in order immediately...

Timothy A Fritz and Jeffrey D Esko 1 Introduction

A powerful approach for studying the relationship of proteoglycan PG structure to function employs inhibitors to block glycosaminoglycan GAG biosynthesis. Although true enzyme-based, active site-directed inhibitors of the glycosyltransferases and sulfotransferases have not yet been described, decoys consisting of P-d-xylose linked to hydrophobic aglycones have been available for some time 1 . As shown over 25 years ago 2 , xylosides block PG assembly by serving as alternate substrates, thereby...

John B Ancsin and Robert Kisilevsky 1 Introduction

Amyloid is a pathological deposit of protein and glycosaminoglycans GAGs that can lead to the destruction of tissue architecture and function 1,2 . To date, 18 unrelated proteins are known precursors of amyloid deposits 3 . Each one is associated with a specific disease, such as Alzheimer's disease, adult-onset diabetes, inflammatory disorders, and some cancers, but regardless of the type of precursor protein involved, all amyloids have common tinctorial and structural characteristics. Fibrils...

Stephanie L Lara Stephen P Evanko and Thomas N Wight

A variety of techniques are available to assess the structure of proteoglycans, their interactions, and their locations and distribution in cells and tissues. In this chapter, we will review the procedures that we have used over the years to examine the morphology of proteoglycans and cite examples of how these techniques have been used to address questions related to the role of these molecules in health and disease. At the outset, we want to stress that all of the procedures we describe were...

Isolation and Characterization of Nervous Tissue Proteoglycans

Nervous tissues contain a variety of proteoglycans 1-3 . In tissues, proteoglycans are present predominantly in extracellular matrices and on cell surfaces. Proteoglycans in extracellular matrices are generally extracted in soluble fractions by physiological buffers without detergents, whereas cell surface proteoglycans that are anchored to the plasma membrane either by transmembrane domains or glycosylphosphatidylinositol GPI linkages are fractionated into membrane fractions. Some...

Analytical and Preparative Strong Anion Exchange HPLC of Heparan Sulfate and Heparin Saccharides

Studies on the structure-function relationships of the complex linear polysaccharides, known as glycosaminoglycans GAGs , are becoming increasingly important as biological functions are established for them. However, structural analysis of GAGs presents a difficult technical problem, particularly in the case of the N-sulfated GAGs heparan sulfate HS and heparin, which display remarkable structural diversity 1 . A widely used and effective approach is to degrade the chains into smaller...

Preparation of Tissue Sections and General Histology Material

Frozen tissue embedding medium, for example, Tissue Tek OCT compound Sakura, Torrence, CA, cat. no. 4583 . 2. Charged histology slides, for example, Fisher Plus slides Fisher Scientific, Pittsburgh, PA, cat. no. 12-550-15 . 3. Cover slips, e.g. Fisher Finest Fisher Scientific, cat. no. 12-548-5P . 4. Slide-staining racks optional , for example, Shandon Sequenza racks and cover plates Shandon, Pittsburgh, PA . 5. Humidified chamber as an alternative to staining racks . 6. Fixation of frozen...

Derivatization of Saccharides with the Fluorophore 2AA

HS and heparin saccharides can be labeled by reaction of their reducing aldehyde functional group with a primary amino group reductive amination . For sulfated saccharides anthranilic acid 2-aminobenzoic acid 2-AA ref. 11 has been found to be effective for the IGS methodology. 2-AA conjugates typically display an excitation maxima in the range 300-320 nm, which is ideal for visualization with a commonly available 312 nm UV source e.g., transilluminators used for visualizing ethidium bromide...

Isolation of Proteoglycans from Cell Cultures and Tissues

Proteoglycan Isolation

Proteoglycans are a class of glycosylated proteins characterized by the presence of glycosaminoglycans as a carbohydrate component, which endow them with unique biological as well as biochemical properties. Therefore, isolation of proteoglycans from various biological sources such as cell cultures and tissues could be achieved by ordinary molecular purification procedures utilizing their general molecular properties and by those taking advantages of the presence of glycosaminoglycan moiety....

Cellulose Acetate Electrophoresis of Glycosaminoglycans

Yanusz Wegrowski and Francois-Xavier Maquart Electrophoresis on cellulose acetate membrane zone electrophoresis is a common method for qualitative and semiquantitative analysis of glycosaminoglycan GAG mixtures. The advantage of this method is its simplicity, rapidity, the possibility of processing several samples at the same time, and the low cost of analysis. Apart from cellulose acetate strips and electrophoresis apparatus, usually applied in diagnostic laboratories for serum protein...

Cell Aggregation Assay

Aggregation buffer For 500 mL of aggregation buffer add 0.2 g of KCl 0.03 g of KH2PO4 4.0 g of NaCl 0.046 g of Na2HPO4 7H2O 5 mL of a 1.0 M HEPES solution 0.175 g of NaHCO3 5.0 g of bovine serum albumin BSA . Next, add water to 500 mL and adjust pH to 7.0. Then, sterile filter and store at 4 C. 2. Heparin solution 1 mg mL porcine heparin Sigma St. Louis, MO in sterile deionized water . 3. Hemocytometer or Coulter counter. 4. Disposable transfer pipets 5-mL capacity Fisher, Pittsburgh, PA .