Iozzo Renato V. Proteoglycan Protocols

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108 Sugumaran and Silbert

25. Some intact proteoglycans are obtained from commercial sources, but they are generally

mixtures. Alternatively, they can be prepared from tissue sources by published techniques,

and may be available from other investigators who have purified them.

2.4. Endogenous Acceptor Substrates

1. Nascent proteoglycans present in microsomal or Golgi preparations are the best substrates

for synthesis of glycosaminoglycans.

2. The native endogenous acceptors are modified by treatment with various detergents to

affect their capacity to serve as substrates by means of undefined changes in their juxta-

position to the enzymes of glycosaminoglycan synthesis (38,39).

3. Specific modifications are made by use of testicular hyaluronidase to remove portions of

glycosaminoglycan, resulting in changes in amounts of polymerization.

4. Bacterial lyases are used for limited periods to eliminate the capacity of the membrane-

bound proteoglycans to act as acceptors by formation of ∆-uronic acid at the nonreducing

ends of the partially stripped glycosaminoglycans. Resistance to this treatment provides a

mechanism to reflect the degree that the nascent proteoglycans may be buried in vesicles

acting as a barrier for access of the lyases (38).

2.5. Enzyme Preparations

Microsomal preparations from the sources listed under Subheading 2.1. contain all

the firmly associated appropriate glycosyl transferases, sulfotransferases, deacetylase,

and epimerases for glycosaminoglycan synthesis plus firmly associated endogenous

nascent proteoglycan primers on which the glycosaminoglycan chains can be initiated

and/or extended and modified by sulfation and epimerization. Attempts to obtain simi-

larly active preparations from many other tissues or cells have been unsuccessful,

apparently due to limited levels of nascent proteoglycan primer, even though the

glycosaminoglycan-synthesizing enzymes are present. Soluble enzymes that appar-

ently lack the membrane-spanning region may also be present in supernatant fractions

from tissue homogenates, serum, or media of cell cultures. Recombinant enzymes have

proven to be valuable in identifying the specificities of some of the transferase reac-

tions (see Chapter Shworak and Rosenberg).

1. Microsomes are prepared from tissues by first suspending 2–5 g of tissue in 4 volumes of

0.25 M sucrose containing protease inhibitors (1 mM phenylmethylsulfonylfluoride, 10

mM EDTA, 1 mM N-ethyl maleimide, 0.5 mg/mL leupeptin and 0.1 mg/mL pepstatin) in

case there might be slight protease modifications of the enzymes (see Note 10), and

homogenizing 3 to 5 times with a Polytron at the maximum speed for 1 min each. The

homogenate is centrifuged at 12,000g (occasionally 10,000g or 20,000g) in a fixed-angle

rotor for 20 min, and the supernatant fluid is then centrifuged at 105,000g for 1 h. The

pellets are suspended in 0.25 M sucrose and recentrifuged at 105,000g for 1 h to wash the

resulting microsomal fraction (see Note 11). Greatest experience has been with microsomal

preparations from chick embryo epiphyseal cartilage (see Note 12) and mouse mastocytomas.

2. Similar procedures are used to obtain a microsomal fraction from lesser amounts of cul-

tured cells except that sonication in short bursts (30 s) at maximal power with a microtip

is utilized for cell disruption.

3. Fractionation of chick embryo epiphyseal cartilage microsomes to provide Golgi is performed

by a sucrose density-gradient procedure that is a minor modification of that utilized by others

for Golgi subfractionation with liver and cultured cells (see Note 13). A microsomal pellet

Glycosaminoglycan-Synthesizing Enzyme Systems 109

from fresh, unfrozen epiphyseal cartilage from 100 seventeen-day-old chick embryos is

resuspended by using a Dounce homogenizer with a loose-fitting pestle in 0.5 mL of 0.25

M sucrose. A 0.2 mL portion of the fresh microsomal suspension is then fractionated on a

six-step sucrose density gradient consisting of 4.0 mL of 55%, 1.4 mL of 40%, 2.2 mL of

35%, 2.2 mL of 30%, 2.0-mL of 25%, and 1.0 mL of 20% (wt/wt) sucrose containing 10

mM Tris-HCl, pH 8.0. After centrifugation for 40 h at 4°C in a Beckman SW-40 rotor,

fractions are collected and used directly or frozen for further use in the assay of various

enzyme activities (see Note 14). Protein is estimated by the method of Lowry et al. (40).

Although Golgi fractions have not been prepared from other sources for investigation of

proteoglycan synthesis, similar fractionations should be applicable.

4. For solubilization of membrane-bound enzymes, microsomal pellets are suspended in 10

mM HEPES buffer, pH 7.2, containing 10 mM MgCl

, 2 mM CaCl

, 1 mM DTT, 20%

glycerol, and protease inhibitors. To this suspension an equal volume of the buffer solution

containing 2% Triton X-100 is added. The suspension is gently mixed for 60 min and cen-

trifuged at 105,000g for 90 min. The supernatant is saved and the pellets are resuspended in

the original buffer. An equal volume of the buffer solution containing 2% Triton X-100 and

2.0 M NaCl is added. After incubation for 60 min with gentle mixing, the suspension is

centrifuged at 105,000g for 90 min. The supernatant is combined with the first supernatant

and used as the solubilized enzyme. By this two-step procedure, 90% or more of the glyco-

syl transferase and sulfotransferase activities are solubilized (see Notes 15, and 16).

Soluble glycosaminoglycan-synthesizing enzyme activities are found in cell culture

medium, serum, and various body fluids, probably as a result of proteolytic cleavage

from in vivo or intact cells presumably between the catalytic domain and transmembrane

domain of the membrane-bound forms of these enzymes.

2.6. Other Reagents

1. Whatman #1 descending paper chromatography using:

Solvent A: ethanol : 1 M ammonium acetate, pH 7.8 (5 : 2)

Solvent B: 1-butanol : acetic acid : 1 M ammonium hydroxide (2 : 3 : 1)

Solvent C: ethyl acetate : acetic acid : water (3 : 1 : 1)

2. Whatman #1 paper electrophoresis using:

Solvent D: 0.08 M pyridine - 0.046 M acetic acid, pH 5.3

3. Methods

3.1. 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 con-

ditions 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

labeled or nonlabeled 0.01–0.2 mM PAPS (see Note 17) in 50 mM MES buffer, pH 6.5, and

15 mM MnCl

together with 1–10 µL of microsomal preparation, in a total volume of

15–25 µL (see Note 18). Incubations are at 37°C for as long as 24 h. Incorporation is

usually linear for the first 3–5 h but then begins to diminish. True polymerization takes

110 Sugumaran and Silbert

place to form sulfated glycosaminoglycan (or nonsulfated glycosaminoglycan if PAPS is

not present) by addition to nascent membrane-bound proteoglycan (see Notes 19 and 20).

2. Incubations for incorporation into exogenous oligosaccharide acceptors are similar except

that better incorporation is obtained when Triton X-100 or other detergent is present (see

Notes 21–23).

3. Incubations using exogenous glycosaminoglycan require detergent for any but minimal

incorporation, and exogenous proteoglycan acceptors have an absolute requirement for

detergent to provide any incorporation.

3.2. Use of Golgi Preparations

1. Reaction mixtures with Golgi fractions of chondroitin sulfate-producing tissues or cells

are essentially the same as with microsomal preparations. However, total activities with

these fractions is usually considerably greater than with the microsomal fractions, and

there is less degradation of substrates.

3.3. Use of Solubilized and/or Purified Enzymes

1. Reaction mixtures with soluble and/or purified enzymes are the same as the reaction with

microsomal preparations using exogenous acceptors.

3.4. Assay of Enzyme Activities

1. Each activity for synthesis of GalNAc/GlcNAc-GlcA-Gal-Gal-Xyl is determined by 2

to 5-h incubations at 37°C of particulate or soluble enzyme preparations in 0.05 M MES

buffer, pH 6.5, 0.015 M MnCl

together with 0.2 mM of the appropriate

C- or

H-labeled

sugar nucleotide and appropriate 0.2–2.0 mM acceptor (see Note 24) in a total volume of

15 - 35 µL. Thus Xyl transferase uses deglycosylated core protein (undetermined molarity

of serine substituent) as acceptor; Xyl-methyl umbelliferyl for Gal I transferase; Gal-Xyl-

methyl umbelliferyl or Gal-methyl for Gal II transferase; Gal-Gal-Xyl-methyl umbelliferyl

or Gal-Gal-methyl for GlcA I transferase; and GlcA-Gal-Gal-Xyl-methyl umbelliferyl or

GlcA-Gal-Gal-methyl for GalNAc I or GlcNAc I transferase. After incubation, each entire

reaction mixture is spotted on Whatman No.1 paper and subjected to descending chroma-

tography in solvent A or C (see Note 25) or paper electrophoresis in solvent D at 70 V/cm

for 45 min (41). When 4-methyl umbelliferyl-linked acceptors are used, the products

move down the paper with solvent A or C, and their locations can be identified by fluo-

rescence, followed by elution and assay by radioactivity. New radioactive spots corre-

sponding to the oligosaccharide products are eluted with water and chromatographed on

Sephadex G-50 for characterization by size and for separation from small amounts of

labeled sugar nucleotide degradation products. Structure is confirmed by incubation with

appropriate glycosidase.

2. Assay of microsomal or Golgi GlcA II, GalNAc II, and GlcNAc II polymerase activities

are made as in step 1 above with or without chondroitin or heparan pentasaccharide,

hexasaccharide, or larger oligosaccharides. The total reaction mixtures are spotted on

Whatman No. 1 paper and chromatographed overnight or longer in solvent B if oligosac-

charide acceptors are present or in solvent A overnight if oligosaccharides are not present.

Labeled products are eluted from the origins (see Note 25), and characterized by

DEAE-cellulose, Sepharose, or Sephadex chromatography. Degradation with appropri-

ate commercially available chondroitin, heparan, or heparin lyases is used to confirm

structure (see Note 26).

3. Assay of soluble/purified enzymes is performed with oligosaccharide acceptors in the

same fashion as above.

Glycosaminoglycan-Synthesizing Enzyme Systems 111

4. Assay of endogenous particulate or soluble sulfotransferases as well as separation and

characterization of products is performed as above using the same incubation conditions

with 3.0 mM PAP

S as substrate.

S-labeled products are subjected to nitrous acid deg-

radation, and to heparan, heparin, or chondroitin lyases and sulfatases, and the disaccha-

ride products then separated by paper chromatography (solvent B), electrophoresis, or

HPLC in order to determine the degree and position of sulfation.

5. N-Deacetylase activity is assayed using [

H]acetyl-labeled E. coli K5 capsular polysaccha-

ride (35) or [

H]acetyl-labeled heparan precursor as the exogenous substrate. Microsomal

or solubilized enzyme protein is incubated at 37°C for 1–2 h with the [

H]acetyl-labeled

substrate in 0.05 M MES buffer, pH 6.5, 0.01 M MnCl

and 0.02% Triton X-100 in a total

volume of 50 µL. When PAPS is added to the incubation mixtures, deacetylation is pro-

moted along with N-sulfation (42). Reaction mixtures are spotted on Whatman No. 1 paper

and subjected to descending chromatography in solvent A. [

H]Acetate released moves

down the paper and can be cut and counted in a scintillation counter.

6. The presence of chondroitin/dermatan sulfate uronyl epimerase is assayed by examina-

tion of the products of microsomal biosynthesis (step 2) for resistance to degradation by

chondroitin AC lyase. However, this is not a quantitative assay. Alternatively the uronyl

C-5 epimerase for chondroitin/dermatan sulfate is assayed based on the formation of

from [C-5-

H]chondroitin (43) when this substrate is incubated with microsomal or solu-

bilized enzyme and 50 mM MES buffer, pH 6.5, 15 mM MnCl

, and 0.5% Nonidet P-40

in 50 µL total volume. After 2 h of incubation at 37°C, the reaction mixtures are extracted

into a toluene-based scintillation fluid containing 10–25% isoamylalcohol. The use of

such a biphasic liquid scintillation system allows the extraction of the radioactive water

into the organic phase containing the scintillant, while the polysaccharide remains in the

aqueous phase (37). The epimerase for heparin/heparan sulfate is measured by incorpora-

tion of

H from

O into the C-5 position of heparin/heparan sulfate or by the release of

O from C-5-

H-labeled N-sulfated heparan/heparan synthesizing systems.

4. Notes

1. Biosynthesis of keratan sulfate glycosaminoglycan has not received much attention. How-

ever, synthesis of the glycosaminoglycan probably occurs in a manner similar to that

shown for other glycosaminoglycans, while synthesis of the linkage region apparently

proceeds in a fashion similar to that of the N-linked or O-linked oligosaccharides found

in glycoproteins. Biosynthesis of hyaluronic acid, a glycosaminoglycan that is not

covalently linked to protein, is different from that of the glycosaminoglycan portions of

proteoglycans. It is not formed within or attached to intracellular membrane structures

(44), but is synthesized at the inner surface of the plasma membrane (45) by pathways

quite distinct from those of proteoglycan glycosaminoglycan formation (46).

2. Incorporation of GalNAc onto linkage region GlcA with microsomal or soluble systems has

been reported in all but one instance (19) to result in α-linked GalNAc rather than β-linked

GalNAc (47). However, the addition of β-GalNAc to a naturally occurring protein that con-

tains unsubstituted GlcA-Gal-Gal-Xyl structures has been reported to be catalyzed by the

chondroitin-polymerizing GalNAc transferase (48). Thus addition of the first GlcNAc of hep-

arin/heparan sulfate but perhaps not the first GalNAc of chondroitin, would appear to require an

enzyme different from the hexosaminyl transferase enzyme for glycosaminoglycan formation.

3. Microsomal preparations from the chick cartilage make only chondroitin 6-sulfate, while

those from skin fibroblast or bovine endothelial cell cultures also make chondroitin 4-sulfate,

dermatan sulfate, and heparan sulfate.

112 Sugumaran and Silbert

4. Microsomal preparation from the rat chondrosarcoma and mast cells make no chon-

droitin 6-sulfate, dermatan sulfate, or heparan sulfate.

5. EHS tumors make heparan sulfate almost exclusively.

6. Smith-degraded proteoglycan (49) and Ser-Gly containing peptides (50) have also been

utilized as substrates for Xyl transferase.

7. The β-glycosidases are commercially available and act sequentially to remove the

non-reducing terminal sugar residues.

8. Commercial sources of chondroitin sulfate are invariably mixtures of chondroitin 4-sul-

fate and chondroitin 6-sulfate having different proportions of each. Chondroitin 4-sulfate

that has no 6-sulfation or dermatan residues can be isolated from mouse mastocytoma

cells or from Swarm rat chondrosarcoma. Chondroitin 6-sulfate free of any 4-sulfate is

not available in any quantity, but can be prepared in small radioactively labeled amounts

by biosynthesis with cartilage microsomal preparations.

9. Commercial heparin and heparan sulfate are mixtures, so that preparations of oligosaccha-

rides will yield variable proportions of N-sulfated, N-acetylated, and O-sulfated products.

10. Microsomes prepared as above are stable for months or even years when stored at -20°C.

Repeated freezing and thawing may modify or reduce but generally does not abolish

activity. We have found no evidence of significant proteoglycan proteases, glycosidases,

or sulfatases, since the products of synthesis with all microsomal preparations that we

have used appear to be stable during incubations. The protease inhibitors are added only

as a special precaution.

11. All the preparations have enzymatic activity for degradation of sugar nucleotides and

particularly PAPS to varying degrees. Sodium fluoride has been used by others to inhibit

these degrading enzymes, but we have found it to be of limited value. However, degrada-

tion can be reduced as much as 10-fold by use of 2,3-dimercaptopropan-1-ol without affect-

ing glycosyl transferases (51). Nevertheless, it is not necessary to use these substances unless

degradation is found to be particularly rapid in a specific microsomal preparation.

12. Approximately 400 fourteen- to seventeen-day old chick embryos are used for microso-

mal preparations from chick embryo epiphyses.

13. Density gradients have been widely utilized with preparations from liver and occasion-

ally from other tissues in order to separate electron microscopically recognizable Golgi.

Liver and other tissues as well as a variety of cultured cell lines have been used for prepa-

ration of Golgi fractions, leading to more specific and detailed characterizations of cis,

medial, trans, and trans network subfractions with various enzymatic activities (52). The

density of the compartments decreases from cis to trans, and the enzymes involved in

glycoprotein (53), glycolipid (54), and glycosaminoglycan synthesis (55) have been

shown to have a sequential spatial arrangement so that the early sugar additions occur in

the denser cis regions of the Golgi proximal to the RER while the later reactions of oli-

gosaccharide termination and glycosaminoglycan polymerization occur in more distal,

less dense medial and trans regions of the Golgi.

14. Enzymatic activities and the products of synthesis are as stable in the Golgi preparations

as they are in the microsomal preparations. Moreover, the degradation of sugar nucle-

otides and PAPS is much less.

15. The only microsomes that we have used for enzyme solubilization were from chick

embryo epiphyseal cartilage. However, we would anticipate that effective solubilization

from other tissues could be accomplished similarly.

16. The glycosyltransferases and sulfotransferases solubilized from microsomal preparations

are much less stable than the naturally occurring soluble transferases. They can only be

Glycosaminoglycan-Synthesizing Enzyme Systems 113

stored for limited periods and have much greater losses of activities upon repeated freez-

ing and thawing.

17. Apparent Km values for sugar nucleotides and PAPS in microsomal preparations

are generally higher by at least 10-fold (0.01–0.2 mM range) than with the purified

enzymes.

18. The small volumes are used in order to maximize the concentrations of expensive radio-

active substrates, and to simplify assay.

19. Unlabeled PAPS can be added subsequent to formation of nonsulfated labeled glycosami-

noglycan. In that case, a 100-fold unlabeled excess is added of whichever sugar nucle-

otide was labeled in the initial incubation. In this way, sulfation of the preformed

glycosaminoglycan can be monitored, since only it will be labeled.

20. Sulfation of chondroitin or chondroitin oligosaccharide is usually in a slightly modified

“all or nothing” pattern rather than random, so that in most cases sulfation yields long,

completely sulfated areas with occasional short, nonsulfated areas and some glycosaminogly-

can that remains completely nonsulfated.

21. The increased incorporation with detergent presumably is due to better substrate penetra-

tion to the microsomal vesicles sites of the biosynthetic enzymes.

22. The use of exogenous acceptors provides for the addition of only one or a few residues rather

than true polymerization. The absence of polymerization on exogenous acceptors reinforces

the concept that endogenous membrane-bound nascent proteoglycan substrates are juxtaposed

firmly with the membrane-bound enzymes, thus permitting true polymerization.

23. Sulfation by addition of PAPS is not as extensive with exogenous acceptors, although it is

still a modified “all or nothing” addition but with much greater areas that are not sulfated.

24. Exogenous substrates are needed for measuring the glycosyl transferases of the linkage

region, since there is insufficient endogenous acceptor at these stages of synthesis.

25. Paper chromatography is the simplest and most rapid assay for multiple samples. After

incubation of substrates and acceptors with purified enzymes, microsomes, or Golgi frac-

tions, an aliquot or the entire reaction mixture is spotted on Whatman No. 1 paper for over-

night descending chromatography in solvent A. With this system, nucleotide sugars, PAPS,

and their degradation products move down the paper while radiolabeled proteoglycan and

glycosaminoglycan products remain at the origin. When oligosaccharides are used as

acceptors, descending chromatography in solvent B is used, since the oligosaccharides

will move down the paper with solvent A. Incubation of the origins with proteases at

37°C or with 0.5 M NaOH at room temperature for a few hours will quantitatively pro-

vide cleavage and consequent solubilization of the glycosaminoglycan portion from

endogenous proteoglycan acceptors. When glycosaminoglycans or oligosaccharides are

used as exogenous acceptors, the products are eluted directly with water, thus providing a

rapid means to separate the products of addition to exogenous acceptors from the prod-

ucts of addition to endogenous nascent proteoglycan. Eluants are then assayed for radio-

activity. A single chromatography paper can be used for as many as 12 simultaneous

assays, and a single large chromatography tank for as many as 12 sheets of chromatogra-

phy paper, thus providing the potential for 144 simultaneous assays of addition to endog-

enous nascent proteoglycans and/or simultaneous assays of addition to exogenous

glycosaminoglycans or oligosaccharides. Since all the solvent systems fix proteoglycan

acceptors to the origin, the intact proteoglycan acceptors cannot be obtained by this tech-

nique. In order to obtain these, 4 M guanidine chloride and occasionally detergents are

added to reaction mixtures that are then chromatographed directly on Sephadex G-50 to

isolate labeled proteoglycans from the labeled substrates.

114 Sugumaran and Silbert

26. Use of these lyases and sulfatases as well as identification of products has been reviewed

in detail (6,32).

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Analyses of Hyaluronan and Chondroitin/Dermatan Sulfate 117

117

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Disaccharide Composition of Hyaluronan

and Chondroitin/Dermatan Sulfate

Analysis with Fluorophore-Assisted Carbohydrate Electrophoresis

Anna H. K. Plaas, L. West, Ronald J. Midura, and Vincent C. Hascall

1. Introduction

The glycosaminoglycan constituents of proteoglycans, heparan sulfate, chondroitin/

dermatan sulfate, or keratan sulfate (1), and the glycosaminoglycan hyaluronan (2) are

important constituents of all tissue matrices. They provide tissue hydration for fluid

flow and molecular transport, charge repulsions or attractions for intermolecular spac-

ing (3) and polyanionic domains for growth factor or integrin signaling (4,5), cell

adhesion, and migration or proliferation (6–8). As a result, studies of extracellular

matrix metabolism in genetic, developmental, or tissue engineering experiments also

include analyses of glycosaminoglycan components. The most widely used proce-

dures for glycosaminoglycan analyses rely largely on the identification and

quantitation of products generated by depolymerization with lyases (9) or hydrolases

(10) by gel electrophoresis (11), nuclear magnetic or mass spectrometry (12–15), high-

performance liquid chromatography (16–21) or capillary zone electrophoresis (22–24).

While sensitive and accurate, such procedures usually require costly or technically

specialized equipment, and involve preparative steps that can lead to loss of products.

We describe here an alternative procedure for the identification and quantitation of gly-

cosaminoglycan lyase products using fluorophore-assisted carbohydrate electrophoresis

or FACE (25,26). It is especially applicable for studies when only small amounts of

tissue or proteoglycans are available and/or a high sample throughout is needed. While

we limit the illustration of this technically simple procedure to chondroitin/dermatan

sulfate and hyaluronan in the chapter, it can also serve as an alternative sensitive and

specific way to determine the identity and quantity of products of heparan sulfate-spe-

cific lyases (9) and keratan sulfate-specific hydrolases (27).

The repeating disaccharide structures of chondroitin/dermatan sulfate and hyaluronan

are illustrated in Fig. 1. In chondroitin and dermatan sulfate, the galNAc

1,4glcA repeats