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

1. Introduction

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 ^ 4)-a-d-N-acetylglucosamine-6-sulfate (1^ 4)-^-d-glucuronic acid (1^ and ^ 4)-a-d-N-sulfoglucosamine (1^ 4)-^-d-glucuronic acid (1^ (see Fig. 1B). Heparin, which contains approx 2.7 sulfate groups per disaccharide unit, is more highly sulfated than HS, which contains less than one sulfate per disaccharide unit.

HS proteoglycans (PGs) are localized on the surface of many mammalian cells and in the extracellular matrix. HS proteoglycans are important for several different biological activities such as cell-cell and cell-protein interactions (2). These biological activities are controlled mainly through the binding of a variety of proteins to the HS chains. Specific sequences in the HS chain are thought to be responsible for the binding of growth factors, protease inhibitors, and adhesion molecules. The use of HS-degrading enzymes can help in separating and identifying biological active oligosaccharides (3).

HS can be degraded enzymatically by using heparin lyases from bacterial sources. The lyase enzymes degrade GAGs by endolytic cleavage (4-6). The enzymes cut glu-cosamine-uronate linkage by elimination (see Fig. 2), leaving a C4-C5 unsaturated bond containing product that can be easily detected by ultraviolet (UV) absorbance. In contrast, mammalian heparanases cleave this linkage by hydrolysis.

Heparin lyases have been isolated from Flavobacterium heparinum (7), Bacteriodes species (8), Bacteriodes heparinolyticus (9), and Prevotella heparinolytica (10). Heparin lyases from F. heparinum have been purified to homogeneity, studied extensively (11), and are available commercially from Sigma and Seikagaku.

Heparin

P Heparan Sulfate oso3"

major (¡¡saccharide sequence oso3"

major (¡¡saccharide sequence coo" ch2oh oh major disaccharide sequence coo" ch2ox >-O J——Q

X = S03' or H, Y = S03", CH3CO or H minor disaccharide sequence

X = S03" or H, Y = S03", CH3CO or H minor disaccharide sequence

Fig. 1. (A) Structure of the major and minor disaccharide sequences of heparin. (B) Structure of the major and minor disaccharide sequences of heparan sulfate.

Three types of heparin lyases have been purified from Flavobacterium: heparin lyase

1, heparin lyase II, and heparin lyase III (see Fig. 2 and Note 1). Heparin lyase I acts primarily on heparin, heparin lyase II cleaves both heparin and HS, and heparin lyase III is active only on HS (see Table 1). The primary linkages cleaved by these enzymes and their relative activities toward heparin and HS are shown in Table 1 and Fig. 3.

From both the DNA and amino acid sequences, there is only 15% alignment between heparin lyase I, II, and III (12). There are certain conserved sequences such as the heparin-binding sites and the calcium-binding regions in heparin lyase I and III. Recently, chemical modification studies and site-directed mutagenisis have been used to help identify critical residues for enzyme activity (13-17). Further studies and the crystal structures of the heparin lyases are needed to help us understand better the relationship between function and structure of these enzymes.

This chapter will explain how the heparin lyase enzymes can be used to degrade both heparin- and HS-containing samples and how to assay the activity of the enzyme. The heparin lyase enzymes can be used to identify the presence of HS/heparin in samples or to purify HS oligosaccharides for structural analysis.

2. Materials

2.1. Enzyme Preparation

1. Heparin Lyase I, II, or III. Enzymes can be ordered from Sigma (St. Louis, MO) and Siekagaku America (Falmouth, MA) (see Notes 1 and 2).

Fig. 2. Eliminative cleavage of GAGs by lyases.

2. Reagents required to make the appropriate buffers:

a. Dibasic sodium phosphate (EM Science, Gibbstown, NJ).

b. Phosphoric acid (Fisher Scientific, Fair Lawn, NJ).

c. Sodium chloride (Fisher Scientific).

3. 500-^L polypropylene microcentrifuge tubes.

2.2. Enzyme Assay

1. HS (bovine kidney HS, sodium salt from Siekagaku America).

2. Heparin lyase solution (see Subheading 3.1.).

4. UV spectrophotometer.

2.3.Sample Digestion

1. HS or heparin samples (see Note 3) or samples containing radiolabeled HS or heparin.

2. Spectropor dialysis membrane (molecular-weight cutoff [MWCO] 1000) (Spectrum, Los Angeles, CA) or Centricon (YM3, MWCO 3000) centrifugal filter units (Millipore, Bedford, MA).

3. 500-^L polypropylene microcentrifuge tubes.

4. Heparin lyase solution.

5. Water baths at 30°C and 35°C for enzyme digestion and at 100°C to inactivate the enzyme reaction.

2.4. Product Analysis

High-performance liquid chromatography (HPLC), capillary electrophoresis (CE), gel-permeation chromatography, or polyacrylamide gel electrophoresis (PAGE) may be used to purify and analyze oligosaccharides prepared from HS/heparin.

Table 1

Activity of Heparin Lyases

Activity and substrate conversion Heparin lyase I Heparin lyase II Heparin lyase III

Heparin0

Percent activity' % Conversion Heparan sulfate6 Percent activity b

10 20

100 40

60 85

100 94

Percent conversion aPorcine mucosal heparin.

'Percent activity = [initial rate on the substrate examined/initial rate on substrate giving the highest activity] x (100).

cPercent conversion = [moles of linkages cleaved/total moles of hexosamine ^ uronic acid linkages] x (100).

dBovine lung heparin. eBovine kidney heparan sulfate.

3. Methods

3.1. Preparation of Lyases for Use

1. Preparation of buffers: The following buffers can be stored at room temperature for over 1 mo (see Note 4).

a. For heparin lyase I, prepare 50 mM sodium phosphate buffer containing 100 mM sodium chloride at pH 7.1. To prepare 1 L of buffer, dissolve 7.1 g of dibasic sodium phosphate and 5.8 g of sodium chloride into 900 mL of distilled water. Adjust the pH to 7.1 with phosphoric acid and bring the volume up to 1 L with distilled water.

b. For heparin lyase II and III, prepare 50 mM sodium phosphate buffer by dissolving 7.1 g of dibasic sodium phosphate in 900 mL of distilled water. For heparin lyase II adjust the pH to 7.1 with concentrated phosphoric acid, and adjust to 7.6 for heparin lyase III. Adjust the final volume to 1 L with distilled water.

2. Aliquot samples:

a. Dissolve 0.1 U of lyophilized enzyme in 100 ^L of the appropriate buffer (see Table 2).

b. Store the enzyme in 10-mU aliquots in 500-^L polypropylene tubes at -70°C (see Note 5).

3.2. Activity Assay for Lyases

1. Add 640 ^L of the appropriate buffer (see Table 2) to a 1-mL quartz cuvet. Warm the cuvet to 30°C in a temperature-controlled UV spectrophotometer (see Note 6).

2. Thaw 10-^L aliquots of the appropriate enzyme solution (see Table 1) at room temperature.

3. Remove 90 ^L of warm buffer out of the cuvet and transfer the solution into the tube containing the enzyme solution. Immediately transfer the entire 100 ^L of buffer and enzyme back into the cuvet, which is incubating at 30°C.

4. Adjust the baseline of the spectrophotometer to zero at 232 nm.

5. Remove the cuvet from the spectrophotometer and add 50 ^L of 20 mg/mL of the appropriate substrate HS/heparin (see Table 1) to the cuvet. Cover the cuvet with Parafilm and

Heparinase
Fig. 3. Primary glycosidic linkages cut by hepain lyases. Abbreviations: X, H or SO3 CH3CO or SO3-. Heparin lyase II cleaves at either glucuronic or iduronic acid residues.

Y, invert two times to mix. Remove the Parafilm and place the cuvet back into the spectrophotometer.

6. Within 30 s after the addition of substrate, begin to measure the absorbance continuously or at 30-s intervals for 2-10 min. Graph absorbance at 232 nm vs time. The initial rate is determined by measuring the slope of the linear portion of absorbance vs time.

7. Calculate the enzyme activity from the initial rate using the extinction coefficient ( e = 3800 Mfor the reaction products (see Note 7). Each product formed has an unsaturated uronic acid residue at its nonreducing terminus that absorbs at 232 nm. The enzyme activity is calculated as

Table 2

Properties of Heparin Lyases and Reaction Conditions

Enzyme

Substrate

X b opt

Heparin lyase II

Heparin lyase III (EC 4.2.2.8)

Heparin

Heparin HS HS

42,800

84,100 70,800

35 35

50 mM NaPO4, 100 mM NaCl, pH 7.1 50 mM NaPO4, pH 7.1 50 mM NaPO4, pH 7.6

aEC is the Enzyme Commission number. 6Topt, optimum temperature for the enzyme.

3.3.Sample Digestion

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