CHAPTER EIGHT

Biological Activities of Heparan Sulfate Muthuvel Arumugam1, Sadhasivam Giji Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, Tamil Nadu, India 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Materials and Methods 2.1 Isolation 2.2 Anticoagulant activity 2.3 Antiproliferative activity 2.4 1H-NMR 3. Results 4. Discussion Acknowledgments References

125 127 127 128 128 128 129 130 134 134

Abstract Heparan sulfate was isolated from two bivalve mollusks such as Tridacna maxima and Perna viridis. The isolated heparin was quantified in crude as well as purified samples and they were estimated as 2.72 and 2.2 g/kg (crude) and 260 and 248 mg/g (purified) in T. maxima and P. viridis, respectively. Both the bivalves showed the anticoagulant activity of the crude and purified sample as 20,128 USP units/kg and 7.4 USP units/mg, 39,000 USP units/kg and 75 USP units/mg, 9460 USP units/kg and 4.3 USP units/mg, and 13,392 USP units/kg and 54 USP units/mg correspondingly in T. maxima and P. viridis. The antiproliferative activity that was studied with pulmonary artery smooth muscle cells using RPMI media reported that the result is in a dose-dependent manner. Among the two clams, P. viridis showed more antiproliferative activity than that of T. maxima.

1. INTRODUCTION Heparan sulfate (HS) has been isolated from tissues of a large number of vertebrate and invertebrate organisms. Invertebrates were first shown to contain a heparin or HS (Burson et al., 1956). An exhaustive assessment Advances in Food and Nutrition Research, Volume 72 ISSN 1043-4526 http://dx.doi.org/10.1016/B978-0-12-800269-8.00008-7

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showed that mollusks are particularly rich source of the sulfated polysaccharides (Nader & Dietrich, 1989) and it amounts to 90% of the total GAGs content of the mollusks. But the heparin/HS isolated from mollusks are structurally different from human heparin and pharmaceutical heparins (Loganathan, Wang, Mallis, & Linhardt, 1990). Heparin and HS have been the subject of intensive study because of their well-recognized ability to bind proteins that regulate a variety of important biological processes. Heparin and HS are comprised of alternating 1 ! 4-linked glucosamine and uronic acid residues. HS is composed of primary monosulfated disaccharides of N-acetyl-D-glucosamine and D-glucuronic acid, while heparin is composed of mainly trisulfated disaccharides of N-sulfoyl-D-glucosamine and L-iduronic acid (Linhardt & Toida, 1996). Recently reported, the diverse biological functions present considerable opportunities for exploiting HS or HS-protein conjugates for developing new classes of anticancer (Shriver, Raguram, & Sasisekharan, 2004), antiviral (Baleux et al., 2009), and improved anticoagulant drugs (Chen, Jones, & Liu, 2007). A method to construct HS is, therefore, critical for developing HS-based therapeutics. Total chemical synthesis is fully capable of preparing short fragments of HS. HS is found on the surface of endothelial cells. This suggests a physiologically relevant role in regulating levels of anticoagulant activity within the blood vessel. Pharmaceutical heparin is a product of mast cells that is isolated from porcine intestinal tissue with a stronger anticoagulant activity than endothelial HS. Heparin is considered a specialized form of HS with higher levels of sulfation per saccharide unit and iduronic acid content. For example, HS contains about 0.6 sulfo groups per disaccharide unit, while heparin contains 2.6 sulfo groups. Further, about 40% of uronic acid in HS is iduronic, while 90% uronic acid in heparin is iduronic. It plays important roles in regulating numerous functions of the blood vessel wall, including blood coagulation, inflammation response, and cell differentiation. HS is a highly sulfated polysaccharide containing glucosamine and glucuronic/ iduronic acid repeating disaccharide units. The unique sulfated saccharide sequences of HS determine its specific functions. Heparin, an analogue of HS, is the most commonly used anticoagulant drug. Because of its wide range of biological functions, HS has become an interesting molecule to biochemists, medicinal chemists, and developmental biologists (Fig. 8.1). Glycosaminoglycans (GAGs) have been isolated from various tissues obtained from a large number of animal species including both vertebrates and invertebrates. Invertebrates were first shown to contain a heparin or HS (Burson et al., 1956). An exhaustive assessment showed that the mollusks are particularly rich source of these sulfated polysaccharides and it often

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Biological Activities of Heparan Sulfate

CH2SO3-

COO-

CH2SO3-

OH

OH

OH

O

O NHSO3-

COOOH

O OH

CH2SO3-

O NHSO3-

OSO3-

CH2SO3COOOH

OH O

O NHSO3-

OSO3-

OH O NHSO3-

Figure 8.1 The structure showing the repeating disaccharide units.

corresponds up to 90% of the total GAG content these organisms (Nader & Dietrich, 1989). Heparin and heparin-like substances have a wide range of important biological activities including inhibition of pulmonary artery smooth muscle cell (PASMC) proliferation. In the normal physiological state, the smooth muscle cells (SMCs) are entering into quiescent growth state in pulmonary arterial walls which is regulated by a balance between inhibitory and mitogenic factors (Garg, Thomson, & Hales, 2000). The major effect of heparin on blood coagulation is to accelerate the normal rate at which antithrombin III neutralizes the proteolytic activities of several serine proteases in the coagulation sequence. The search for the new sources of heparin with low toxicity prompted many scientists to focus their attention toward marine animals. Earlier studies have shown that heparin-like compounds are also present in some invertebrates (Cassaro & Dietrich, 1977; Patel, Ehrlich, Stivala, & Singh, 1980). A substance denoted “mactin” with anticoagulant activity and structural similarities to mammalian heparins was isolated from the mollusks Cyprinia islandica and Mactrus pussula. Another compound from the clam Mercenaria mercenaria also exhibited several structural similarities to heparin. Previously conducted detailed investigations on the heparin isolated from the clams (Pejler, Danielsson, Bjork, & Lindahl, 1987) A. brasiliana and T. mactroides found that the clam heparin preparations have basically the same structure as mammalian heparins, but notable differences included the greater chain length of clam heparin. Further heparin from some mollusks was reported to show difference in their anticoagulant activity (Dietrich et al., 1989). The present study aimed to find out the source of heparin and discuss their antiproliferative and anticoagulant activity. This chapter reports the isolation of GAGs and their structural features with special reference to heparin/HS from marine bivalve mollusks.

2. MATERIALS AND METHODS 2.1. Isolation The standard procedure was followed for the extraction of heparin/HS, with suitable modification (Arumugam & Shanmugam, 2004) for the

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defating and deproteinization of T. maxima and P. viridis treated with CPC. The purification of the crude HS was done by the using the anionic resin (Amberlite IRA-900 Cl). The purified GAGs were converted into heparin sodium/HS salts by using cationic resin (Amberlite IR-120 Na+) and the recovered precipitate was taken for further analyses (Nishino, Yokoyama, Dobashi, Fujihara, & Nagumo, 1989).

2.2. Anticoagulant activity Heparin readily catalyzes the inactivation of factor Xa by anti-thrombin III. Factor Xa inactivation was used in this study to assess the anticoagulant activity of the GAGs prepared from both mussels using a Heparin Assay Kit (Sigma). In this assay, when both factor Xa and anti-thrombin III are present in excess, the inhibition of factor Xa is directly proportional to the limiting concentration of heparin. Thus, residual factor Xa activity, measured with factor Xa-specific chromogenic substrate, is inversely proportional to the heparin concentration (Teien, Lie, & Abilguard, 1976).

2.3. Antiproliferative activity The antiproliferative activity of samples was determined by the MTT assay (Mosmann, 1983). The isolated bovine PASMCs were seeded at 1.5  104 cells/well into a 6-well tissue culture plate, grown for 2 days; then growth was arrested at the end of 48 h by reducing the serum concentration of the medium from 0.1% to 10%. The media was then changed to the experimental samples which contained either standard media (RPMI1640 with 10% fetal bovine serum (FBS) (Sigma, St. Louis, Mo)), growth arrest media (RPMI with 0.1% FBS), or standard media with oligomers/ heparin (5 μg/ml). All media contained streptomycin (100 μg/ml), Penicillin (100 U/ml), and amphotericin B (1.25 μg/ml). After 4–5 days of growth, the cells were lifted with trypsin/EDTA and then counted using a Coulter Counter. Results are presented as mean  standard error of the mean. Comparisons among groups were made with a factorial analysis of variance (ANOVA), using the STATE VIEW software package (Brainpower, Inc., to Calabasas, CA) for Macintosh computers.

2.4. 1H-NMR NMR spectroscopy was analyzed on samples dissolved in D2O to remove exchangeable protons and transferred to Shigemi tubes. 1H-NMR experiments were performed on an AMX-400 at 400 MHZ.

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Biological Activities of Heparan Sulfate

3. RESULTS The amount of HS was estimated as 2.72 g/kg of dry tissue in T. maxima and 2.2 g/kg in P. viridis (crude). After purification by using the Amberlite anion exchange resin, the yield was found to be 260 and 248 mg/g, respectively, in T. maxima and P. viridis, as presented in Table 8.1. In the anticoagulant assay (Table 8.2), the yield and anticoagulant activity of the T. maxima crude sample were reported to be 20,128 USP units/kg and 7.4 USP units/mg; whereas the purified sample showed 39,000 USP units/kg and 75 USP units/mg of the sample, respectively. Likewise, in the case of P. viridis, the yield and anticoagulant activity were estimated as 9460 USP units/kg and 4.3 USP units/mg in the crude sample and 13,392 USP units/kg and 54 USP units/mg in the purified sample, respectively. The effect of HS (GAGs) isolated from P. viridis and T. maxima and Upjohn heparin grown in RPMI medium containing 0.1% and 10% FBS is depicted in Fig. 8.2. The heparin/HS isolated from P. viridis recorded increasing inhibition over the growing cells when the concentration increased from 1.0 to 100 μg, i.e., the percentage cell mean in the case of 1.0 μg was found to be 61.764  3.660; in 10 μg, it was 33.064  3.507; Table 8.1 The yield of crude and purified heparan sulfate complex of T. maxima and P. viridis Net yield S. no.

Source

Crude (g/kg)

Purified (mg/g)

1.

Tridacna maxima

2.72

260

2.

Perna viridis

2.2

248

Table 8.2 The anticoagulant activities of the heparan sulfate extracted from T. maxima and P. viridis Anticoagulant assay (USP method) Crude

Purified

S. no.

Origin

Activity (IU/mg)

Yield (IU/kg)

Activity USP (units/mg)

Yield USP (units/kg)

1.

Tridacna maxima

7.4

20,128

75

39,000

2.

Perna viridis

4.3

9460

54

13,392

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Muthuvel Arumugam and Sadhasivam Giji

Interaction bar plot for % growth Effect: Fish HP-final results Error bars: ± 1 Standard error (s) 180 160 140

Cell mean

120 100 80 60 40 20 0

THP100mg

THP10mg

THP1mg

PHP100mg

PHP10mg

PHP1mg

UHP100mg

UHP10mg

UHP1mg

FBS0.1%

-40

FBS10%

-20

Cell

Figure 8.2 The antiproliferative effect of heparin and heparin like glycosaminoglycans from P. virdis (PHP) & T. maxima (THP) and Upjohn heparin.

and in 100 μg concentration, it was 15.071  4.609, showing an increasing influence over the cell growth. The inhibition of P. viridis is comparatively more even than that of Upjohn heparin, the standard used in the present study, which showed the percentage mean cell growth as follows: 1 μg— 72.533  11.200, 10 μg—8.809  7.940, and 100 μg—19.168  5.921, respectively. But on the contrary, the heparin and heparin-like GAGs extracted from T. maxima were found to promote the cell growth in lower concentrations, i.e., 1 μg—134.939  26.468% and 10 μg—123.934  25.325%. But at the same time, at higher concentration it was also found to be reducing the growth (at 100 μg concentration, the mean cell growth was only 77.429  18.923%). 1 H-NMR analysis revealed the presence of signals corresponding to GlcNac (N-acetyl methyl at 1.99 ppm and H-1 at 5.4 ppm) in both the samples (Figs. 8.3 and 8.4).

4. DISCUSSION In the present investigation, the yield of crude HS was found to be 2.72 g/kg and 2.2 g/kg in T. maxima and P. viridis, respectively, whereas the HS yield was 2.2–2.8 g/kg, 1.8–2.5 g/kg, and 2.7–3.8 g/kg in A. brasiliana, D. striatus, and T. mactroides, respectively (Dietrich et al.,

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Biological Activities of Heparan Sulfate

6

5

4

3

2

1

0

ppm

1

Figure 8.3 H-NMR spectroscopy of heparan sulfate from Tridacna maxima.

6

5

4

3

2

1

0

ppm

1

Figure 8.4 H-NMR spectroscopy of heparan sulfate from Perna viridis.

1989). At the same time, the yield of heparin and other sulfated mucopolysaccharides from thymus is 274 μg/kg only (Straus, Nader, Bianchini, & Osima, 1981). Likewise, the sulfated mucopolysaccharides were isolated by using quaternary ammonium salts (Barlow, Coen, & Mozen, 1964)

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and the yield was reported to be 170, 174, 843, 307, and 1090 μg/kg dry tissue in different molluscan species such as Aulocombia ater, Perna perna, Mesodesma donacium, Loligo brasiliense, and Octopus species, respectively. In the present study, the crude sample of T. maxima and P. viridis showed 7.4 USP units/mg and 4.3 USP units/mg of anticoagulant activity, respectively. But in the purified sample of T. maxima and P. viridis, the anticoagulant activity was found to be higher (75 USP units/mg and 54 USP units/ mg, respectively) than that of the crude sample which is further higher than that of K. opima (39.7 BP units/mg), sheep heparin (59 USP/mg), fin back whale Balaenoptera physalus (40–70 USP units/mg), H. pugilinus (26 USP units/mg) (Barlow et al., 1964), and HS anticoagulant activity (Nader et al., 1983) in Pomacea sp., T. gibbus, A. brasiliana, beef pancreas (

Biological activities of heparan sulfate.

Heparan sulfate was isolated from two bivalve mollusks such as Tridacna maxima and Perna viridis. The isolated heparin was quantified in crude as well...
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