Prev. Nutr. Food Sci. 2014;19(4):307-313 http://dx.doi.org/10.3746/pnf.2014.19.4.307 pISSN 2287-1098ㆍeISSN 2287-8602
Structural Effects of Sulfated-Glycoproteins from Stichopus japonicus on the Nitric Oxide Secretion Ability of RAW 264.7 Cells Rong-An Cao1,2, Su-Han Lee3, and SangGuan You2 1
College of Food Science, Heilongjiang Bayi Agricultural University, Daqing 163319, China Department of Marine Food Science and Technology, Gangneung-Wonju National University, Gangwon 210-702, Korea 3 Department of Food Technology and Service, Eulji University, Gyeonggi 461-713, Korea 2
ABSTRACT: The effect of various levels of proteins, sulfates, and molecular weight (Mw) of a sulfated-glycoprotein (NF3) from a sea cucumber, Stichopus japonicus, on nitric oxide (NO) releasing capacity from RAW 264.7 cells was investigated. The NF3 derivatives had various amounts of proteins (4.8∼11.2%) and sulfates (6.8∼25.2%) as well as different Mw 3 3 (640.3×10 ∼109.2×10 g/mol). NF3 was able to stimulate RAW 264.7 cells to release NO with lower protein contents, indicating that the protein moiety was not an important factor to stimulate macrophages. On the other hand, the NO inducing capacity was significantly reduced with decreased levels of sulfates and Mw, implying that sulfates and Mw played a pivotal role in activating RAW 264.7 cells. It was not clear why sulfates and a certain range of Mw were essential for stimulating macrophages. It appeared that certain levels of sulfates and Mw of sulfated-glycoproteins were required to bind to the surface receptors on RAW 264.7 cells. Keywords: Stichopus japonicus, sulfated-glycoproteins, structure-bioactivity, nitric oxide, RAW 264.7
INTRODUCTION The sulfated fucans (SF) are water-soluble, complex, and heterogeneous macromolecules, predominantly found in the cell wall of marine brown algae and in the body wall of marine invertebrates (1). They have been reported to exhibit various biological and pharmacological activities, such as anticoagulant, antitumor, and immunomodulating activities (2-4). According to Soeda et al. (5), the SF derivatives with various sulfate contents stimulated tissue plasminogen activator (t-PA)-induced plasma clot lysis and prevented the formation of fibrin polymers, and such activities increased proportionally with the degree of sulfation. It was also reported that the over-sulfated fucans possessed higher macrophage-stimulating activities than native SF, and thus more effectively up- regulated cytokine induction from the macrophage cells (6,7). However, partially desulfated fucans with sulfate contents of less than 20% showed drastic decreases in both anticoagulant and anticancer activities (8). In a study of Nishino et al. (9), the Mw of SF from Ecklonia kurome was related to their anti-
coagulant activity. The authors found that SF with Mw 3 3 ranging from 10×10 to 300×10 g/mol showed the most potent anticoagulant activities, suggesting that a specific range of sugar- chains and a comfortable conformation were required to bind the thrombin for the potent anticoagulant activity. Therefore, the biological activities of SF appeared to be closely related to their compositional and macromolecular structures. Indeed, a basic understanding of both the primary and secondary structures of SF may lead to successful interpretation of their bioactivities. Sea cucumbers, known as trepang, beche-de-mer or gamat, are marine invertebrates belonging to Echinodermata, Holothuroidea, and Aspidochirotida, and are widely found in the benthic area and deep-seas worldwide (10). They are flexible, elongated, worm-like organisms with a leathery skin and gelatinous body. Sea cucumbers are extensively consumed in China, Japan, Korea and other Asian countries as traditional and nutritional foods and medicinal resources (11). Various components, such as triterpene glycosides, peptides, fatty acids, and SF, are present in the body walls of sea cu-
Received 11 November 2014; Accepted 24 November 2014; Published online 31 December 2014 Correspondence to Su-Han Lee, Tel: +82-31-740-7196, E-mail:
[email protected] Copyright © 2014 by The Korean Society of Food Science and Nutrition. All rights Reserved. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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cumbers (10). Among the various constituents, SF are major edible parts of sea cucumbers (12), and have been broadly studied because of their numerous bioactivities including anticoagulant (13), anti-inflammatory (14), antithrombotic (15), antitumor (16), and immunomodulatory (17) activities. Several studies have been performed for the structure analysis of SF from sea cucumbers using various techniques such as surface-enhanced raman scattering, gas chromatography-mass spectrometry (GC-MS), fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance (NMR) spectroscopy after some modifications of sulfated polysaccharides (18,19). The reported backbones of SF from sea cucumbers consisted of (1→3)-linked fuco-pyranosides and/or branched (1→3)-linked fuco-pyranosides substituted at C-4 with a fuco-pyranose, and sulfate substitutions occurred at C-2, C-4 or C-2 and C-4 (18,19). In our previous study, the soluble compounds were extracted from a sea cucumber, Stichopus japonicus, followed by the removal of free proteins. Despite the free protein removal, the compounds included large amount of proteins bound to the SF. Actually, the protein-sulfated fucan (PSF) complex is the major ingested compound from sea cucumbers when they are consumed. However, the bio-activity studies of sea cucumbers constituents have been focused on the bio-polymers of either proteins or SF itself and not on the complex form of PSF. Upon the investigation of the PSF from sea cucumbers in our previous studies, the crude and fractionated PSF significantly improved the proliferation of murine macrophage cells (RAW 264.7), and strongly stimulated the RAW 264.7 cells to induce the production of NO and some cytokines such as cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-, interleukin (IL)-10, and IL-6, suggesting that they were strong immunostimulating agents. Among the crude and fractionated fractions, fraction F3 having higher SF (76.6%) exhibited relatively strong immunomodulatory activity. Therefore, in the current study, a further investigation regarding a correlation between the molecular structure and the immunomodulatory activity was performed after the sulfate and protein contents as well as the Mw of the F3 fraction were systematically changed.
MATERIALS AND METHODS Samples and reagents The live specimens of sea cucumber S. japonicus were collected from the east coast of Korea. The body wall of the sea cucumber was carefully separated from other tissues, washed with tap water, minced, and lyophilized. The dried sample was ground to powder using a grinder, sieved (
