Determination of sialic acids in immune system cells (coelomocytes) of sea urchin, Paracentrotus lividus, using capillary LC-ESI-MS/MS.

Coelomocytes are considered to be immune effectors of sea urchins. Coelomocytes are the freely circulating cells in the body fluid contained in echinoderm coelom and mediate the cellular defence responses to immune challenges by phagocytosis, encapsulation, cytotoxicity and the production of antimicrobial agents. Coelomocytes have the ability to recognize self from non-self. Considering that sialic acids play important roles in immunity, we determined the presence of sialic acid types in coelomocytes of Paracentrotus lividus. Homogenized coelomocytes were kept in 2 M aqueous acetic acid at 80 °C for 3 h to liberate sialic acids. Sialic acids were determined by derivatization with 1,2-diamino-4,5-methylenediaoxy-benzene dihydrochloride (DMB) followed by capillary liquid-chromatography-electrospray ionization/tandem mass spectrometry (CapLC-ESI-MS/MS). Standard sialic acids; Neu5Ac, Neu5Gc, KDN and bovine submaxillary mucin showing a variety of sialic acids were used to confirm sialic acids types. We found ten different types of sialic acids (Neu5Gc, Neu5Ac, Neu5Gc9Ac, Neu5Gc8Ac, Neu5,9Ac2, Neu5,7Ac2, Neu5,8Ac2, Neu5,7,9Ac3, Neu5Gc7,9Ac2, Neu5Gc7Ac) isolated in limited amounts from total coelomocyte population. Neu5Gc type of sialic acids in coelomocytes was the most abundant type sialic acid when compared with other types. This is the first report on the presence of sialic acid types in coelomocytes of P. lividus using CapLC-ESI-MS/MS-Ion Trap system (Capillary Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry).

Introduction

Sialic acids are a diverse family of monosaccharides widely expressed on all cell surfaces of vertebrates and higher invertebrates (such as starfish and sea urchin), and on some bacteria that interact with vertebrates [1]. The expression of sialic acids is highly conserved in deuterostomes, i.e., from echinoderms to humans [2]. Sialic acids are biosynthesized by almost all organisms as a 9-carbon carboxylated monosaccharide and found most frequently as the terminal sugars of cell surface glycolipids and glycoproteins [3], [4], [5], [6]. Because of their terminal location and negative charge, sialic acids have numerous roles in many aspect of immunity [1]. These monosaccharides have the potential to contribute or inhibit many intermolecular and intercellular interactions [4], [7]. Sialic acids also contribute to many features of the cell surface and frequently serve as ligands for receptor-mediated intercellular interactions, host cell-pathogen recognition processes and lymphocyte-endothelial cell interactions [8], [9]. Many earlier studies removed sialic acids from immune cell surfaces using sialidases and observed changes in behavior of such cells [1]. Sialic acids are crucial in the development of vertebrates, as observed that mouse embryos engineered to deficiency of sialic acid expression die after they birth [10]. In pathogenic diseases, including some inflammations, particular microbes recognize cell surface sialic acids when invading host cells [11], [12]. For example, binding of influenza A and B viruses to host cell is accomplished by the specific recognition of the host sialic acid linkage by the virus. Sialylated molecules on Aspergillus fumigatus conidia (fungal pathogen) are recognized by cultured mouse macrophages and lung epithelial cell lines and enhance the uptake of conidia into the endosomal system of the host cell [13]. Sialic acid residues can also mask recognition sites such as galactose residues on glycoproteins to prevent their in vivo removal by asiaologlycoprotein receptors [14]. In certain cancers, changes in sialic acid amounts, types and linkages have been associated with tumorigenesis and cancer metastasis [12]. More than 50 naturally occurring sialic acid [15], [16], [17] derivatives of the three main forms, N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) and 2-keto-3-deoxy-nonulosonic acid (KDN) were identified [6], [18]. The great structural diversity in the sialic acids originates from different substitutions on the amino group at carbon 5 and hydroxyl groups at carbons 4, 7, 8 and 9 by acetyl groups (O-acetylation) [3], [19], [20].

There have been many reports concerning the analysis of specific sialic acids in animals. Although small amounts of glycoconjugates with sialic acids have been reported in certain protostome animals such as insect [21], [22], [23], [24], these sugars are found in largest amount and exhibit greatest structural variability in deuterostomia, predominantly in the echinodermata and hemichordata [25].

Echinoderms have a potential immune system for their survival [26]. Many species, living in coastal and estuarine waters, are directly exposed to potentially pathogenic microorganisms and have developed defence responses mainly based on immunocytes (coelomocytes) and humoral factors contained in the coelomic fluid [27], [28], [29], [30]. In echinoids, sea urchin, Paracentrotus lividus, at least four main coelomocyte types including a subpopulation of macrophage-like cell, phagocytic cells that circulate in the coelomic fluid have been described: amoebocytes, vibratile cells, red and uncolored spherulocytes [26], [30], [31], [32], [33], [34], [35]. Cell-based immunity is carried by the coelomocytes, a morphologically heterogeneous population of free roaming cells that are capable of recognizing and neutralizing pathogens. Coelomocytes present diverse functions which include phagocytosis, encapsulation, clotting, cytotoxicity and wound healing [31], [35].

The cell-based immunity system is able to distinguish between self and non-self by oversialylation and undersialylation of their cell surface (but so far not examined in echinoids) [36]. In terms of their immune systems, echinoderms represent the same basic types of responses that most multicellular (including vertebrates) animals do. The coelomocytes of echinoderms can recognize self from non-self and, if a foreign material (e.g., microorganism/pathogen) enters the body cavity, they can readily neutralize it and dispose of it [32], [34], [35]. Due to multifarious roles of sialic acids in immunity [1], sialic acid is an ideal points of investigation in coelomocytes of echinoids. Until recently, there has been no study on the determination and function of sialic acid and their types in immune system cells, particularly in the echinoderm coelomocytes.

Materials and methods

Sea urchins

Sea urchins of the species P. lividus were collected from the near-shore waters of the northern Izmir coastline, Foça, Turkey.

Coelomocyte collection for sialic acid analysis

Necessary quantities of the coelomic fluid with coelomocytes were immediately taken from the coelomic cavity without feeding sea urchins. As previously described [37], [38], the coelomic fluid from each animal was collected by removing Aristotle's lantern, or the mouth structure, and aliquoted into 3 ml volumes and then diluted with isosmotic anticoagulant solution (20 mM Tris, 0.5 M NaCl, 70 mM EDTA pH 7.5) (ISO-EDTA). After centrifugation (1000 × g for 10 min at 4 °C), coelomocytes were washed three times in ISO-EDTA and then fixed in methanol either at −20 °C or −80 °C.

Coelomocyte preparation for microscopy

Coelomocytes were suspended in coelomocyte culture (CCM: 0.5 M NaCl, 5 mM MgCl2, 1 mM EGTA, 20 mM HEPES, pH 7.4). Cell suspensions (100 μl) were settled onto glass coverslips. Cells in fresh preparations were viewed on a Leica DM 4000B microscope using a 63× NA 1.4 apochromatic objective lens coupled to a DP71 camera (Olympus).

Materials

All chemicals and solvents were of the highest grade commercially available (LC-MS grade). Standart sialic acids; Neu5Ac, Neu5Gc and KDN (A9646, G9793 and 60714) were purchased from Sigma (Sigma-Aldrich Chemie GmbH Riedstrasse 2 D-89555 STEINHEIM). DMB (1,2-diamino-4,5-methylenediaoxy-benzene dihydrochloride) was obtained from Sigma (D4784). Sialic acids were determined by labeling with DMB. Mucin (purchased from Sigma-M-3895 Bovine Submaxillary Gland) was used in the present study as a model for sialic acid compounds. Salivary mucin is a collection of proteins that contain large amounts of O-glycosidically linked carbohydrate chains. Mucin isolated from bovine submaxillary gland using as a standard compound contains most members of sialic acid family [4], [39]. The amino group of sialic acids in mucin is usually substituted with acetyl or glycolyl groups. Hydroxyl groups of sialic acids are also sometimes substituted with acetyl, lactyl and other groups [40].

Liberation of sialic acids from mucin glycoprotein and derivatization of standards

For the confirmation of molecular species of sialic acids, mucin (1 mg) was mixed with 20 μL 2 M aqueous acetic acid. The mixture (1 mg/20 μL) was kept at 80 °C for 3 h for releasing sialic acids. The solution (20 μL) containing sialic acids was mixed with 7 mM DMB, 0.75 M β-mercaptoethanol, and 8 mM sodium hydrosulfite in 1.4 M aqueous acetic acid. The mixture was incubated at 60 °C for 150 min in the dark. After cooling, a portion was analyzed using LC-ESI-MS/MS.

Standards; Neu5Ac, Neu5Gc and KDN (1.25 μg/mL) were directly derivatized with DMB and reaction mixture directly injected to LC-ESI-MS/MS system.

Liberation and derivatization of sialic acids from coelomocytes

Coelomocytes were homogenized by sonication (Bandelin-Sonorex) at 4 °C in 2 M aqueous acetic acid solution (1 mg of cells/5 μL). The solution was used for sialic acid analysis. Homogenizated 40 μL sample solutions (1 mg/5 μL) were kept for 3 h at 80 °C. After hydrolysis, the mixtures were derivatized with DMB in the same manner as above. The mixtures were centrifuged at 16,300 rpm for 10 min at 4 °C. Supernatant solution containing the derivatizated sialic acids was pipetted to HPLC vial insert (250 μL). The injection volumes of both samples were 0.5 μL for analyzing by CapLC-ESI-MS/MS with ion trap.

Capillary LC-ESI-MS/MS/Ion trap parameters

LC was performed using an Agilent 1200 Capillary HPLC system with an ODS capillary column (Agilent ZORBAX SB-C18 150 × 0.5 mm, 5 μm, USA) delivering 20 μL/min of the eluent. Elution was performed by isocratic mode using mixture of methanol–acetonitrile–water (7.5:5:87.5, v/v). The column was kept at 30 °C during the analysis. The samples were stored at 5 °C in temperature controlled autosampler board (Agilent G1377A). The column of LC system was connected to an electrospray ion source (ESI positive). All mass spectrometric measurements were performed on a HCT Ultra ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ionization (ESI) source. Spectrometric conditions such as the ion optics voltages, nebulizer gas and dry gas flow rates, and the dry gas temperature were controlled by EsquireControl software 6.1. Both the nebulizer gas and the dry gas were nitrogen, and helium (He) (99.9%) was used as the collision gas in the ion trap. The ion source parameters were set as follows: dry temperature 310 °C, nebulizer pressure 15.0 psi, and dry gas flow 5 l/min. MS/MS spectra were carried out by collision-induced dissociation (CID). All mass spectra were acquired in the mass range 200–600 m/z, with a scan speed of 26,000 m/z per second. Data analysis was carried out using Data Analysis software (v.3.4, Bruker Daltonics).

Results

To determine whether substituted sialic acids exists on the circulating coelomocytes, 2 M acetic acid treatment was used to release sialic acids from glycoconjugates without destroying the substitutions that may be present on the sialic acid molecule. Released sialic acids were then derivatized with 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB) and separated by CapLC-ESI-MS/MS and retention times-CID fragments were compared with standards: an acid hydrolysate of bovine submaxillary mucin (BSM) which is a rich source of sialic acids and O-acetylated sialic acids [4] and other standard sialic acids (Neu5Ac, Neu5Gc and KDN). Sialic acids derivatized with DMB (Fig. 1 ) in all samples were presented with dehydrated ion ([M + H-18]+ and pseudomolecular ion [M + H]+ by MS in the positive ion mode. The molecular mass of sialic acid derivatives, and thereby the number and type of the substituent is reflected by pseudomolecular ion [M + H]+. An important CID (collision-induced dissociation) fragment is the result of H2O elimination, [M + H-18]+. Other product ions obtained from CID using MRM (multiple reaction monitoring) analysis also provides useful information regarding the identity and location substituents of sialic acids, even without knowing the relative retention times of standard sialic acid derivatives [4]. But, this approach is limited in ion trap systems due to an inherently low mass cutoff (LMCO) derived from CID. The ion trap fails to trap the ions at lower end of the m/z range when using CID for tandem mass spectrometry (MS/MS) [41]. Therefore, [M + H-18]+ ions obtained from MRM of sialic acids are very useful for screening the chromatogram and the spectrum in MS/MS system [15].

Fig. 1

Schema of the reaction of 1,2-diamino-4,5-methylenediaoxy-benzene (DMB) derivatization of sialic acids.

Twelve different sialic acids were detected in BSM by DMB derivatization-CapLC-ESI-MS/MS (data not shown). The peaks for Neu5Ac, Neu5Gc, Neu5,9Ac2 along with additional sialic acid peaks were assigned by comparison of the elution times and CID fragments ([M + H-18]+ ions and others (m/z)) as reported by Refs. [4], [15] using bovine submaxillary mucin and other sialic acid-containing glycoconjugates.

We found the presence of ten different types of sialic acids (Neu5Gc, Neu5Ac, Neu5Gc7Ac, Neu5Gc8Ac, Neu5,7Ac2, Neu5Gc9Ac, Neu5,8Ac2, Neu5,9Ac2, Neu5Gc7,9Ac2, Neu5,7,9Ac3) isolated in limited amounts of total coelomocyte population (Fig. 2 ) by comparison with the data obtained for the analysis of sialic acids of BSM (data not shown) and standard sialic acids (KDN, Neu5Gc and Neu5Ac) (Fig. 3 ). The peaks were identified by extracted ion chromatogram. The highly abundant fragment ions were used for quantification and others used for confirmation of sialic acid derivatives. All DMB derivatives of sialic acids in our samples show clear molecular ions at [M + H]+ and [M + H-18]+. The fragment pattern of the MS/MS spectrum of sialic acids in total coelomocyte population (see Table 1 ) was consistent with that of DMB derivatives of sialic acids in mucin and the previous reports [4], [20].

Fig. 2

LC-ESI-MS/MS mass spectrum and chromatogram of DMB-Sialic acid species (ten types) found in total coelomocyte population. Monitored of molecular ions [M + H]+ as labeled (♦) by software, and CID (collision-induced dissociation) fragments [M + H-H2O]+ at m/z; 424 for Neu5Gc, 408 for Neu5Ac, 466 for Neu5Gc7Ac, Neu5Gc8Ac and Neu5Gc9Ac, 450 for Neu5,7Ac2, Neu5,8Ac2 and Neu5,9Ac2, 492 for Neu5,7,9Ac3, 508 for Neu5Gc7,9Ac2.

Fig. 3

LC-ESI-MS/MS mass spectrum and chromatogram of DMB-Standard Sialic acid species (KDN, Neu5Gc and Neu5Ac). Monitored of molecular ions [M + H]+ as labeled (♦) by software, and CID (collision-induced dissociation) fragments [M + H-H2O]+ at m/z.

Table 1

Characteristic ions [M + H]+ and CID (collision-induced dissociation) fragments of DMB-Sialic acid derivatives obtained from coelomocytes, generated and analyzed by LC-ESI-MS/MS. (RT, retention time).

Sialic acids	RT	[M + H]+	CID fragments
			[M + H-H2O]+	Fragments (m/z)
Neu5Gc	7.3	442	424	313-295-283-229
Neu5Gc7Ac	9.9	484	466	313-295-229
Neu5Ac	10.0	426	408	313-295-283-229
Neu5Gc8Ac	10.8	484	466	313-295-229
Neu5,7Ac2	11.9	468	450	313-295-283-229
Neu5Gc9Ac	13.4	484	466	313-295-229
Neu5,8Ac2	15.2	468	450	313-295-229
Neu5,9Ac2	19.7	468	450	313-295-229
Neu5Gc7,9Ac2	24.2	526	508	313-295-229
Neu5,7,9Ac3	28.0	510	492	313-295-229

The most abundant sialic acids were determined as Neu5Gc in coelomocytes. Mass spectral data revealed that the peaks of Neu5Ac and O-acetylated sialic acids were lower mass than Neu5Gc. Di-O-acetyl derivatives of Neu5Gc (Neu5Gc7,9Ac2) and Neu5Ac (Neu5,7,9Ac3) were observed respectively at m/z = 526 [M + H]+, m/z = 508 [M + H-18]+ and m/z = 510 [M + H]+, m/z = 492 [M + H-18]+ in the mass chromatogram on total coelomocyte population. 7, 8, 9-O-acetylated of Neu5Ac was obtained at m/z = 468 [M + H]+, m/z = 450 [M + H-18]+. Also, Neu5Gc type of sialic acids and its acetylated compounds (Neu5Gc7Ac, Neu5Gc8Ac and Neu5Gc9Ac) were identified at m/z = 484 [M + H]+, m/z = 466 [M + H-18]+. The distributions of sialic acids in total coelomocyte population were shown Fig. 4 . KDN is a structurally related α-keto acid that is also found in glycoconjugates [13]. However, under the conditions used in this study, KDN was not found both coelomocytes and BSM. Hence, Neu5Gc was established as the predominant sialic acid on circulating coelomocytes of P. lividus.

Fig. 4

Distribution of sialic acids amounts measured by LC-ESI-MS/MS. Sialic acid contents of total coelomocyte population were demonstrated by average peak area of MRM chromatograms of their types. Data are presented as mean ± standard deviation (S.D.) of peak area from three replicated experiments.

Discussion

Although small amounts of sialic acid have been reported in insects [21], [22], [23] and a gastropod [24], they are not found generally in glycoconjugates of protostome animals [42]. However, within the deuterostomate lineage of the animal kingdom, both the echinoderms and vertebrates possess significant amounts of sialylated glycoconjugates and frequently display a considerable number of sialic acid types. The structural variety among the sialic acids results from a number of enzyme-catalyzed modifications of N-acetylneuraminic acid occurring at different stages of glycoconjugate biosynthesis [43]. N-Glycolylneuraminic acid (Neu5Gc) is one of the most common sialic acids and formally results from the hydroxylation of the methyl group of the 5-N-acetyl moiety of Neu5Ac. Interestingly, no Neu5Gc has been found in human and chicken tissues, although small amounts have been detected in tumors [9]. In the marine deuterostomes, including sea urchins and starfish, Neu5Gc is often main and predominant sialic acid and is widely distributed in glycoproteins and gangliosides of especially oocyte and sperm cells, suggesting that it is involved in a number of important biological functions in these marine invertebrates [9], [25], [44], [45]. In immune cells of sea urchin, P. lividus, Neu5Gc was the most abundant type sialic acid when compared with other found sialic acid types. Cell surface glycoconjugates which include Neu5Gc may help to protect tissues from attack by certain pathogen-derived toxic enzymes. In order to aid the digestion of the cell surface glycoconjugates, several microorganisms secrete sialidase, which may facilitate bacterial infection. Since the cleavage rate of Neu5Gc by bacterial and viral sialidases is generally lower than that of Neu5Ac, the expression of Neu5Gc may be an adaptation to retard the effects of sialidases [46]. Neu5Ac is the most common ligand for virus binding but Neu5Gc and O-acetylated sialic acids were also found to be specific ligands, for example for influenza C and corona viruses [16]. The presence of Neu5Gc and its derivatives with O-acetyl groups in immune system cells of sea urchin may be related to their habitat, providing a first line of defense against sialidase-containing microorganisms. According to the results, our data may provide an explanation of why 9-O-acetylated Neu5Gc (Neu5Gc9Ac) was found more than other O-acetylated sialic acids in immune cells of P. lividus (Fig. 4).

Naturally occurring sialic acids can be O-acetylated at all their hydroxyl groups at positions C4, C7, C8 and C9 of these carboxylated sugars [47]. One sialic acid molecule may carry one or several, up to there, O-acetyl groups but they mostly are mono-O-acetylated derivatives. Biological effect depends on the position relative to the 9-carbon scaffold and whether it is the Neu5Ac or Neu5Gc [2], [47]. O-acetylated sialic acids seem to be involved in many biological phenomena [2]. Given variable expression of O-acetyl groups on sialic acids and their diverse effects in all immune reactions [1]. O-acetylation of sialic acids can be developmentally regulated in immune systems or create new biological functions [3], [6]. They therefore revealed to be important in cell differentiation, neural development, non-immunogenic protection and immuno reactivity [47]. However, high levels of 9-O-acetylated sialic acids were detected on developing and peripheral resting and activated T-cells [48]. As another example, side chain O-acetylated sialic acids are also expressed human leukocytes, where they can be used as markers for specific subsets of T-helper cell. For several subsets of T-lymphocytes O-acetylation of sialic acids is known to be a differentiation and maturation marker [47], [48]. This may be one of reasons why immune cells, circulating coelomocytes, contain such a wide variety of O-acetylated sialic acids (Neu5Gc9Ac, Neu5Gc8Ac, Neu5,9Ac2, Neu5,7Ac2, Neu5,8Ac2, Neu5,7,9Ac3, Neu5Gc7,9Ac2, Neu5Gc7Ac). And also, O-acetylation of sialic acids may be a novel marker for finding subsets of coelomocytes like T-cells.

In conclusion, the investigation of the immune system of echinoderms should help in understanding the cellular and humoral interactions that control host defense in this group of marine animals and mammals. Our results are also important for a phylogenetic point of view because they may contribute to the understanding of the immune phenomena of higher animals, including mammals. Moreover, they may lead to possible analogies or homologies with immune reactions in vertebrates.