Biochemical studies on sphingolipids of Artemia franciscana: complex neutral glycosphingolipids

Brine shrimp are primitive crustacean arthropodal model organisms, second to daphnia, which can survive in high-salinity environments. Their oviposited cysts, cuticle-covered diapausing eggs, are highly resistant to dryness. To elucidate specialties of brine shrimp, this study characterized glycosphingolipids, which are signal transduction-associated material. A group of novel and complex fucosyl glycosphingolipids were separated and identified from cysts of the brine shrimp Artemia franciscana by repeated lipid extraction, alkaline methanolysis, acid treatment, successive column chromatography, and post-source decay measurements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Structures of the glycosphingolipids were elucidated by conventional structural characterization and mass spectrometry, and the compounds were identified as GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer. These compounds also contained a branching, non-arthro-series disaccharide with an α-GlcNAc terminus, similar to that found in a previously reported ceramide hexasaccharide (III3(GlcNAcα2Fucα)-At4Cer). The glycans within these complex GSLs are longer than reported glycans of the animal kingdom containing α-GlcNAc terminus. These complex GSLs as well as the longest GSL with ten sugar residues, ceramide decasaccharide (CDeS), contain the fucosylated LacdiNAc sequence reported to associate with parasitism/immunosuppression and the α-GlcNAc terminus reported to show a certain antibacterial effect in other reports. CDeS, the longest GSL of this species, was found in the highest amount, which indicates that CDeS may be functionally important.


Introduction
Glycosphingolipid (GSL) is an amphipathic compound consisting of a sugar chain and a ceramide composed of a fatty acid and sphingoid base. GSLs are ubiquitous on the outer surface of the plasma membrane in animal cells, aggregated into patches called microdomains, and play an essential role in intercellular interaction and recognition [1][2][3]. However, a comprehensive understanding of GSL function has not yet been attained because of the structural complexity of the sugar chain and ceramide moiety [4,5].
The study of invertebrate GSLs increases our understanding of human GSLs, helps determine which types of sphingolipids are essential for living animals, and clarifies evolutionary relationships between phyla in the animal kingdom. In the phylum Arthropoda, structural analyses of GSLs have been performed with flies [6,7], and a characteristic arthro-series sugar chain (GlcNAcβ3Manβ4GlcCer; At 3 Cer) has been characterized. Functional analyses of GSLs by knockout experiments of egghead and brainiac have shown that At 3 Cer is essential for insect development [8]. Furthermore, At 3 Cer was detected in flies, including Lucilia caesar and Calliphora vicina, as well as in arthropods (the millipede Parafontaria laminata armigera) and crustaceans (Euphausia superba, Macrobrachium nipponense) [5]. We have further demonstrated the existence of At 3 Cer and some shorter GSLs, including GlcCer (CMS), mactosylceramide (MacCer, CDS), II 3 Fucα-MacCer (nonarthro-CTS), At 4 Cer (CTeS), II 3 (Glc-NAcα2Fucα)-MacCer (nonarthro-CTeS), III 3 Fucα-At 4 Cer (CPS), and III 3 (GlcNAcα2Fucα)-At 4 Cer (CHS), in cysts of the brine shrimp Artemia franciscana [9]. However, it is more difficult to identify structural aspects of the sugar chain in complex GSLs than it is in shorter GSLs. Identifying the sugar chain in certain complex GSLs, which may be mixed with GSLs bound to similar oligosaccharides, depends on faint differences in polarity to determine long sugar sequences and the positions of substituted sugar residues at branching points. Several relatively complicated strategies have been employed to identify the structures of complex neutral GSLs, such as successive enzymatic degradation with exoglycosidase, chemical degradation to liberate partial oligosaccharides followed by Gas-liquid chromatography (GC) analysis, and fast atom bombardment (FAB) mass analysis of permethylated GSL to elucidate sugar sequences. In fact, there are relatively few reports that detail the structural analyses of complex GSLs in invertebrates. Complex GSLs have been identified in the green-bottle fly L. caesar and the blowfly C. vicina [10,11], the human blood fluke Schistosoma mansoni [12][13][14], the bivalves Corbicula sandai [15] and Hyriopsis schlegeli [16], the seawater bivalve Meretrix lusoria [17], and the oyster Ostrea gigas [5].
The current study uses post-source decay (PSD) measurements coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the structures of complex GSLs in diapausing eggs (cysts) of the brine shrimp A. franciscana, a crustacean arthropod harvested from the Great Salt Lake. These complex GSLs contain seven to ten sugar chains and exhibit a hybrid structure of core arthro-series sugar chains with a branching non-arthroseries disaccharide (GlcNAcα2Fucα).
Gas chromatography GC analyses of GSL components, such as sugars, fatty acids, and sphingoids, and methylation procedures were performed as described previously [9]. For the GC analysis, the following amount of GSL was used: 0.2 mg for sugar component analysis, fatty acid analysis, and methylation study, and 0.3 mg for sphingoid analysis.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) MALDI-TOF MS analysis was performed using a Shimadzu Axima Confidence MALDI Mass Spectrometer with a nitrogen laser (337 nm). The matrix α-cyano-4-hydroxycinnamic acid (CHCA: high-purity mass-spectrometric grade) was purchased from Shimadzu GLC (Tokyo, Japan). About 4 μg of GSL was loaded on to a MALDI plate and air-dried. Subsequently, 1 μL of CHCA dissolved in 50 % ethanol at a saturating concentration was loaded 4 times on the dried GSL. External mass calibration was provided by the [M+Na] + ions of angiotensin I (1296.96 mass units; Sigma-Aldrich Co., USA) and bradykinin fragments I-V (573.31 mass units; Sigma-Aldrich Co., USA).

Results
Purified neutral glycosphingolipids Figure 1 shows a developed TLC plate with separated GSLs. The GSLs with less than six sugar residues, corresponding to lanes 2-9 in Fig. 1a, have been previously reported [9]. The yields of purified GSLs obtained from 1.8 kg of brine shrimp  (Fig. 1b). Crude COS 2 (1.4 mg) and CNS (∼1 mg) were separated with an eluent of ammoniacal propanol. However, it was revealed in subsequent MALDI-TOF MS spectra that both COS 2 and CNS preparations contained silicic acid. Further purification using a Sep-pak C18 cartridge was performed and the resulting lipids were again analyzed by MALDI-TOF MS. The non-GSL fraction was analyzed on the basis of the mass spectrum.

Aliphatic components
Aliphatic components such as fatty acids and sphingoids were identified by GC ( Table 2). The majority of the fatty acids were saturated with chain lengths from C 16 to C 24 , with C 22 being the most predominant. The monoenoic acid of C 22 was common in all of the GSLs. In some GSLs, the odd-numbered saturated fatty acids C 21 and C 23 were also detected in low amounts. The sphingoid components of the GSLs were composed of d16:1 and d17:1. In each case, the amount of d16:1 was approximately double that of d17:1.

MALDI-TOF MS analysis
The positive in-source decay mode of MALDI-TOF MS confirmed the putative structures of the six purified GSLs (Fig. 3). Each GSL mass spectrum contained peaks attributed to two major monoisotopic [M+Na] + ion species, the ceramide moieties of d16:    Fig. 1d).

Discussion
Structural characterization of complex GSLs can be a painstaking process because the results obtained by different analyses must converge on a single structure. For example, characterization by liquid chromatography-mass spectrometry (LC-MS) and tandem MS (MS-MS) provides accurate mass values of ceramide moieties, fatty acids, and sphingoids, but not of sugar species. It is also possible that liquid chromatographic separation would not be able to fully separate GSLs with different sugar chains of identical molecular weight. The conventional analyses, which consist of GC and MS, can be used to determine sugar species and molar ratios without overlooking the possibility of GSLs with different sugar chains of identical molecular weight. Another benefit of the conventional analysis is the ability to determine branching structures by  Black arrows indicate mass differences between fragments with a ceramide molecular group. Gray arrows indicate mass differences between fragments without a ceramide molecular group. All fragments were detected as sodium adducts. The spectrum of b was expanded by a factor of 5 in the low-molecular-weight region hydrolysis. No matter the analysis, it is difficult to characterize complex GSLs because each result is also complex. A few decades ago, characterization of a complex GSL required 10 mg of isolated GLS for chemical degradation, which liberated shorter GSLs, followed by GC analysis to determine sugar sequence and exoglycosidase treatment or chromic acid oxidation to determine anomeric configurations. In this study, we conducted MALDI PSD analysis to determine sugar sequence and 1 H-NMR spectroscopy to identify anomeric configurations by using only 1-mg samples of each complex GSL separated from A. franciscana. Although PSD spectra of complex GSLs were confusing, sugar sequence analysis using sequences speculated from fragments, with the ceramide moiety observed in the high mass region, were consistent with sequences speculated from mere glycan fragments observed in the low mass region.
This study examined the structures of complex GSLs separated from the brine shrimp A. franciscana. Of them, a fucosylated LacdiNAc trisaccharide structure (GalNAcβ1-4[Fucα1-3]GlcNAcβ) was found in CHpS 2 , COS 2 , CNS, and CDeS, similar to a previous report on CHS [9]. The trisaccharide structure is an analog of Lewis X and is also found in GSLs from the parasite S. mansoni [25] and in the carbohydrate portion of a human immunosuppressive glycoprotein, glycodelin [26].
The arthro-series core structure and a branching nonarthro-series disaccharide were also found in CHpS 2 , CNS, and CDeS, as described in previous reports on nAtCTeS and CHS. The α-anomeric GlcNAc residue is rare, and there have been few reports of this residue among the carbohydrate portions of GSLs and glycoproteins. A human gastric mucin with an α-anomeric GlcNAc residue could arrest the proliferation of Helicobacter pylori [27].
We speculate that CHpS 2 is elongated with an additional Fucα group, forming COS, and that COS is further elongated with branching GalNAcβ to form CNS. Although we could not separate COS as a direct precursor of CNS, we believe it exists in the biosynthetic pathway (Fig. 6). CNS is then finally elongated with an additional GlcNAcα to form CDeS. However, another branching residue on CHpS 1 and COS 1 indicates other biosynthetic pathways. A. franciscana GSLs with α-anomeric GlcNAc at the nonreducing end do not seem to be elongated with further saccharides, which suggests the action of a "capping" mechanism. This theory is further supported by the observation that all of the αanomeric GlcNAc residues were reported at the nonreducing end [9, 22-24, 28, 29]. Capping by an amino sugar in A. franciscana GSLs may be similar to the capping action of the 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) residue in the rainbow trout Salmo gairdneri [30].
A series of structural analyses, including this study, has established the existence of sphingomyelin as a sphingophospholipid and GlcCer, MacCer, At 3 Cer, II 3 Fucα-MacCer (nonarthro-CTS), At 4 Cer, II 3 (GlcNAcα2Fucα)-MacCer (nonarthro-CTeS), III 3 Fucα-At 4 Cer (CPS), III 3 (GlcNAcα2-Fucα)-At 4 Cer (CHS), CHpS 1 , CHpS 2 , COS 1 , COS 2 , CNS, and CDeS as GSLs of A. franciscana. The complex GSLs reported this time are as the novel fucomannolipids. However, we confirmed that gangliosides and their functional alternative materials (other acidic GSLs) were below the limit of detection. In mammals, acidic GSLs such as gangliosides play important roles in the formation of the plasma membrane surface environment for signal transduction. It is interesting that A. franciscana can survive without significant amounts of acidic GSLs.
Performing structural analyses of complex GSLs in A. franciscana cysts, which are developmentally diapaused gastrulae, we established a GSL profile during embryonic development. Similar GSL analyses have been carried out in other species, including the frog Xenopus laevis [31], the chicken Gallus gallus domesticus [32], and the mouse Mus musculus [33]. However, all of these studies were conducted during the neurula stage or later, emphasizing the importance of timing in our study. A. franciscana is a very suitable organism for studying GSL profiles during early embryogenesis, as they require 5 days from fertilization to the gastrula stage.
In the brine shrimp A. franciscana, there are two reproduction patterns: oviparity, which generates diapausing eggs (cysts), and ovoviviparity, which generates nauplii. Oviparous eggs are resistant to dryness due to the existence of the heat shock protein p26 [34], which seems to be induced by environmental factors than by temperature. Nambu et al. [35] conducted experiments showing that certain conditions of light/dark cycles and temperature could affect the reproduction pattern of brine shrimp. GSLs have been shown to be involved in signaling, and signal transduction is, at least in part, responsible for the choice of oviparity or ovoviviparity. Therefore, this study, which provides a comprehensive analysis of GSLs in embryonic A. franciscana, might be of major significance in studies of embryogenesis and signal transduction.