Structural Analysis and Anti-Complement Activity of Polysaccharides from Kjellmaniella crsaaifolia

Two polysaccharides, named KCA and KCW, were extracted from Kjellmaniella crassifolia using dilute hydrochloric acid and water, respectively. Composition analysis showed that these polysaccharides predominantly consisted of fucose, with galactose, mannose and glucuronic acid as minor components. After degradation and partial desulfation, electrospray ionization mass spectrometry (ESI-MS) was performed, which showed that the polysaccharides consisted of sulfated fucooligosaccharides, sulfated galactofucooligosaccharides and methyl glycosides of mono-sulfated/multi-sulfated fucooligosaccharides. The structures of the oligomeric fragments were further characterized by electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS2 and ESI-CID-MS3). Moreover, the activity of KCA and KCW against the hemolytic activity of both the classical and alternative complement pathways was determined. The activity of KCA was found to be similar to KCW, suggesting that the method of extraction did not influence the activity. In addition, the degraded polysaccharides (DKCA and DKCW) displayed lower activity levels than the crude polysaccharides (KCA and KCW), indicating that molecular weight had an effect on activity. Moreover, the desulfated fractions (ds-DKCA and ds-DKCW) showed less or no activity, which confirmed that sulfate was important for activity. In conclusion, polysaccharides from K. crassifolia may be good candidates for the treatment of diseases involving the complement pathway.

degraded slightly during the extraction process. Only trace contents of UA and protein were found. Interestingly, the molecular weight of DKCA was higher than DKCW, indicating that KCW was more sensitive to degradation. Desulfation resulted in a substantial decrease in the molecular weights for both ds-DKCW and ds-DKCA, indicating that the parent compounds were likely to be highly sulfated. In addition, the ratios of other monosaccharides, such as rhamnose (Rha), glucose (Glc) and xylose (Xyl), were increased.

IR Analysis
The IR spectra ( Figure 1) showed that KCA, DKCA, KCW and DKCW had the same infrared absorption properties, suggesting that they contained the same functional groups. The band at 1260 cm −1 corresponded to the S=O stretching vibration, and the band at approximately 845 cm −1 was assigned to the C-O-S vibration, suggesting that the presence of the sulfate group was mainly at the C-4 axial position on fucose [15][16][17]. Thus, it was concluded that KCW and KCA were mainly sulfated at C-4 on fucose. After desulfation, the intense band at 1260 cm −1 vanished, indicating that ds-DKCW and ds-DKCA lost a high amount of sulfate during the process of desulfation, which was confirmed by the results in Table 1.

MS Analysis of Structure
MS is an important tool for the analysis of heteropolysaccharides because of its speed and sensitivity. Though there have been many studies on the structural features of heteropolysaccharides [5,6,[18][19][20][21][22][23], it was previously not possible to analyze heteropolysaccharides that contained large, highly charged molecules. Thus, heteropolysaccharides needed to be degraded. The ESI-MS spectrum of ds-DKCW determined in the present study was shown in Figure 2a The ESI-MS spectrum of ds-DKCA is shown in Figure 2b. The fragment ions were similar to those of ds-DKCW. However, ds-DKCA mainly consisted of methyl glycosides of mono-sulfated fucooligosaccharides and had few multi-sulfated fucooligosaccharides or sulfated galactofucooligosaccharides.
To confirm the above hypothesis, the fragmentation pattern for the singly-charged ion at m/z 681.208 was characterized by ESI-CID-MS 3 and was found to correspond to the ion [Fuc4SO3Na-Na] − , as shown in Figure 4b. One set of fragment ions at 225.014, 371.075 and 517.136 assigned as B-type ions arose from the glycosidic bond cleavage from the reducing end, suggesting that the sulfate was located at the non-reducing end. Anastyuk et al. and Saad and Leary [18,25] reported that, when an oligosaccharide's sulfate group was close to the glycosidic linkage spatially, it would undergo easier B-type fragmentation, which indicated that the sulfate group was substituted at C-2. Another set of less intense fragment ions, 389.086 and 535.147, corresponded to Y-type ions, suggesting the sulfate was substituted at the reducing end. In addition, the characteristic ion at m/z 607.170 was a 0,3 X3-type ion, suggesting that the linkage was a 1→3 linkage. No 0,2 A-type ions were detected, which confirmed that the linkage between Fuc and Fuc was 3-linked. In sum, Fuc 4 SO 3 Na consisted primarily of Fuc(2SO3Na)→Fuc→Fuc→Fuc, and, to a lesser degree, of Fuc→Fuc→Fuc→Fuc(2 or 4SO3Na).
The fragmentation pattern for the doubly charged ion at m/z 383.103 (−2) was also elucidated by ESI-CID-MS 3 in Figure 4c. The characteristic ion at m/z 361.090 (−2) ( 2,4 A5) arose from the loss of C2H4O (44 Da) from the ion at m/z 383.103 (−2) ( 2,5 A5), suggesting that the reducing end was a Gal residue. In addition, it also indicated that the linkage between Fuc and Gal was a 1→4 linkage. Moreover, a set of fragment ions at m/z 243.014, 389.086 and 535.147 were determined to be C-type ions, suggesting that the sulfate was located at the non-reducing end. This glycosidic cleavage observation was not consistent with the previous study [18,25], indicating that the sulfate was at C-4. Therefore, it was concluded that Gal(Fuc)4SO3Na was Fuc(4SO3Na)→Fuc→Fuc→Fuc→Gal.

Anti-Complement Activity
As shown in Figure 7a-d, the effects of the polysaccharides on activation of human complement through the classical pathway (Figure 7a,b) and the alternative pathway (Figure 7c,d) were examined in 1:10-diluted NHS, with heparin used as a reference. The complement group (i.e., positive control) displayed a 93.11% ± 2.96% activation of the classical complement pathway. The activities of KCA, KCW, DKCA, DKCW and heparin were dose-dependent, while ds-DKCA and ds-DKCW showed little or no activity (Figure 7a,b). The activities of KCA and KCW reached a plateau at a concentration of 10 μg/mL, while DKCA plateaued at 50 μg/mL. In addition, the concentration that resulted in 50% inhibition of the classical complement pathway (CH50) for DKCW was approximately 218 μg/mL, which was lower than heparin. Therefore, KCA, KCW, and DKCA were more potent than heparin in inhibiting activation of the classical pathway. On the other hand, the concentrations of KCA, KCW, DKCA, DKCW and heparin that resulted in 50% inhibition of the alternative pathway (AP50) were 4.83, 18.60, 24.50, 19.97 and 137.25 μg/mL, respectively (Figure 7c,d). This finding indicated that KCA, KCW, DKCA and DKCW were more potent than heparin in inhibiting activation of the alternative pathway.
KCA and KCW displayed similar activity levels against the two complement pathways, indicating that the extraction methods did not affect the activity levels. The degraded polysaccharides DKCA and DKCW exhibited weaker activity compared to the crude polysaccharides KCA and KCW, which suggested, as others have reported, that the change in molecular weight influenced the anti-complement activity of the two compounds [9,26]. In addition, the finding that ds-DKCA and ds-DKCW showed little or no activity against the two pathways suggested that sulfate was important for anti-complement activity, which was in agreement with other reports [13,27,28].
In sum, the polysaccharides from Kjellmaniella crassifolia may be potent drugs that are capable of suppressing complement activation.

Preparation of Polysaccharides
K. crassifolia was collected in Rongcheng, Shandong Province, China, in June of 2013. The polysaccharides were extracted from K. crassifolia as previously described [29]. Briefly, the dried algae were cut into pieces, and the polysaccharides were extracted three times in water at room temperature for 2 h. The solution was dialyzed against water and distilled water. Finally, the polysaccharide was concentrated and precipitated with ethanol. The resultant precipitate was named KCW. In an alternative extraction, the dried algae were cut and extracted with 0.1 M HCl at room temperature for 2 h. The solution was neutralized, concentrated, dialyzed and precipitated with ethanol. This precipitate was named KCA.

Preparation of Low Molecular Weight Fucoidans and Their Desulfated Mixtures
The crude polysaccharides KCA and KCW were degraded using hydrogen dioxide and ascorbic acid to obtain low molecular weight polysaccharides, as previously described [21]. Briefly, crude polysaccharide (1 g) was dissolved in water (100 mL). Ascorbic acid (0.5 g) and hydrogen dioxide (0.3 mL) were then added, and the solution was stirred for 2 h at room temperature. The degraded polysaccharides (e.g., DKCA and DKCW) were obtained after ultrafiltration, concentration and lyophilization.
The desulfation of DKCA and DKCW was performed according to the modified method of Nagasawa et al. [30]. Desulfation was carried out using its pyridinium salt. Briefly, the sample was dissolved in distilled water (10 mL) and mixed with cationic resin for 3 h. After filtration, the solution was neutralized with pyridinium and lyophilized. The polysaccharide was then dissolved in 20 mL of a 9:1 ratio of dimethyl sulfoxide: methanol (v:v) at 80 °C for 5 h. The desulfated solution was dialyzed and lyophilized to give desulfated products (e.g., ds-DKCA and ds-DKCW).

Composition Methods
Total sugar and fucose content were determined according to the method of Dubois et al. [31] and Gibbons [32], using fucose as a standard. The level of sulfation was analyzed by the barium chloride-gelatin method of Kawai et al. [33]. The uronic acid (UA) content was estimated with a modified carbazole method using D-glucuronic acid as a standard [34]. The protein level was determined according to the method of Bradford et al. [35]. For the determination of sugar composition, the acid-hydrolyzed glycoses were converted into their 1-phenyl-3-methyl-5-pyrazolone derivatives (PMP) and separated by HPLC chromatography [36]. The molecular weight of the samples was assayed by a HP-GPC system on a TSK gel PWxl 3000 column (7 μm, 7.8 × 300 mm) eluted with 0.2 M Na2SO4 at a flow rate of 0.5 mL min −1 at 30 °C [37].

Spectroscopic Analysis
Infrared spectra (IR) were recorded from polysaccharide powder in KBr pellets on a Nicolet-360 FTIR spectrometer between 400 and 4000 cm −1 (36 scans, at a resolution of 6 cm −1 ).
MS was performed on a LTQ ORBITRAR XL (Thermo Scientific, Waltham, MA, USA). Samples, dissolved in CH3CN-H2O (1:1, v:v), were introduced into the MS at a flow rate of 5 μL min −1 in the negative ionization mode. The capillary voltage was set to −3000 V, the cone voltage was set to −50 V, the source temperature was set to 80 °C, and the desolvation temperature was set to 150 °C. The collision energy was optimized between 20 and 50 eV. All spectra were analyzed by Xcalibur (Thermo Scientific, Waltham, MA, USA).
To assay sample inhibition of the alternative complement pathway, 150 μL of various dilutions of tested samples were mixed with 150 μL of 1:10-diluted NHS and 200 μL of rabbit erythrocytes (ER). The mixture was then incubated for 30 min at 37 °C. Cell lysis was determined by the same method as described above for the classical pathway. Controls for 100% lysis, sample control, complement and blank were included. The percent inhibition was calculated using the following equation: inhibition of ER lysis (%) = (Acomplement − [Asample − Asample control])/Acomplement × 100.

Conclusions
In this study, two polysaccharides, KCW and KCA, were extracted from K. crassifolia using water and dilute hydrochloric acid, respectively, and their degraded fractions and desulfated fractions were prepared. The chemical compositions indicated that KCA had higher contents of sulfate and Fuc and less of other monosaccharides than KCW. To elucidate the structural features of KCA and KCW, they were degraded and partly desulfated. The desulfated mixtures were determined by ESI-MS. Both were found to contain sulfated fucooligosaccharides, sulfated galactofucooligosaccharides and methyl glycosides of mono-sulfated/multi-sulfated fucooligosaccharides. The major difference between ds-DKCA and ds-DKCW was the intensity of the fragment ions. In addition, the structural features of oligomeric fragments were characterized by ESI-CID-MS 2 and ESI-CID-MS 3 . It was shown that the polysaccharides had a backbone of 3-linked Fuc residues sulfated at C-2, C-4 or C-2 and C-4. Some oligomers had the 4-linked Gal residue at the reducing terminus. Some fucooligomers were interspersed by Gal residues. Moreover, activities of the polysaccharides against the classical and alternative complement pathways were measured. The crude polysaccharides KCA and KCW had the highest activity levels, while the desulfated fractions ds-DKCA and ds-DKCW had little or no activity. These data suggested that the change in molecular weight and sulfate content influenced the activity levels. In summary, polysaccharides from K. crassifolia may be a good candidate drug for anti-complement therapy.