Branched Chondroitin Sulfate Oligosaccharides Derived from the Sea Cucumber Acaudina molpadioides Stimulate Neurite Outgrowth

Fucosylated chondroitin sulfate (FCS) from the sea cucumber Acaudina molpadioides (FCSAm) is the first one that was reported to be branched by disaccharide GalNAc-(α1,2)-Fuc3S4S (15%) and sulfated Fuc (85%). Here, four size-homogenous fractions, and seven oligosaccharides, were separated from its β-eliminative depolymerized products. Detailed NMR spectroscopic and MS analyses revealed the oligomers as hexa-, hepta-, octa-, and nonasaccharide, which further confirmed the precise structure of native FCSAm: it was composed of the CS-E-like backbone with a full content of sulfation at O-4 and O-6 of GalNAc in the disaccharide repeating unit, and the branches consisting of sulfated fucose (Fuc4S and Fuc2S4S) and heterodisaccharide [GalNAc-(α1,2)-Fuc3S4S]. Pharmacologically, FCSAm and its depolymerized derivatives, including fractions and oligosaccharides, showed potent neurite outgrowth-promoting activity in a chain length-dependent manner. A comparison of analyses among oligosaccharides revealed that the sulfate pattern of the Fuc branches, instead of the heterodisaccharide, could affect the promotion intensity. Fuc2S4S and the saccharide length endowed the neurite outgrowth stimulation activity most.


Introduction
Neurons are highly differentiated terminal cells that cannot regenerate. If compounds can promote neuronal growth, it will be meaningful for treatment of neuronal damage and degeneration. Chondroitin sulfate (CS) is a class of glycosaminoglycans and is involved in cell division and neuronal development. CS proteoglycans (CS-PGs) were defined as inhibitory molecules of neuron growth in early experiments, and the use of chondroitinase ABC could attenuate the inhibitory effect on neuron growth [1][2][3]. However, some researches have reported that CS could promote neuronal growth [4]. According to the sulfation sites on the saccharide chain, CS could be classified as type A, B, C, D, E, et al. ( Figure 1A). CS-A, CS-B, and CS-C were considered to inhibit neuron growth, while CS-D and CS-E exhibited neurite outgrowth-promoting activity. Some reports also proved that CS-E strongly inhibited nerve regeneration [5][6][7][8][9][10]. It has been known that commercially available CS retains heterogeneity, including CS-A (50-80% of A-type unit), CS-C (50-70% of C-type unit), CS-D (20-40% of D-type unit) and CS-E (63.6% of E-type unit). The low purity of saccharides may contribute to the contradictory results [11]. In regard to the CS analogs from invertebrates, the keratan sulfate disaccharidebranched CS-E from a clam, Mactra chinensis, stimulated the neurite outgrowth of hippocampal neurons [11]. A fucosylated CS from the sea cucumber Apostichopus japonicus exhibited neurite outgrowth-promoting activity [12].  [14][15][16][17][18][19]. Structural differences of FCS from different sea cucumber species are embodied in the size, the composition, the sulfate substitution of branches ( Figure 1B). Besides structure, another aspect of FCS that appeals to researchers is that FCS has wide-ranging biological activities, such as anti-coagulant, anti-thrombotic, anti-virus, and anti-cancer effects [13,[20][21][22]. Previous research proposed that the FCS from the sea cucumber A. japonicus exhibited neurite outgrowth-promoting activity, and recognized the potential roles of the fucose branch in neurite outgrowth. A chemically synthesized FCS trisaccharide that is composed of a CS-E disaccharide unit with a sulfated fucose branch also exhibited comparable stimulation activity [12]. The effects of the FCS originated from other sea cucumbers on neurite's growth, and the structure-function relationships remain unresolved and deserve detailed investigations.
FCSAm was obtained from the sea cucumber Acaudina molpadioides in our previous work [23]. Oligosaccharides from the β-eliminative depolymerized FCSAm revealed that its backbone has a full content of type E, which was branched by monosaccharide [Fuc2S4S and Fuc4S], and the disaccharide [GalNAc-(α1,2)-Fuc3S4S] on O-3 of each GlcA. This is the first report of such a disaccharide branch in natural FCS. FCSAm showed the variety of branches with sulfated substitutions, as well as the novel disaccharide.
In this work, further investigation on this unique CS analog (FCSAm) was conducted. From its β-eliminative depolymerized products, the size-homogenous fractions with a higher degree of polymerization were obtained. Subsequently, charge-separation by SAX-HPLC was employed to yield hexa-, hepta-, octa-, and nonasaccharide. These oligosaccharides clearly revealed the structural features of natural FCSAm, and further verified the characteristics of its branches which is in line with our previous report. In view of the novelty of FCSAm, and the structural diversity of its derivatives, including the size-homogenous fractions and oligosaccharides, the effects on neurite outgrowth were evaluated and the structure-activity relationships were also discussed.

Preparation of Homogenous Fractions and Oligosaccharides from the Depolymerized FCSAm
β-Eliminative depolymerization has been established as an effective method for deciphering the structures of FCS from sea cucumbers. Here the FCSAm was employed as the In regard to the CS analogs from invertebrates, the keratan sulfate disaccharidebranched CS-E from a clam, Mactra chinensis, stimulated the neurite outgrowth of hippocampal neurons [11]. A fucosylated CS from the sea cucumber Apostichopus japonicus exhibited neurite outgrowth-promoting activity [12].  [14][15][16][17][18][19]. Structural differences of FCS from different sea cucumber species are embodied in the size, the composition, the sulfate substitution of branches ( Figure 1B). Besides structure, another aspect of FCS that appeals to researchers is that FCS has wide-ranging biological activities, such as anti-coagulant, anti-thrombotic, anti-virus, and anti-cancer effects [13,[20][21][22]. Previous research proposed that the FCS from the sea cucumber A. japonicus exhibited neurite outgrowth-promoting activity, and recognized the potential roles of the fucose branch in neurite outgrowth. A chemically synthesized FCS trisaccharide that is composed of a CS-E disaccharide unit with a sulfated fucose branch also exhibited comparable stimulation activity [12]. The effects of the FCS originated from other sea cucumbers on neurite's growth, and the structure-function relationships remain unresolved and deserve detailed investigations.
FCS Am was obtained from the sea cucumber Acaudina molpadioides in our previous work [23]. Oligosaccharides from the β-eliminative depolymerized FCS Am revealed that its backbone has a full content of type E, which was branched by monosaccharide [Fuc 2S4S and Fuc 4S ], and the disaccharide [GalNAc-(α1,2)-Fuc 3S4S ] on O-3 of each GlcA. This is the first report of such a disaccharide branch in natural FCS. FCS Am showed the variety of branches with sulfated substitutions, as well as the novel disaccharide.
In this work, further investigation on this unique CS analog (FCS Am ) was conducted. From its β-eliminative depolymerized products, the size-homogenous fractions with a higher degree of polymerization were obtained. Subsequently, charge-separation by SAX-HPLC was employed to yield hexa-, hepta-, octa-, and nonasaccharide. These oligosaccharides clearly revealed the structural features of natural FCS Am , and further verified the characteristics of its branches which is in line with our previous report. In view of the novelty of FCS Am , and the structural diversity of its derivatives, including the size-homogenous fractions and oligosaccharides, the effects on neurite outgrowth were evaluated and the structure-activity relationships were also discussed.

Preparation of Homogenous Fractions and Oligosaccharides from the Depolymerized FCS Am
β-Eliminative depolymerization has been established as an effective method for deciphering the structures of FCS from sea cucumbers. Here the FCS Am was employed as the depolymerization process described in Section 3.2 to prepare its low-molecular-weight product, dFCS Am . As shown in Figure 2A, dFCS Am was composed of a series of fractions with different sizes, reflected by its HPGPC profile. For the sake of their structural identification, dFCS Am was fractionated by GPC using Bio-Gel P-10 & P-6 columns, until each fraction exhibited a relatively single and symmetric peak on the HPGPC analysis chromatogram. Finally, five fractions (F1-F5) were obtained (Figure 2A).
Mar. Drugs 2022, 20, x 3 of 16 depolymerization process described in Section 3.2 to prepare its low-molecular-weight product, dFCSAm. As shown in Figure 2A, dFCSAm was composed of a series of fractions with different sizes, reflected by its HPGPC profile. For the sake of their structural identification, dFCSAm was fractionated by GPC using Bio-Gel P-10 & P-6 columns, until each fraction exhibited a relatively single and symmetric peak on the HPGPC analysis chromatogram. Finally, five fractions (F1-F5) were obtained ( Figure 2A). F1 and F2 have been further analyzed in our previous work. Both fractions consisted of the oligomers with the same or approximate size. From F1, three trisaccharides were obtained. And from F2, two tetra-, one penta-, and two hexa-saccharides were purified by the aid of SAX separation (oligosaccharides 1-7). For the fractions with larger size, theoretically, its oligosaccharide composition should be more complex and variable. Though F3-F5 presented the homogeneities in size, further analysis based on the charge difference showed the characteristic of a multi-component. In this work, F3 was further charge-separated on the Dionex Ionpac™ AS11-HC Semi-prep column repeatedly to yield oligosaccharides 8-13. Chromatographic analysis indicated that 8-13 running over the SAX column presented a single chromatographic peak, respectively ( Figure 2B). The same separation strategy has been adopted for fraction F4, while, unfortunately, the separated components are mixtures as well.

Structural Elucidation of the Oligosaccharides
The structures of oligosaccharides 8-13 were elucidated by the aid of their 1D & 2D NMR spectra and ESI-Q-TOF MS spectra.
The spectra of compound 8 indicated it was a hexasaccharide with the structure of L-Fuc2S4S-(α1,3)-L-Δ 4,5 GlcA-(α1,3)-D-GalNAc4S6S-(β1,4)-[L-Fuc2S4S-(α1,3)-]-D-GlcA-(β1,3)-D-GalNAc4S6S-ol, the same as the one we have reported before [18]. The distinctive methyl groups signaling at ~2.0 ppm (-COCH3) and ~1.3 ppm (-CH3) in the 1 H NMR spectrum revealed that 8 contained two GalNAc residues and two Fuc residues. The anomeric protons at δH 5.68 and 5.50 ppm, derived from the two Fuc residues, indicated the sulfation at O-2 and O-4 positions. The downfield shift signals of the respective protons (I2, 4.49 ppm; I4, 4.85 ppm; dI2, 4.42 ppm; dI4, 4.69 ppm) reconfirmed the substitution of sulfate esters. For oligosaccharide 9, as shown in Figure 3, the appearance of one more set of anomeric signals (at the region of δH 5.0-5.7 ppm, δC 100-106 ppm), accompanying with one more methyl signal at δH 2.09 ppm, indicated that 9 possessed one more GalNAc residue (marked as A') than that in 8. Another signal at δH 5.40 ppm was from the anomeric proton of Fuc residue (designated as II) with the sulfation at O-3 and O-4 positions, which were confirmed by the resonance signals (II3: 4.74, 76.7 ppm; II4: 4.91, 81.9 ppm). Every resonance of each residue was determined by the HSQC spectrum ( Figure 4). The correlation of A'1 (δH 5.09 ppm) and II2 (δC 75.1 ppm) in the HMBC spectrum indicated that residue A'1 connected to II by an α1,2 linkage, and this linkage could be reconfirmed by the correlation of A'1 (δH 5.09 ppm) and II2 (δH 4.04 ppm) in the ROESY spectrum ( Figure 4). Thus, 9 was proved to be a heptasaccharide with the structure of D-GalNAc-(α1,2)-L- F1 and F2 have been further analyzed in our previous work. Both fractions consisted of the oligomers with the same or approximate size. From F1, three trisaccharides were obtained. And from F2, two tetra-, one penta-, and two hexa-saccharides were purified by the aid of SAX separation (oligosaccharides 1-7). For the fractions with larger size, theoretically, its oligosaccharide composition should be more complex and variable. Though F3-F5 presented the homogeneities in size, further analysis based on the charge difference showed the characteristic of a multi-component. In this work, F3 was further charge-separated on the Dionex Ionpac™ AS11-HC Semi-prep column repeatedly to yield oligosaccharides 8-13. Chromatographic analysis indicated that 8-13 running over the SAX column presented a single chromatographic peak, respectively ( Figure 2B). The same separation strategy has been adopted for fraction F4, while, unfortunately, the separated components are mixtures as well.  Figure 3, the appearance of one more set of anomeric signals (at the region of δ H 5.0-5.7 ppm, δ C 100-106 ppm), accompanying with one more methyl signal at δ H 2.09 ppm, indicated that 9 possessed one more GalNAc residue (marked as A') than that in 8. Another signal at δ H 5.40 ppm was from the anomeric proton of Fuc residue (designated as II) with the sulfation at O-3 and O-4 positions, which were confirmed by the resonance signals (II3: 4.74, 76.7 ppm; II4: 4.91, 81.9 ppm). Every resonance of each residue was determined by the HSQC spectrum ( Figure 4). The correlation of A'1 (δ H 5.09 ppm) and II2 (δ C 75.1 ppm) in the HMBC spectrum indicated that residue A'1 connected to II by an α1,2 linkage, and this linkage could be reconfirmed by the correlation of A'1 (δ H 5.09 ppm) and II2 (δ H 4.04 ppm) in the ROESY spectrum ( Figure 4). Thus    The 1D spectra of 10-12 occurred similarly in the number of characteristic signals, including the methyl groups (two -COCH3 of GalNAc, δH ~2.0 ppm, and three -CH3 of Fuc, δH ~1.3 ppm), the anomeric resonances (three signals, at δH 5.0-5.7 ppm; seven signals at δC 99-107 ppm), and the two carbon signals of C-2 of GalNAc residues (δC ~ 54 ppm)   The 1D spectra of 10-12 occurred similarly in the number of characteristic signals, including the methyl groups (two -COCH3 of GalNAc, δH ~2.0 ppm, and three -CH3 of Fuc, δH ~1.3 ppm), the anomeric resonances (three signals, at δH 5.0-5.7 ppm; seven signals at δC 99-107 ppm), and the two carbon signals of C-2 of GalNAc residues (δC ~ 54 ppm) The 1D spectra of 10-12 occurred similarly in the number of characteristic signals, including the methyl groups (two -COCH 3 of GalNAc, δ H~2 .0 ppm, and three -CH 3 of Fuc, δ H~1 .3 ppm), the anomeric resonances (three signals, at δ H 5.0-5.7 ppm; seven signals at δ C 99-107 ppm), and the two carbon signals of C-2 of GalNAc residues (δ C~5 4 ppm) ( Figure 5). This suggested that 10-12 shared the common skeleton of an octasaccharide.

Structural Elucidation of the Oligosaccharides
Detailed analyses based on their 2D NMR spectra lead to the full assignments of all the signals, which are shown in Figure 5 and Table 1. The structural differences between 10-12 existed in the sulfation form of the Fuc branches. According to the downfield shift signals of H2/C2 and H4/C4 (marked in Italic, Table 1), three Fuc 2S4S (dI, I, and rI represented the Fuc 2S4S locating at the non-reducing end, the middle, and the reducing end, respectively) were determined in oligosaccharide 10, while in 11 and 12, an Fuc 4S at the middle and the nonreducing end were elucidated and designated as III and dIII, respectively. The structures of 10-12 were determined as L- The full assignments are displayed in Table 1. The structures of 8-12 were also confirmed according to the accurate molecular mass information of the excimer ion peaks, as shown in Table 2.

Structural Confirmation of the Native FCS Am
In our last work, from the size-homogeneous fractions with low degree of polymerization that separated from the β-eliminative depolymerized product dFCS Am , we have elucidated a series of purified oligosaccharides, including tri-, tetra-, penta-, and hexasaccharides [23]. The branches contained the mono-and disaccharide [Fuc 4S , Fuc 2S4S , and GalNAc-(α1,2)-Fuc 3S4S ]. Here, we have purified and elucidated the oligomers 8-13 consisting of hexa-, hepta-, octa-, and nonasaccharide from the fraction F3 with a higher degree of polymerization. 8-13 presented a continuation with the structural features summarized before which are composed of the central core of 3)-D-GalNAc 4S6S -(β1,4)-D-GlcA-(β1, and the sulfated branches [Fuc 4S , Fuc 2S4S , and GalNAc-(α1,2)-Fuc 3S4S ]. Oligosaccharides that contain the heterodisaccharide branch were confirmed again. These results further supported our structural deduction of the natural FCS Am : its backbone consists of a repeating disaccharide unit of GlcA and GalNAc linked alternatively by β1,4 and β1,3, wherein all the GalNAc residues were sulfated at O-4 and O-6, the same as CS-E; all the GlcA residues were branched and all the branches glycosylated at O-3 of GlcA, instead of any other sites; the branch types included sulfated fucose (Fuc 4S and Fuc 2S4S ) and heterodisaccharide [GalNAc-(α1,2)-Fuc 3S4S ]; the disaccharide branches distributed randomly, but there was no case that such disaccharide branches adjacently distributed in the saccharide chain. All of the reducing end residues (Fuc) of the heterodisaccharide branch were sulfated both at O-3 and O-4, while no sulfation occurred on any sites of the non-reducing end residues (GalNAc) (Figure 8). The sulfation pattern of the heterodisaccharide branch seemed conserved, markedly differing from that reported in the FCS from the sea cucumbers Holothuria nobilis and Ludwigothurea grisea [17,19]. the branch types included sulfated fucose (Fuc4S and Fuc2S4S) and heterodisaccharide [Gal-NAc-(α1,2)-Fuc3S4S]; the disaccharide branches distributed randomly, but there was no case that such disaccharide branches adjacently distributed in the saccharide chain. All of the reducing end residues (Fuc) of the heterodisaccharide branch were sulfated both at O-3 and O-4, while no sulfation occurred on any sites of the non-reducing end residues (Gal-NAc) (Figure 8). The sulfation pattern of the heterodisaccharide branch seemed conserved, markedly differing from that reported in the FCS from the sea cucumbers Holothuria nobilis and Ludwigothurea grisea [17,19].

Neurite Outgrowth-Promoting Activity of FCSAm and Its Derivatives
FCSAm and a variety of its derivatives, including the size-homogenous fractions with different molecular weights and the purified oligosaccharides with different sulfate substitutions, laid the substance that is fundamental for the further analysis of the influence of structural characteristics on the neurite outgrowth-promoting activity.
As shown in Figure 9 and Table 3, natural FCSAm and its depolymerized product dFCSAm showed significant activity in promoting neurite outgrowth. Compared with blank control and CS-E groups, the length increased by 50% at the concentration of 50 μg/mL. Meanwhile, FCSAm, dFCSAm and CS-E showed an aggregation phenomenon under the condition ( Figure 9C). While for the size-homogenous fractions F2-F5 obtained after GPC treatment, they all showed the promotion activities, and the promoting effects increased with the increase of molecular weight from 1.5 to 4.4 kDa. Among them, F5 exhibited more than two-times the outgrowth promoting activity than CS-E, and no obvious

Neurite Outgrowth-Promoting Activity of FCS Am and Its Derivatives
FCS Am and a variety of its derivatives, including the size-homogenous fractions with different molecular weights and the purified oligosaccharides with different sulfate substitutions, laid the substance that is fundamental for the further analysis of the influence of structural characteristics on the neurite outgrowth-promoting activity.
As shown in Figure 9 and Table 3, natural FCS Am and its depolymerized product dFCS Am showed significant activity in promoting neurite outgrowth. Compared with blank control and CS-E groups, the length increased by 50% at the concentration of 50 µg/mL. Meanwhile, FCS Am , dFCS Am and CS-E showed an aggregation phenomenon under the condition ( Figure 9C). While for the size-homogenous fractions F2-F5 obtained after GPC treatment, they all showed the promotion activities, and the promoting effects increased with the increase of molecular weight from 1.5 to 4.4 kDa. Among them, F5 exhibited more than two-times the outgrowth promoting activity than CS-E, and no obvious aggregation phenomenon occurred. In contrast, FCS Am did not show a significantly stronger growth promoting effect than fractions, as expected, indicating that an optimum chain length is required for promotion. For dFCS Am , the presence of the saccharides with large Mw (Figure 2A) should be responsible for the weaker promotion effect than F5.
Mar. Drugs 2022, 20, x 11 of 16 aggregation phenomenon occurred. In contrast, FCSAm did not show a significantly stronger growth promoting effect than fractions, as expected, indicating that an optimum chain length is required for promotion. For dFCSAm, the presence of the saccharides with large Mw (Figure 2A) should be responsible for the weaker promotion effect than F5.  A, B). Cells were cultured for 2 days in plates coated with PDL (50 μg/mL) alone (control) or size-homogeneous fractions F2-F5 (50 μg/mL). The length (n = 50-100 cells) of the longest neurite (mean ± SD) was measured by Image J software and expressed as percentage of growth relative to control (* p < 0.05, ** p < 0.01); the aggregation phenomenon of FCSAm, and dFCSAm, and neurite and cell body aggregation are indicated by yellow and green arrows, respectively (C). Scale bar, 200 μm.  Figure 9. Effects of FCS Am , dFCS Am , and the size-homogeneous fractions F2-F5 on neurite outgrowth (n = 4-6) (A,B). Cells were cultured for 2 days in plates coated with PDL (50 µg/mL) alone (control) or size-homogeneous fractions F2-F5 (50 µg/mL). The length (n = 50-100 cells) of the longest neurite (mean ± SD) was measured by Image J software and expressed as percentage of growth relative to control (* p < 0.05, ** p < 0.01); the aggregation phenomenon of FCS Am , and dFCS Am , and neurite and cell body aggregation are indicated by yellow and green arrows, respectively (C). Scale bar, 200 µm.  [23]. c Data was from the Certification of Analysis provided by the manufacturer.
For the purified oligosaccharides from tri-to nonasaccharides, different concentrations (4.4, 13.3, 40 µM) of oligosaccharides were coated on PDL to evaluate the effects. As shown in Figure 10 and Table 4, compared with the control, all of the oligosaccharides showed neurite outgrowth-promoting activity to different degrees, which basically acted in a concentration-dependent manner. In response to the trisaccharide 1, the length of neurons increased by approximately 50%, which was consistent with the previous report [12]. In addition, at the same concentration, the activity of oligosaccharides on neuron growth increased with the increase of the chain length. By comparing the structural characteristics and activity effects of 10, 11 and 12, the sulfate ester enhanced the activity at low (4.4 µM) and medium (13.3 µM) concentrations. Moreover, the residue Fuc 2S4S , instead of Fuc 4S , located at the middle of octasaccharide (10-12) showed greater promotion. By contrast, whether the branch located at non-reducing end is Fuc 2S4S or Fuc 4S , the octasaccharides  acteristics and activity effects of 10, 11 and 12, the sulfate ester enhanced the activity at low (4.4 μM) and medium (13.3 μM) concentrations. Moreover, the residue Fuc2S4S, instead of Fuc4S, located at the middle of octasaccharide (10-12) showed greater promotion. By contrast, whether the branch located at non-reducing end is Fuc2S4S or Fuc4S, the octasaccharides (10 and 12) showed a similar enhancement effect on neurite outgrowth. For the novel disaccharide branch [GalNAc-(α1,2)-Fuc3S4S], it did not show an obvious difference in the promotion or inhibition effects, which could be concluded from the comparison of analyses of 1 & 3, 8 & 9 and 10-13. Figure 10. Effects of oligosaccharides 1, 3-6, and 8-13 from dFCSAm on neurite outgrowth (n = 3). Neurons were cultured in 96 plates for 2 days in plates coated with PDL (50 μg/mL) alone (control) Figure 10. Effects of oligosaccharides 1, 3-6, and 8-13 from dFCS Am on neurite outgrowth (n = 3). Neurons were cultured in 96 plates for 2 days in plates coated with PDL (50 µg/mL) alone (control) or with oligosaccharides. The neurons were fixed and visualized with 4% paraformaldehyde and TritonX-100, and incubated with tublin Ш (green) and DAPI (blue) (A). The length (n = 50-100 cells) of the longest neurite (mean ± SD) was measured by Image J software and expressed as length, relative to control (*, # p < 0.05, **, ## p < 0.01, or *** p < 0.001) (B). Scale bar, 200 µm.

Depolymerization of FCS Am
The β-eliminative depolymerization of FCS Am was employed according to an established method. Specifically, 6.5 g FCS Am was dissolved in 97.5 mL of water and then reacted with 245 mL of benzethonium chloride solution (62.5 mg/mL) under stirring at room temperature. After centrifugation (4000 rpm × 15 min) and dryness under reduced pressure at 40 • C to constant weight, 17.5 g benzethonium salts of FCS Am were obtained. They were then dissolved in 88 mL dimethyl formamide, and 6.23 mL benzyl chloride was added at 35 • C for 24 h. After that, 53.7 mL of freshly prepared EtONa/EtOH (0.16 M) was added into the reaction system for another 30 min at room temperature at 300 rpm. Subsequently, 120 mL of saturated sodium chloride solution and 960 mL EtOH were added and the precipitate was collected by centrifugation. In order to remove the benzyl groups, the resulting precipitate was dissolved in 360 mL H 2 O, and 16 mL freshly prepared 6M NaOH was added into the solution at 25 • C for 30 min. Meanwhile, 1385 mg of sodium borohydride was added to the solution to reduce the hemiacetals of the resulting saccharides. After that, the solution was neutralized by HCl. Finally, the β-eliminative depolymerized product was obtained after desalting by Sephadex G-25.

Preparation of Size-Homogenous Fractions and Oligosaccharides from Depolymerizated FCS Am
dFCS Am was subjected to a Bio-Gel P-10 column and eluted by 0.2 M sodium chloride solution in batches. The sample in tubes was detected at the UV wavelength of 232 nm, and according to the absorption values, the elution curve was plotted. After repeating separation using Bio-Gel P-10 or P-6 column, the fractions with a similar size were pooled and lyophilized separately. The obtained five size-homogenous fractions were designated as F1~F5 in order of size from lowest to highest. F3 was further purified by strong anion exchange chromatography using a Dionex Ionpac AS11-HC Semi-prep column. The gradient elution program was as follows: 0−60 min, 0−60% elution B (2 M NaCl, pH 3.5); elution A was H 2 O (pH 3.5). The flow rate was 3.5 mL/min. The preparation was employed under the online UV detection at λ 232 nm . Next, the oligosaccharides were desalted by GPC on a Bio-Gel P-2 or Sephadex G-25 column and lyophilized.

Structural Elucidation of the Oligosaccharides
The structures of the oligosaccharides were determined by 1D & 2D NMR spectra and ESI-Q-TOF-MS data, which were recorded at 298 K with a Bruker Advance 600/800 MHz spectrometer equipped with a 13 C/ 1 H dual probe in FT mode, and with a 6540 UHD Accurate-Mass Q-TOF LC/MS spectrometer. For the NMR data acquisition, each sample was dissolved in deuterium oxide (D, 99.9%).

Neuronal Cultures and Coating Plates
Neurobasal medium contained B27 supplement (1×), 2 mM glutamax, penicillin and streptomycin (1×). The plates were pre-coated with 50 µg/mL PDL at 37 • C for 6 h, washed three times with double distilled water and then coated with the sample overnight at 37 • C. The cortical tissue of rats at embryonic day (E) 18 were isolated under a dissecting microscope, removed quickly, and placed in cold PBS and gently chopped, then incubated in a 1 mg/mL papain and DNase I (20 IU/mL) solution at 37 • C for 30 min. Supplementing with neurobasal medium, the cells were eventually plated at 10,000/cm 2 into plates. The cultures were maintained at 37 • C in a humidified 5% CO 2 atmosphere [24][25][26].

Immunofluorescence
After 2 days, neurons were fixed with 4% paraformaldehyde for 30 min, washed with PBS three times, incubated in 0.2% Triton X-100 for 15 min, blocked with 1% BSA and incubated overnight with anti-tublin Ш antibody at 4 • C. The second day, the cells were washed in PBS three times. The second antibody was then incubated at room temperature for 1 h and then rinsed in PBS three times. The nuclei were stained with DAPI for 10 min. The neurons were washed three times with PBS, and then, the cell morphology was detected on an IncuCyte S3 live-cell analysis system [12,27].

Statistical Analysis
Only cells with neurites longer than one cell body diameter were counted. The length of the longest neurite (50-100 cells) were measured by Image J software and expressed as percentage of growth or length, relative to control (* p < 0.05, ** p < 0.01, or *** p < 0.001). The percentage of growth rate was calculated as follows: growth rate (%) = (length of tested group-length of control group)/control group × 100%. All data were expressed as mean ± SD. A statistical analysis was performed using IBM SPSS Statistics 20. Statistical significances were evaluated with a two-sided unpaired Student's t-test for two-group comparisons, and a one-way ANOVA followed by the Dunnett post hoc test for multigroup comparisons. p < 0.05 was considered statistically significant [12,28].

Conclusions
Fucosylated chondroitin sulfate (FCS) from the sea cucumber Acaudina molpadioides has been obtained (FCS Am ), and its precise structure was preliminary investigated in our previous work. An unusual disaccharide branch [GalNAc-(α1,2)-Fuc 3S4S ] was first found in natural FCS, and sulfated fucose Fuc 2S4S and Fuc 4S were also elucidated as the branches linked to O-3 of GlcA in the central core. In view of the novelty and variety of the branch type of FCS Am , further investigation of structure and activity were conducted in this study. From the glycosidic bond-selectively depolymerized product (dFCS Am ), several size-homogenous fractions (F2-F5) with the molecular weight increasing from 1.5 kDa to 4.4 kDa were obtained by GPC separation. Furthermore, seven oligosaccharides were purified after repeat chargeseparation from fraction F3. A detailed analyses of the NMR and MS spectroscopic data clearly presented their structures as hexa-, hepta-, octa-, and nonasaccharide. Combined with the oligomers including tri-, tetra-, penta-, and hexasaccharide obtained from fractions F1 and F2, the precise structure of native FCS Am was confirmed. Its CS-E-like backbone was composed of the repeating disaccharide unit [3)-GalNAc 4S6S -(β1,4)-GlcA-(β1,], where all the GlcA residues were branched by sulfated fucose [Fuc 2S4S (60%) and Fuc 2S4S (25%)] and heterodisaccharide [GalNAc-(α1,2)-Fuc 3S4S (15%)]. The disaccharide branches dispersively distributed in the saccharide chain rather than clustered.
Based on the fractions and oligosaccharides with rich structural features, the effects on the cortical neuron outgrowth were evaluated. The natural FCS Am and its depolymerized products dFCS Am showed neurite outgrowth promotion and cell aggregation simultaneously. For the size-homogonous fraction (F2-F5), F2 could stimulate the outgrowth, and the promoting effects increased with the increase of molecular weight. A comparison of analyses among the purified oligosaccharides also showed that the promoting activity was positively correlated with chain length, while an optimum chain length is required for maximum growth without aggregation. In addition, the branch Fuc 2S4S significantly contributed to the promotion, whereas the novel disaccharide branch [GalNAc-(α1,2)-Fuc 3S4S ] did not show obvious difference in promotion or inhibition effects. The results expanded the range of FCS activity analysis and provided some guidance for drug research in neuro-systemic diseases.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/md20100653/s1, Figure S1. 1H/13C NMR spectra of oligosaccharide 9; Figures S2-S4. HSQC spectrum and signal assign-ments of oligosaccharide 10-12; Figure S5. MS spectra and signal assignments of oligosaccharides 8-13. Data Availability Statement: All data presented in this study are available from the corresponding author on reasonable request.

Conflicts of Interest:
The authors declare that they have no conflict of interest.