Structural Characterization and Anti-Osteoporosis Effects of a Novel Sialoglycopeptide from Tuna Eggs

Several sialoglycopeptides were isolated from several fish eggs and exerted anti-osteoporosis effects. However, few papers have explored sialoglycopeptide from tuna eggs (T-ES). Here, a novel T-ES was prepared through extraction with KCl solution and subsequent enzymolysis. Pure T-ES was obtained through DEAE-Sepharose ion exchange chromatography and sephacryl S-300 gel filtration chromatography. The T-ES was composed of 14.07% protein, 73.54% hexose, and 8.28% Neu5Ac, with a molecular weight of 9481 Da. The backbone carbohydrate in the T-ES was →4)-β-D-GlcN-(1→3)-α-D-GalN-(1→3)-β-D-Glc-(1→2)-α-D-Gal-(1→2)-α-D-Gal-(1→3)-α-D-Man-(1→, with two branches of β-D-GlcN-(1→ and α-D-GalN-(1→ linking at o-4 in →2,4)-α-D-Gal-(1→. Neu5Ac in the T-ES was linked to the branch of α-D-GlcN-(1→. A peptide chain, Ala-Asp-Asn-Lys-Ser*-Met-Ile that was connected to the carbohydrate chain through O-glycosylation at the –OH of serine. Furthermore, in vitro data revealed that T-ES could remarkably enhance bone density, bone biomechanical properties, and bone microstructure in SAMP mice. The T-ES elevated serum osteogenesis-related markers and reduced bone resorption-related markers in serum and urine. The present study’s results demonstrated that T-ES, a novel sialoglycopeptide, showed significant anti-osteoporosis effects, which will accelerate the utilization of T-ES as an alternative marine drug or functional food for anti-osteoporosis.


Introduction
Osteoporosis is a systemic bone disease characterized by bone microstructure destruction [1].It is caused by a decrease in bone density and a deterioration of bone microstructure, leading to bone mass reduction, bone strength decrease, and bone brittleness increase [2,3].Resulting from the high global prevalence and disability, the World Health Organization defines osteoporosis as the one of the three major senile diseases and frequently occurring disease in the world [4].It was reported that there were 158 million people aged over 50 who suffered from osteoporosis worldwide in 2010 and this number would double by 2040 [5].In China, it was estimated that the age-standardized prevalence of osteoporosis was 6.46% and 29.13%, respectively, for men and women aged 50 years and older [6], and the number of osteoporosis or bone loss patients would account for 533 million people in 2050 [7].Therefore, effective treatment of osteoporosis has become one of the important and significant research areas.
Sialoglycoprotein/sialoglycopeptide is composed of sialic acids (5-N-acetyl-neuraminic acid (Neu5Ac) or 5-N-glycolyl-neuraminic acid (Neu5Gc)), carbohydrate chains, and protein/peptide chains.In the compound, sialic acids are located at the terminal monosaccharide, and the hydroxyl of anomeric carbon in carbohydrates is linked to the hydroxyl (O-glycopeptide bond) or the acylamino (N-glycopeptide bond) of specific amino acids in acids in protein/peptides [8].Some papers have reported that several sialoglycoprotein/sialoglycopeptide were isolated and characterized from fish eggs, including rainbow trout, medaka fish, pacific herring, and Gadus morhua [9][10][11][12][13].Moreover, the structure of sialoglycoprotein/sialoglycopeptide was distinguished between the various fish species, showing the specific carbohydrate chain, peptide chain, glycopeptides bone, and the kinds of sialic acids.Several studies also proved that sialoglycoprotein/sialoglycopeptide from fish eggs exhibited anti-osteoporosis activities [12,14].
As one of the world's three major nutritious fishes, tuna is a deep-sea economic fish with annual catches exceeding 6 million tons.Tuna eggs contain abundant protein, phospholipids, unsaturated fatty acids, etc.Our previous experiments proved that the concentration of sialoglycopeptide from tuna eggs (T-ES) accounts for 4% of the egg.Two antioxidative peptides were isolated from tuna eggs [15].However, there are few reports on T-ES and its bioactivities.In this study, a novel sialoglycopeptide was isolated and purified from tuna eggs, and its structure profile was clarified.Furthermore, the anti-osteoporosis effects of T-ES were also investigated in senescence-accelerated mouse prone (SAMP6) mice.This research will provide a theoretical basis for application in the prevention and treatment of senile osteoporosis.

Isolation and Purification of T-ES and General Property Description
As shown in Figure 1A, four fractions were obtained through polarity separation with NaCl.According to the phenol-sulfuric acid method, T-ES existed in fraction 1 (W1, 9.36%).Purified T-ES was separated using a sephacryl S-300 column and its yield was 13.52%.The weight-average molecular weight of T-ES was 9481 Da (Figure 1B).

Glycosidic Bond Type of T-ES
N-glycosylation and O-glycosylation are the main linkages between carbohydrate and peptide.As shown in Figure 1E, T-ES with NaOH showed a peak at 20.44 min using the HPLC method, while T-ES without NaOH represented a peak at 17.71 min.These data suggest that T-ES was deduced to be a glycoprotein with O-glycosidic linkages between hydroxyamino acids and polysaccharides.The same peaks occurred at 24.75 min for the T-ES with and without PNGase F treatment (Figure 1F), suggesting an unchanged glycoprotein with N-glycosylation in T-ES suffered with PNGase F. These data indicate that T-ES has O-glycosylation between the peptides and carbohydrates, rather than Nglycosylation.It was reported that N-linked glycoproteins existed both in sialoglycopeptide from Gadus morhua eggs and in zebrafish egg chorion [16,17], while O-linked glycan units were present in neuraminic acid-rich glycoprotein from rainbow trout eggs [18].These indicate that the glycosidic bond between carbohydrate chain and peptide chain is different among different species.

FT-IR, Methylation and NMR Spectroscopy Analysis for Carbohydrate Chain
We used FT-IR to examine the chemical functional groups of the carbohydrate chain in T-ES, and information about the absorption peaks from 3600 to 3200 cm −1 (characteristic peaks of carbohydrates) is displayed in Figure 2A.The stretching vibration of -OH had a prominent peak at 3392 cm −1 , which was the characteristic peak of the carbohydrate [19].The peak at 2935 cm −1 might be attributed to C-H stretching vibration [20].The C=O stretching vibrations were responsible for the absorption peaks at 1733 cm −1 and 1540 cm −1 [19].The variable vibration of C-H had a peak at 1457 cm −1 [21].The C-O molecules were responsible for the prominent peaks at 1419 cm -1 and 1147 cm -1 [22].The variable vibration of O-H had a peak at 1114 cm -1 [21].The prominent peak at 873 cm −1 might be attributed to C-H variable vibration in the pyranose ring [20].

MS Spectrometry for Peptide Chain
Amino acid sequences were analyzed using LC-MS/MS and the results are shown at Figure 3, Supplementary Figures S20-S23, and Supplementary Table  The experiments using NaOH treatment (Figure 1E) suggested that serine might exist in the O-glycosylation of hydroxyamino acids.This result was similar to that of the sialoglycoprotein isolated from rainbow trout eggs [10].In summary, combining Neu5Ac, peptide, and glycan moieties, we speculated the structural outline of T-ES is as shown in Figure 4.

T-ES Increased Bone Density
Bone mineral density is the golden standard in the early diagnosis, therapeutic, and prognosis of osteoporosis [23].As shown in Table 3, a high dosage of T-ES caused significant increases in the bone density of both the femur and of tibia by 40.75%, and 1.14-fold compared with SAMP mice (p < 0.05, Table 1).Moreover, tibial bone density in the T-ES-L group was markedly increased by 54.90% compared with the model group (p < 0.05).In addition, there were no significant differences of femoral bone density or tibial bone density between the alendronate (ALN) group and T-ES-H group (p > 0.05), suggesting that the effect of the high dosage of T-ES on enhancing bone density was equivalent to that of the ALN group.These indicate that T-ES can improve senile osteoporosis.

T-ES Enhanced Bone Biomechanical Properties
Bone biomechanical properties are of great significance in evaluating osteoporosis [24].The max. load and max.deflection of both the femur and of tibia were significantly elevated with high dosage of T-ES compared to the SAMP6 mice (p < 0.05, Table 1).Moreover, a low dosage of T-ES caused remarkable increases in femoral max.load, tibial max.load, and tibial max.deflection (p < 0.05).These results indicate that T-ES can enhance bone strength and quality, and lessen fracture risk.

T-ES Improved Bone Microstructure
As shown in Table 3 and Figure 5, the SAMP6 mice treated with the high dosage of T-ES showed significant increases in femoral trabecular thickness, trabecular number, connectivity density, cortical bone thickness, and remarkable reductions in femoral trabecular separation and structural model index compared with the SAMP6 mice (p < 0.05).Similarly, the aforementioned parameters of the tibia were also improved in the T-ES-H group compared with the model group (p < 0.05).Furthermore, the low dosage of T-ES also dramatically increased femoral trabecular thickness and tibial cortical bone thickness, and decreased tibial trabecular separation and the structural model index (p < 0.05).Interestingly, the increase in femoral trabecular number was markedly greater in the T-ES-H group than that found in the ALN group (p < 0.05).These results indicate that T-ES can improve osteoporosis-induced destruction of bone microstructure, and promote the repair of bone tissue.

T-ES Elevated Serum Osteogenesis-Related Parameters
As an osteogenesis biomarker, BALP, an osteoblasts-secreted extracellular enzyme, can maintain bone growth and regeneration [25].The high dosage of T-ES caused significant increase in serum BALP concentration in SAMP6 mice (p < 0.05).PICP is the essential protein for collagen synthesis in bone tissue, which is synthesized by osteoblasts [26].PICP can reflect the activity of osteoblasts and the synthesis rate of type I collagen [27].Both low and high dosages of T-ES increased serum PICP concentration in SAMP6 mice (p < 0.05).BGP can maintain bone mineralization rate, which has important value for diagnosing osteoporosis [28].Serum BGP levels were dramatically elevated in both the T-ES-L group and T-ES-H group compared with the model group (p < 0.05).Interestingly, the effects of the high dosage of T-ES on increasing serum BGP were better than those of ALN.These results indicate that T-ES can elevate serum osteogenesis-related parameters.
and decreased tibial trabecular separation and the structural model index (p < 0.05).Interestingly, the increase in femoral trabecular number was markedly greater in the group than that found in the ALN group (p < 0.05).These results indicate that T-ES can improve osteoporosis-induced destruction of bone microstructure, and promote the repair of bone tissue.The ratio of OPG to RANKL is closely related to the generation of osteoclasts, which resolves the rate of bone resorption [29].In the present study, the ratio of OPG to RANKL is significantly decreased in T-ES-H group compared with the model group (p < 0.05).Cath-k, MMP-9, and CTX-I are the critical enzymes that effect bone resorption, which are secreted by mature osteoclasts [30].The mice treated with the high dosage of T-ES showed remarkable serum Cath-k, MMP-9, and CTX-I concentrations compared with the SAMP6 mice.Xia et al. reported that sialoglycoproteins isolated from the eggs of Carassius auratus exhibited anti-osteoporotic activity through increasing the OPG/RANKL ratio [12].Zhao et al. reported that Gadus morhua eggs' sialoglycoprotein prevents high bone turnover by controlling the OPG/RANKL/TRAF6 pathway [14].
Ca and P are the dominant components of bone mineral [31], DPD is an important sector of collagen I [32].The three parameters are the important indicators in evaluating bone loss [32].In our study, urinary Ca, P, and DPD concentrations in the T-ES-H group were distinctly decreased compared with the model group.These results indicate that T-ES can mitigate osteoporosis through reducing bone resorption-related parameters.

HPLC-MS Spectrometry
The peptide chain of T-ES was analyzed according to the method in [39].Briefly, T-ES was dissolved in Tris-HCl/dithiothreitol (100 mM, pH 8.5, 37 C) for 2 h and subsequently in iodoacetamide for 15 min.After centrifugation, the precipitate was washed using Tris-HCl/NH 4 HCO 3 and hydrolyzed using pancreatin.The peptide fragment was analyzed with an HPLC-MS system (Easy-nLC1200 and Q Exactive, Thermo, USA).The data were identified and quantified using PEAKS Studio 8.5 (Bioinformatics Solutions Inc., Waterloo, ON, Canada).

Animals' Experiments
Male SAMP6 mice (12 w, 18 ± 2 g), one kind of senescence accelerated mice, were purchased from Beijing Zhishan Co. Ltd. (SCXK-2018-0010).The mice were housed in individual cages ( .Before the end of the experiments, the mice were fed in individual metabolism cages to collect urine.After 120 days of continuous feeding, the mice were anesthetized using diethyl ether and then sacrificed to obtain blood, femurs, and tibias for subsequent analysis.

Bone Mineral Density Measurement
Bone mineral density of the whole left-side femur and the whole left tibia were detected using a Dual-energy X-ray bone densitometer (GK99, I'acn, Italy).

Bone Biomechanics Determination
Bone biomechanics of the left-side femur and tibia were determined using a universal material testing machine (5500, Instron, Boston, MA, USA).Briefly, bone samples were gently thawed from −80 • C to 25 • C, and the bone biomechanics were determined with a material testing machine (5 mm/min load velocity, 15 mm spacing) using the three-point bending method.

Bone Microstructure Observation
The distal end of the right-side femur and the proximal end of the right-side tibia in SAMP6 mice were fixed with neutral formalin, washed with running water, and subsequently scanned using a Micro-CT scanner (CT80, Scanco, Wangen-Brüttisellen, Switzerland), with 70 kV voltage, 114 µA electricity, and 10 µm distinguishability.The threedimensional image was reestablished using the GPU NRecon Server (local) V1.7.4.2.Several parameters were assessed, including trabecular thickness, trabecular number, trabecular separation, connectivity density, structural model index, and cortical bone thickness.

Determination of Osteogenesis-and Bone Resorption-Related Markers in Serum and Urine
The blood was centrifuged to obtain the serum.Serum osteogenesis-related markers (BALP, PICP, and BGP) and bone resorption-related markers (RANKL, OPG, MMP-9, CTX) were determined using ELISA kits (Invitrogen, Carlsbad, CA, USA).Urinary Ca and P levels were measured using commercial kits (Jiancheng, Nanjing, China), and DPD content was determined using an ELISA kit (Invitrogen, Carlsbad, CA, USA), respectively.

Statistical Analysis
Data are expressed as the mean ± standard deviation (SD).One-way analysis of variance (ANOVA) followed by Dunnett's T3 post-hoc test was performed for significant difference among the four groups with SPSS 21.0 software.Statistical significance was considered at p < 0.05.

Conclusions
In conclusion, a novel T-ES was extracted using KCl solution and purified using DEAE-Sepharose fast flow ion exchange chromatoe graphy followed by sephacryl S-300 gel chromatography.The structure of the T-ES was as follows: the backbone carbo-

Figure 1 .
Figure 1.Isolation, purification, and general property analysis of T-ES.(A), Stepwise elution curve of T-ES on a DEAE-52 ion exchange column; (B), Purification of T-ES by Automatic Gel Purification System and its molecular weight; (C), HPLC analysis for Neu5Ac in T-ES; (D), GM analysis for monosaccharide in T-ES; (E), HPLC analysis for T-ES treated with/without NaOH; (F), HPLC analysis for T-ES treated with/without PNGase.

Figure 1 .
Figure 1.Isolation, purification, and general property analysis of T-ES.(A), Stepwise elution curve of T-ES on a DEAE-52 ion exchange column; (B), Purification of T-ES by Automatic Gel Purification System and its molecular weight; (C), HPLC analysis for Neu5Ac in T-ES; (D), GM analysis for monosaccharide in T-ES; (E), HPLC analysis for T-ES treated with/without NaOH; (F), HPLC analysis for T-ES treated with/without PNGase.

1
. There were five peptides in the T-ES.The area of peptide No. 4 greatly exceeded that of the other peptides.The sequence of the peptide No.4 chain in the T-ES was Ala-Asp-Asn-Lys-Ser*-Met-Ile.

Figure 3 .
Figure 3. Two-stage mass spectrum of a peptide segment of T-ES.Ser* represents the O-glycosylation between Ser and sugars.In summary, combining Neu5Ac, peptide, and glycan moieties, we speculated the structural outline of T-ES is as shown in Figure 4.

Figure 3 .
Figure 3. Two-stage mass spectrum of a peptide segment of T-ES.Ser* represents the O-glycosylation between Ser and sugars.

Figure 3 .
Figure 3. Two-stage mass spectrum of a peptide segment of T-ES.Ser* represents the O-glycosylation between Ser and sugars.In summary, combining Neu5Ac, peptide, and glycan moieties, we speculated the structural outline of T-ES is as shown in Figure4.

Figure 4 .
Figure 4.Chemical structure of T-ES.(A), T-ES structure; (B), the three-position structure of T-ES.Figure 4. Chemical structure of T-ES.(A), T-ES structure; (B), the three-position structure of T-ES.

Figure 4 .
Figure 4.Chemical structure of T-ES.(A), T-ES structure; (B), the three-position structure of T-ES.Figure 4. Chemical structure of T-ES.(A), T-ES structure; (B), the three-position structure of T-ES.

Figure 5 .
Figure 5. Effects of T-ES on the microstructure of femoral and tibial tissues.(A,a), femoral microstructure in the model group; (B,b), femoral microstructure in the ALN group; (C,c), femoral microstructure in the T-ES-L group; (D,d), femoral microstructure in the T-ES-H group; (E,e), tibial microstructure in the model group; (F,f), tibial microstructure in the ALN group; (G,g), tibial microstructure in the T-ES-L group; (H,h), tibial microstructure in the T-ES-H group.

Figure 5 .
Figure 5. Effects of T-ES on the microstructure of femoral and tibial tissues.(A,a), femoral microstructure in the model group; (B,b), femoral microstructure in the ALN group; (C,c), femoral microstructure in the T-ES-L group; (D,d), femoral microstructure in the T-ES-H group; (E,e), tibial microstructure in the model group; (F,f), tibial microstructure in the ALN group; (G,g), tibial microstructure in the T-ES-L group; (H,h), tibial microstructure in the T-ES-H group.

2. 10
. T-ES Reduced Bone Resorption-Related Parameters in Serum and Urine RANKL and OPG are pivotal factors stimulating osteoclasts differentiation and activization.

Funding:
This research was supported by the Zhejiang Province of China (2021ZJ004), the Science and Technology Project of Zhoushan (2020C21020), and the Natural Science Foundation of China (41806182).Institutional Review Board Statement:The animal experiments were reviewed and approved by the Ethics Committee at Zhejiang Ocean University (No. 2021061).

Table 1 .
Component characterization of monosaccharide in T-ES.

Table 2 .
Analysis of T-ES methylation.

Table 3 .
Effects of T-ES on osteoporosis-related parameters in SAMP6 mice.
12/12 h of light/dark, 23 ± 1 • C).The animal experiments were reviewed and approved by the Ethics Committee at Zhejiang Ocean University (No. 2021061).After 8 weeks of feeding, the SAMP6 mice were randomly divided into four groups (10 mice per group): model group (intragastric administration with 10 mL/kg of normal saline), ALN group (1 mg/kg of ALN), low dosage of T-ES group (T-ES-L, 40 mg/kg of T-ES), and high dosage of T-ES group (T-ES-H, 160 mg/kg of T-ES)