2022 Volume 28 Issue 1 Pages 67-74
Lanzhou lily has been confirmed as health care product and medicine because of its great ornamental and edible medicinal values. At present, the research on Lanzhou lily glycoprotein has not been reported in the literature. In this study, the glycoprotein components of Lanzhou lily were extracted and purified, and its structural characterization and immunological activity were studied. The results showed that Lanzhou lily glycoprotein (LGP-1) was linked by O-glycopeptide bonds and had characteristic absorption peaks of carbohydrate and protein. Moreover, LGP-1 possessed great immunological activity. When RAW264.7 was cultured for 48 hours with the glycoprotein concentration of 100 µg/mL, it could significantly promote cell proliferation and activate RAW264.7 to release NO and TNF-α. This confirms that LGP-1 has the potential to become a functional food.
Lanzhou lily (Lilium davidii var. unicolor Salisb.) is the only dual-use sweet lily in China. Because of its high carbohydrate and low crude fiber contents, it is one of the important economic crops of Lanzhou and surrounding areas (Wang et al., 2018). As confirmed by reports, Lanzhou Lily, as a medicinal plant; has revealed various biological activities such as anti-oxidation (Li et al., 2020a), anti-tumor (Han et al., 2013) and immunity enhancement (Sun et al., 2014).
As a binding protein, glycoprotein is formed by covalent bond between carbohydrates and polypeptides or proteins (Yun et al., 2018). It is a macromolecular substance playing a wide biological role in living organisms. Studies have revealed that more than 80% of proteins are glycoproteins, including many hormones, immunoglobulins, structural proteins, carrier proteins and receptors (Li et al., 2013). They play various biological activities, such as antioxidant, immune regulation and anti-tumor (Dan et al., 2018).
Currently, most of researches on active ingredients in Lanzhou lily focuses on polysaccharides, alkaloids and flavonoids, and no research on glycoprotein components has been reported yet (Farnsworth et al., 2010; Li et al., 2020b). Therefore, its glycoprotein was selected as the research object and its structure was characterized. Moreover, its immunological activity was studied at the cellular level, which provided a basic research for the further development and utilization of Lanzhou Lily glycoprotein.
Materials Lanzhou lily, lily planting was cultured in Xiguoyuan Town, Qilihe District, Lanzhou City, Gansu Province, China.
Extraction and Purification of LGP Fresh Lanzhou lily were as follow. They were first cleaned, dried, crushed and sifted through 100 mesh sieve and then defatted by petroleum ether (Yantai, China) reflux. It was then extracted with 0.01 mol/L PBS buffer solution of pH 7.4 under 200 W ultrasound, and extracted for 2 h at 50 °C water bath (1:4, w/v); centrifuged at 4 000 rpm and concentrated at 40 °C in supernatant. Free protein was removed with Sevag reagent (Butanol: Chloroform = 1:4, v/v). Ammonium sulfate was then added for salting out, and the precipitate was collected after centrifugation. The precipitate was reconstituted with distilled water, dialyzed, and the dialysis end point was measured with 10% BaCl2 solution. To obtain crude extraction of LGP, the desalted samples were vacuum freeze. The yield of LGP was calculated as following:
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The dried glycoprotein was dissolved in PBS buffer solution of pH 7.4, applied to the Sephadex G-200 column (Solarbio, Beijing), and eluted with ultrapure water at a flow rate of 0.5 mL/min. The sample size was 20 µL and was used to detect the outflow of carbohydrate and protein.
The sample was applied to the DEAE-cellulose 52 column (Solarbio, Beijing) and eluted with a gradient of NaCl solution (0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mol/L) to detect and collect the efflux of carbohydrate and protein. After dialysis with deionized water and vacuum freeze-drying, the purified LGP samples were obtained.
In this paper, the purified LGP-1 served as the main research topic, and both of structural characterization and immunological activity were studied.
Evidence of homogeneity The HPLC system (Ultimate 3000 HPLC, DIONEX, America) and the chromatographic column Silversit C18, 250 × 416 mm (5 µm) were established. The UV detection wavelength was set to 280 nm, and the mobile phase was double distilled water with the injection volume of 20 µL. The column temperature was 30 °C and the flow rates was 0.7 mL/min. The chromatographic curves of samples were recorded (Feng et al., 2008).
To detect carbohydrate and protein efflux and to plot outflow curves. LGP-1 was dissolved and applied to the surface of Sephadex G-100 column at an elution rate of 0.5 mL/min.
Gel electrophoresis was performed with the sample of LGP-1 containing 0.1% SDS on 10% polyacrylamide gel. The electrophoresis was carried out at 100 V, 30 mA for 2.5 h and stained with Coomassie brilliant blue R-250.
Determination of carbohydrate and protein content The standard curves were prepared by using Glucose and Bovine Serum Albumin (BSA) as standards, and the carbohydrate and protein contents were determined by both phenol-sulfuric acid and Coomassie Brilliant Blue G-250 (Bio-Asia Biotechnology) methods, respectively.
FT-IR FT-IR (Nicolet is10 FT-IR, Thermo, America) analysis was conducted using KBr disc method. After being grinded and pressed, a small amount of fully dried LGP-1was involved in KBr. It was scanned 16 times in the range of 400–400 cm−1 with a resolution of 4 cm−1.
Monosaccharide composition analysis The monosaccharide composition was determined by GC-MS (Thermo. America), and the experimental procedure was performed based on the methods available in the laboratory.
Pre-column derivatization: Accurately weighed 20.0 mg LGP-1 was added to 4 mol/L TFA and acidified for 10 h and then dried. After drying, hydroxylamine hydrochloride (Wuhan, China) and pyridine (Tianjin, China) were added and the reaction was conducted for 30 min. Later on, acetic anhydride (Yantai, China) was added for 30 min, dried, and dissolved in chloroform. The reaction was performed under the protection of N2.
GC condition: The column was selected from capillary column TR-5ms SQC (30 mm × 0.25 mm × 0.25 µm), and high purity helium (He) was used as carrier gas. The column flow rate was 1.0 mL/min. The sample temperature was 250 °C and the single injection volume was 0.2 µL. Heating program: After holding at 120 °C for 3 min, the temperature was raised to 250 °C at a heating rate of 5 °C/min for 5 min.
MS condition: The ionization mode was an electron ionization (EI) source with 70 eV of electron energy and the ion source temperature of 250 °C with quadrupole of 150 °C and the scanning mass range of 33–550 m/z. The standard monosaccharide derivatization methods and GC-MS conditions were the same as the one mentioned above.
Amino acid composition analysis Accurately weighed 11.0 mg of purified LGP was dissolved with 6N HCl, and acidified at 110 °C for 22 h under N2 protection. After cooling, it was transferred to 10 mL volumetric flasks for constant volume. It was then dried again with 2.0 mL of 55 °C N2. Later on, 1mL of distilled water was added and then dried again for the third time. It was dissolved in 1.0 mL of deionized water (0.02 mol/L HCl), mixed, and filtered through a 0.45 µm filter. The injection volume was 20 µL and was determined by Hitachi L-8900 Amino Acid Analyzer (Hitachi Limited, Japan), and the standard amino acid was used as a control.
Molecular weight determination The molecular weight of LGP-1 was established by SEC-LLS. The used detector is a multi-angle laser light scattering (DAWN Eos, Wyatt Technology Co., USA, λ = 690 nm), and the chromatographic column was Ultrahydrogel™ column (7.8 × 300 mm, Waters, USA), with the refractive index detector (DAWN Eos, Wyatt Technology Co., USA). The sample concentration was 2.0 mg/mL, and the sample was filtered by a 0.2 µm pore size filter before detection (Zastre et al., 2002).
Circular dichroism To determine the secondary structure of the protein, LGP-1 was prepared into 1.0 mg/mL solution with ultrapure water, and its asymmetry was observed by CD (JASCO, Japan) (Zadali et al., 2020).
Glycopeptide linkage types analysis A certain mass of LGP-1 was weighed and added to 10.0 mL, 0.2 mol/L NaOH solution system. It was then incubated for 5h at 55 °C, and scanned in the wavelength range of 190–400 nm with UV-VIS (LabTech, America). The equal concentration of LGP-1 was treated in the absence of NaOH solution and was subjected to scanning of the ultraviolet absorption spectrum in the same wavelength range.
Scanning electron microscopy An appropriate amount of LGP-1 was applied to the sample holder, and then the floating sample was blown off with an ear-washing ball, and the apparent morphology was observed with an Ultra Plus thermal field emission scanning electron microscope (Zeiss, Germany).
Cell viability assay RAW 264.7 was cultured in a 96-well plate at 2 × 104 density, and the sterilized LGP-1 was formulated into a gradient concentration solution of 200, 100, 50, 25, 12.5 µg/mL, and added to a 96-well plate. Later on, six groups of parallel experiments for each concentration were set up, and cultured in three times gradients of 24, 48 and 72 h respectively. The cell viability was measured by CCK-8 kit (Solarbio, Beijing). Enzyme mark instrument (Epoch Life Science, America) was used to measure absorbance, and the experiment was repeated for three times.
The used cell viability formula was as follows: (Chen et al., 2016a)
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ADosing: Absorbance of wells with cells, CCK-8 solution and drug solution;
AUnmedicated: Absorbance of wells with cells, CCK-8 solution without drug solution;
Acontrol: Absorbance of wells with medium and CCK-8 solution without cells.
Determination of NO release Cell culture and seeding methods are the same for cell viability determination, and six groups of parallel experiments were set up. Used the BCA Protein Assay Kit to determine the protein concentration in the cell sample, and calculated the NO content in the cell per unit protein. NO release was detected by NO Assay Kit (Solarbio, Beijing), and experiment was repeated for three times.
The NO content was calculated as follows: (Chen et al., 2016b)
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AExperiment: Absorbance of dosing hole;
AStandard: Absorption of standard;
AControl: Blank control group.
Determination of TNF-α content Cell culture and seeding methods were the same for cell viability determination, and six groups of parallel experiments ware set up. Used the BCA Protein Assay Kit to determine the protein concentration in the cell sample, and calculated the NO content in the cell per unit protein. The TNF-α content in the culture supernatant was determined by Mouse TNF-α ELISA Kit (Solarbio, Beijing) (Wang and Zhang., 2018), and experiment was repeated for three times.
Extraction, purification and purity identification of LGP Fresh bulbs of Lanzhou lily were degreased, and the buffer solution was extracted and salted out to obtain the crude LGP. The results showed that the yield of LGP was 2.09%. After purification by Sephadex G-100 and DEAE-cellulose 52 column chromatography, two glycoprotein components named LGP-1 and LGP-2 were obtained (Fig. 1A and B), and were named according to the elution concentration.
Purification and purity identification of LGP. (A) LGP was applied to the Sephadex G-200 column and eluted with ultrapure water at a flow rate of 0.5 mL/min. (B) LGP was applied to the DEAE-cellulose 52 column and eluted with a gradient of NaCl solution (0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mol/L). (C) The HPLC chromatogram of LGP-1 was a single peak. (D) Elution pattern of LGP-1 on Sephadex G-100 column, a single elution peak was observed. (E) Protein band of LGP-1 in SDS-PAGE and it was revealed only one protein band.
The HPLC chromatogram of LGP-1 was a single peak with a retention time of 2.51 min (Fig. 1C). After elution of LGP-1 with Sephadex G-100, a single elution peak was observed by measuring the efflux of carbohydrate and protein (Fig. 1D). In addition, in the SDS-PAGE electrophoresis diagram, LGP-1 also revealed only one protein band (Fig. 1E). The above results showed that the purified LGP-1 was a homogeneous component.
Chemical structure characterization of LGP-1 The standard curve was drawn with glucose and Bovine Serum Albumin and the linear equations were y = 0.0162x-0.0438 (R2 = 0.9991) and y = 0.0079x-0.0022 (R2 = 0.9986). The calculated carbohydrate content in LGP-1 was 55.80% and the protein content was 35.28%.
The molecular weight of LGP-1 was measured by SEC-LLS, and was found to be 36.39 kDa with the Mn of 18.66 kDa and a dispersion coefficient (Mw/Mn) of 1.950.
The infrared absorption spectrum of LGP-1 in the range of 4 000–400 cm−1 is presented in Fig. 2(A). As it can be observed, LGP-1 exhibits typical absorption peaks of carbohydrate and protein. The strong and broad absorption peaks near 3 400 cm−1 are the stretching vibration peaks of O-H and the stretching vibration peaks of N-H groups, which are characteristics of absorption peaks of carbohydrate and protein (Cui et al., 2014). The two peaks appearing between 2 900 and 3000 cm−1 are the stretching vibration and bending vibration of C-H (Li et al., 2013). As pointed out by Razmkhah et al., the infrared absorption between 1 700–1 600 cm−1 and 1 600–1 500 cm−1 is attributed to Amide I (the stretching vibration of C=O and C-N) and Amide II (mainly the bending vibration of N-H) (Razmkhah et al., 2017). The absorption peaks at 1 204.82 cm−1 and 1 135.99 cm-1 are caused by the stretching vibration of the C-N group (Li et al., 2013).
Chemical structure characterization of LGP-1. (A) The FT-IR of LGP-1 showed obvious characteristic absorption peaks of carbohydrates and proteins. (B) GC-MS results show that LGP-1 is composed of Man and Glc, and its molar ratio is 1.07: 1.01. (C) CD spectrogram of LGP-1 shows that the main chain structure of LGP-1 is β-sheet. (D) Ultraviolet scanning spectrogram of LGP-1 before and after β-eliminate, indicate that O-linked glycopeptide bonds are contained in LGP-1. (E) SEM photographs of LGP-1. a) ×1 000, b) ×10 000, c) ×50 000 and d) Size distribution map of LGP-1.
LGP-1 was hydrolyzed by trifluoroacetic acid, derivatized with saccharin acetyl, and analyzed by GC-MS. The chromatogram results are shown in Fig. 2(B). LGP-1 consists of Man and Glc, and its molar ratio is 1.07 : 1.01.
The far ultraviolet region of the CD (185–245nm) can reflect the conformation of the protein backbone. The secondary structure of the protein is different, and the resulting spectra are also different. The α-helical generally revealed two negative grooves at 209 nm and 222 nm, which is called a double groove curve; the β-sheet has a negative groove around 215 nm and a small positive peak around 195 nm. A random coil has a negative groove around 200 nm, and a positive peak appears around 220 nm. It can be seen from Fig. 2(C) that the CD of LGP-1 has a positive peak at 195nm and a significant negative groove between 210 and 220nm, indicating that the main chain structure of LGP-1 is β-sheet (Zadali et al., 2020).
After treatment with 0.2 mol/L NaOH solution, the significant increase of the absorbance of LGP-1 near 240 nm was observed (Fig. 2D), indicating that after the dilution with alkali treatment, the β-elimination reaction occurred, that is, the reaction produced α-aminobutenoic acid or α-aminoacrylic acid, resulting in a significant change in absorbance at 240 nm. The results indicate that O-linked glycopeptide bonds are contained in LGP-1.
Observation at low magnification using SEM revealed that LGP-1 exhibited a relatively aggregated sheet-like structure, and at 50 000-fold observation (Fig. 2E(a–c)), it was found that LGP-1 had an ellipsoid of a relatively uniform size and a smooth surface. As a result of the particle size distribution chart, it was found that the particle diameter of LGP-1 was mainly distributed in the range of 0.3 to 0.9 µm, with the average value of 0.51 µm (Fig. 2E(d)).
The acid-hydrolyzed LGP-1 was analyzed in an amino acid automatic analyzer. The results showed that LGP-1 consisted of 16 amino acids, and the mass ratio is shown in Table 1. The high content of Asp, Gly and Pro was observed in LGP-1 and the absence of Met was remarked. Furthermore, for the amino acid composition of LGP-1, the high ratio of Ser and Thr constituting the O-glycopeptide bond was also remarked. Generally, LGP-1 contains 27.52% of essential amino acids (EAA).
| Amino acid species | Concent (nmol/20µL) | Amino acid species | Concent (nmol/20µL) | Amino acid species | Concent (nmol/20µL) |
|---|---|---|---|---|---|
| Asp | 2.755 | Ile | 0.502 | Cys | 0.412 |
| Thr | 1.176 | Leu | 1.436 | Arg | 0.769 |
| Ser | 1.751 | Tyr | 0.424 | Pro | 2.263 |
| Glu | 1.459 | Phe | 0.740 | Val | 1.462 |
| Gly | 2.226 | Lys | 1.307 | Met | – |
| Ala | 1.386 | His | 0.286 | ||
| AA | 5.602 | TAA | 20.354 | EAA/TAA | 27.52% |
Cell viability assay Fig. 3(A) showed the effect of LGP-1 on the viability of RAW264.7. The results showed that LGP-1 promoted the significant proliferation of RAW 264.7 and revealed a concentration-dependent effect. From the sample concentration point of view, the effect of LGP-1 on the cell viability of RAW 264.7 was significantly different from that of the control group at each concentration gradient (12.5, 25, 50, 100 and 200 µg/mL) (p < 0.05). However, there was no significant difference in loading concentration of 200µg/mL compared to 100 µg/mL, indicating that 100 µg/mL was the optimal concentration to promote RAW 264.7 proliferation. When the culture time was 48h, the concentration of 100 µg/mL of LGP-1 promoted the cell viability to a maximum of 188.21%.
Detection of cell viability of LGP-1. (A)Effect of LGP-1 on cell viability of RAW 264.7. When the culture time is 48h, the optimal concentration to promote the proliferation of RAW264.7 is 100 µg/mL, and the cell viability reaches 188.21%. (B) Effect of LGP-1 treatment for 48 h on NO emission. When the incubation time is 48h, the NO release amount of 100 µg/mL reaches 21.47 µmol/L. (C) Effect of LGP-1treatment for48 h on TNF-α emission. When the incubation time is 48 h, the NO release amount of 100 µg/mL reaches 21.47 µmol/L. (* p < 0.05, ** p < 0.01)
Based on that, the treatment time of RAW264.7 by LGP-1 was 48 h, and the release of NO and TNF was detected. The effect of LGP-1 on NO release from RAW 264.7 was shown in Fig. 3(B). The results showed that LGP-1 significantly promoted the ability of RAW 264.7 to release NO and showed a concentration-dependent relationship. From the concentration analysis, it was found that the five concentrations of LGP-1 were significantly different from the control group (p < 0.05), indicating that LGP-1 with different loading concentrations had the ability to activate NO in RAW 264.7. When the loading concentration was 100 µg/mL, the release amount of NO was maximized, which also indicated that 100 µg/mL was the optimum concentration to promote the NO release ability of RAW 264.7. When the culture time reached 48 h, the release amount of NO reached the maximum point at 21.47 µmol/L.
The effect of LGP-1 on TNF-α content is shown in Fig. 3(C). The results showed that the released amount of TNF-α reached a maximum value of 1 716.30 pg/mL when the concentration of NO was 100 µg/mL and the culture time was 48 h.
As a unique plant resource in Lanzhou, Lanzhou lily has brought huge economic benefits to surrounding growers and distributors. It has played a significant role in driving the economic development of Lanzhou and has gradually become a new term for Lanzhou. However, the development of its resources is still relatively primitive, and the study of its active ingredients is mainly based on polysaccharides. As an important biologically active macromolecule in the body, glycoprotein plays an important role in anti-oxidation, anti-tumor and hypoglycemic. Especially in the process of immunomodulation, many immunoregulatory factors are glycoproteins.
In this paper, Glycoprotein in Lanzhou lily was extracted and purified by Sephadex G-200 and DEAE-Cellulose 52 columns, and its structure and immunological activity were studied. After extraction and purification, two glycoprotein components were obtained. The homogeneity test results showed that LGP-1 was a homogeneous component sample and was linked by O-glycopeptide bonds.
As a kind of biologically active macromolecule existing in living organisms, glycoprotein has multiple biological activities. It plays an important role in immune regulation, and has a good development prospect in the prevention and treatment of some chronic diseases (Fan et al., 2014). In this paper, RAW 264.7 was employed as an experimental cell to investigate the immunological activity of LGP-1 on this cell.
Macrophages are one of the important immune cells playing a major role in immune function. They can not only directly phagocyte and eliminate foreign pathogenic microorganisms, but can also exert their immunological activities by secreting specific cytokines. Among them, NO and TNF-α are cytokines released by activated macrophages and play an irreplaceable role in the process of immune regulation. Therefore, the role of LGP-1 in immunoregulation can be reflected by measuring the cell viability, NO and TNF-α secretion of RAW264.7 (Barbara et al., 2021; Biswas and Mantovani, 2010).
Results of cell viability assay showed that LGP-1 can promote the proliferation of RAW264.7, and activate the cells to release cytokines such as NO and TNF-α to exert their immunomodulatory activities. The highest immunological activity observed when the sample reached 100 µg/mL at the culture time of 48 h.
This study provided a theoretical basis for the further development of the lily industry in Lanzhou, and promotes the development of the lily planting industry in Lanzhou. It also revealed that Glycoprotein in Lanzhou lily has great immunological activity for further development and being used into functional food.
Funding This work was supported by The National Natural Science Foundation of China (51873175).
Conflict of interest There are no conflicts of interest to declare.
Glycoprotein in Lanzhou lily
PBSPhosphate buffer saline
HPLCHigh performance liquid chromatography
FT-IRFourier transform infrared spectrometer
GC-MSGas chromatography-mass spectrometry
CDCircular dichroism
TFATrifluoroacetate
UV-VISUltraviolet-visible spectrometer
SEMScanning electron microscope
