- Email: [email protected]
The impact of N-glycosylation on conformation and stability of immunoglobulin Y from egg yolk
International Journal of Biological Macromolecules 96 (2017) 129–136
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
The impact of N-glycosylation on conformation and stability of immunoglobulin Y from egg yolk Long Sheng, Zhenjiao He, Jiahui Chen, Yaofa Liu, Meihu Ma, Zhaoxia Cai ∗ National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China
a r t i c l e
i n f o
Article history: Received 5 October 2016 Received in revised form 8 December 2016 Accepted 9 December 2016 Available online 14 December 2016 Keywords: Immunoglobulin Y N-glycosylation Conformation
a b s t r a c t Immunoglobulin Y (IgY) is a new therapeutic antibody, and its applications in industry are very broad. To provide insight into the effects of N-glycosylation on IgY, its conformation and stability were studied. In this research, IgY was extracted from egg yolk and then digested by peptide-N4-(Nacetyl-beta-glucosaminyl) asparagine-amidase. SDS-PAGE and infrared absorption spectrum showed that carbohydrates were distinctly reduced after enzymolysis. The circular dichroism spectrum indicated that the IgY molecule became more flexible and disordered after removal of N-glycan. The fluorescence intensity revealed that Trp residues were buried in a more hydrophobic environment after disposal of N-glycan. Storage stability decreased with the removal of oligosaccharide chains based on size-exclusion chromatography analysis. Deglycosylated IgY exhibited less resistance to guanidine hydrochlorideinduced unfolding. After deglycosylation, IgY was more sensitive to pepsin. Therefore, N-glycosylation played an important role in the maintenance of the structure and stability of IgY. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Chicken egg yolk immunoglobulin, also known as immunoglobulin Y (IgY), has attracted considerable attention due to its ability to prevent and control disease. Compared with mammalian immunoglobulin G (IgG), IgY provides plenty of advantages, including cost-effectiveness, convenience and high yield [1]. In the formation of the chicken egg, serum IgY from the laying hen is deposited in large quantities in the egg yolk to protect the developing embryo from potential pathogens [2]. IgY/IgG can be divided into two functional parts, the antigen binding fragment (Fab) and crystalline fragment (Fc) [3]. Oral administration of the IgY antibody has been shown to be effective against a variety of intestinal pathogens, such as bovine and human rotaviruses, enterotoxigenic Escherichia coli (ETEC), Salmonella spp., Edwardsiella tarda, Yersinia ruckeri, Staphylococcus and Pseudomonas [4]. Glycosylation is one of the most important post-translational modifications, and has a significant effect on the structure and functions of proteins. Oligosaccharides, especially the N-glycan of glycoprotein play an important role in cell recognition, signal transduction and receptor modulation. Hodoniczky, et al. reported
∗ Corresponding author at: College of Food Science and Technology of Huazhong Agricultural University, Wuhan, Hubei Province, PR China. E-mail address: [email protected] (Z. Cai). http://dx.doi.org/10.1016/j.ijbiomac.2016.12.043 0141-8130/© 2016 Elsevier B.V. All rights reserved.
that IgGs mediated antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity only when N-glycan was involved [5]. Due to the unique physical and chemical properties of therapeutic proteins, they are prone to a number of changes such as oxidation, deamination, aggregation, misfolding, and adsorption during storage [6]. These modifications could result in the potential loss of therapeutic efficacy or unwanted immune reactions [7]. Glycosylation is a vital factor for protein folding and structural integrity [8]. The Fc portion of IgG possesses one conserved glycosylation site at Asn297 in each of the CH 2 domains, where complex biantennary type N-glycan is essential for the conformation of IgG [9]. Sondermann, et al. reported that N-acetylneuraminic acid induced significant structural changes in the IgG molecule [10]. Ioannou, et al. found that glycosylation at Asn215 could restrain the exposure of hydrophobic surface area and cause aggregation of ␣-galactosidase A [11]. Weintraub, et al. found that hormothyrin easily gathered after deglycosylation [12]. Presently, the research concerning N-linked glycosylation of mammalian IgG has focused on galactosylated modification at a specific site [13]. As a glycoprotein, the effects of N-glycosylation on conformation and stability of IgY were poorly understood. IgY contained monoglucosylated oligomannose-type oligosaccharides, oligomannose-type oligosaccharides, and biantennary complextype oligosaccharides [14]. Suzuki and Lee found that each heavy chain contained two potential N-glycosylation sites located on
130
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136
the CH 2 and CH 3 domains in chicken IgG [15]. However, there is still a lack of information about the effects of N-glycosylation on the structure and stability of IgY. The glycans, which are bulky hydrophilic polymers, contribute to the high solubility of the protein and increase its stability against proteolysis. Moreover, the covalent binding of the glycans to the protein surface may inherently enhance the thermal and kinetic stability of the protein [16]. To prepare deglycosylated protein, approaches including chemistry methods, loci mutation and in vitro enzymatic hydrolysis are used. In vitro enzymatic hydrolysis is a relatively easy and low-cost procedure [17]. Generally, peptide-N4-(N-acetylbeta-glucosaminyl) asparagine-amidase (PNGase F) is the most frequently used enzyme for N-linked carbohydrate release because of its specific cleavage capability under mild conditions between the innermost core N-acetylglucosamine in the carbohydrate structures and its holding asparagine residue [18]. Thus, a basic understanding of the effect of the structural features of proteins on their physicochemical stability can provide fundamental solutions to the formation of IgY. In this paper, the impact of N-glycosylation on the conformation and stability of IgY from egg yolk was investigated. Deglycosylated IgY was prepared from IgY by PNGase F treatment and purified by chromatography. The changes in protein conformation before and after deglycosylation were analyzed by spectrum technology. Meanwhile, the stability of the protein molecule such as storage stability, anti-enzyme ability and resistance of guanidine hydrochloride denaturation were also explored.
and diluted with two volumes of PBS (pH 7.5). Afterwards, 3.5% PEG 6000 (w/v) of the total volume was added to dilute yolk. The mixture was rolled for 20 min at 4 ◦ C and centrifuged at 10,000g for 20 min at 4 ◦ C. Hereafter, 8.5% PEG 6000 (w/v) of the total volume was added to the supernatant, rolled and centrifuged as the above way. The precipitate was collected and dissolved in 50 mM phosphate buffer (pH 7.5). A total of 12.5% PEG 6000 (w/v) was added to the dissolved solution followed by 20 min rolling at 4 ◦ C. The mixture was centrifuged at 10,000g for 20 min at 4 ◦ C, and the precipitate was collected for further purification by HiPREPTM 26/60 SEPHACRYL S-300 HR (GE, USA) gel permeation chromatography. The sample injection volume was 3–5% of the column volume. Twenty mM phosphate buffer (pH 7.5) was used as a mobile phase at a flow rate of 1 mL/min. Protein detection was at 280 nm. Samples were collected and freeze-dried (Model: FreeZone6L, Labconco, America). 2.2. Purification of deglycosylated IgY PNGase F (New England Biolabs, cat. P0705L) was used to remove the N-glycan by incubating 1 mg of IgY with 800 units of PNGase F at 37 ◦ C for 48 h. After digestion, DEAE Sepharose FF (GE, USA) chromatography was used for purification. The sample injection volume was 1 mL. A total of 50 mM phosphate buffer (pH 7.5) was used as a mobile phase at a flow rate of 2 mL/min. After the first eluting peak ended, 1 M NaCl dissolved in 50 mM PBS (pH 7.5) was used as a mobile phase at a flow rate of 2 mL/min. Protein detection was at 280 nm. Samples were collected and freeze-dried.
2. Materials and methods 2.3. SDS-PAGE 2.1. Purification of IgY from egg yolk Fresh eggs laid within 24 h were purchased from a local hennery (Jiufeng Farm, Wuhan, China). IgY was prepared from egg yolk according to a previously reported method [19]. The eggshell was carefully cracked, and the yolk was transferred to a “yolk spoon” to remove as much egg white as possible. The yolk was transferred to a filter paper and rolled to remove remaining egg white, and then the yolk membrane was cut with a lancet. The yolk was collected
SDS-PAGE was performed under both reducing and nonreducing conditions. SDS gel electrophoresis was performed in 1.0 M Tris-HCl buffer (pH 6.8) for stacking gel (4% acrylamide) and 1.5 M Tris-HCl buffer (pH 8.8) for separating gel (12% acryl-amide). Migration was performed at 80 V in the stacking gel and at 120 V in the separating gel. Ten g protein sample was loaded on the gel. After migration, the gel was stained with Coomassie brilliant blue R-250 composed of 25% ethanol and mixed with 8% acetic acid for 30 min.
Fig. 1. The anion exchange chromatography of isolation and enrichment of deglycosylated IgY.
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136
131
The destaining step was carried out after immersion of the gels in 7% acetic acid. 2.4. Fourier transform infrared spectrum FT-IR spectrum of representative sample was collected in KBr pellets on an infrared spectrometer (Perkin-Elmer 16 PC spectrometer, Boston, USA) over a wavelength range of 500–4000 cm−1 . 2.5. Circular dichroism spectrum Circular dichroism (CD) spectra were taken using a Jasco-810 CD spectropolarimeter (Easton, MD). Far-UV CD spectra of 0.2 mg/mL of protein in 50 mM phosphate buffer (pH 7.5) were recorded at 25 ◦ C in the range from 190 to 250 nm with a spectral resolution of 0.1 nm. The scan speed was 100 nm/s and the response time was 0.125 s with a bandwidth of 1 nm. Quartz cells with an optical path of 0.1 cm were used. Typically, 16 scans were accumulated and subsequently averaged. A total of three spectra were accumulated and averaged. 2.6. Fluorescence spectrum The fluorescence intensity was measured at an excitation wavelength of 295 nm and an emission wavelength from 300 to 450 nm using a RF-1501 Fluorescence spectrophotometer (Shimadzu, Kyoto, Japan). 2.7. Size-exclusion liquid chromatography (SEC-HPLC) Separation of proteins was carried out on a Tosoh TSKgel super SW3000 column on a Waters e2695 high-pressure liquid chromatograph. The column temperature was 22 ◦ C and 50 mM phosphate buffer (pH 7.5) was used as a mobile phase at a flow rate of 0.9 mL/min. The sample concentration and injection volume were 1.0 mg/mL and 5 L, respectively. Protein detection was at 280 nm. 2.8. Second derivative UV absorbance spectroscopy UV spectra of the antibodies were recorded using a NanoDrop 2000 UV–vis spectrophotometer during protein unfolding. An 80 L aliquot of a 0.5 mg/mL sample with 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 or 4.0 M of guanidine hydrochloride (GdnHCl) was loaded into a 1 cm pathlength quartz cuvette. Samples were equilibrated in GdnHCl solutions for 5 min before the UV measurement. Spectra were collected from 240 to 320 nm at room temperature and a buffer blank spectrum was subtracted. Origin 7.0 software was used to calculate second derivative spectra. 2.9. Anti-pepsin activity The protein sample was digested by pepsin digestion in 20 mM sodium acetate, pH 4.0 buffer at 37 ◦ C; the protein concentration during digestion was 1 mg/mL. The ratio of protein to pepsin (Sigma) was 40:1 (w/w) and 10:1 (w/w). Digested samples were collected at 15 and 30 min, and at 1, 1.5, 2, 4, 6, 8, 16, and 24 h. The digestion was quenched by adding 2 M tris base with 1/20 of the total volume. Native SDS-PAGE was used to measure the digestion of protein. 3. Results and discussion 3.1. Preparation of deglycosylation IgY Due to incomplete enzymolysis, the products still had a few IgY remaining. Anion exchange chromatography was used to separated
Fig. 2. The SDS-PAGE analysis of IgY and deglycosylated IgY. Lane 1, protein marker; lane 2, IgY; lane 3, deglycosylated IgY.
deglycosylated IgY from residual native IgY. As shown in Fig. 1, two elution peaks appeared with increased NaCl concentration. The component of peak 1 belonged to protein incapable of binding with the weak anion-exchange packing. Peak 2 only appeared under a high concentration of NaCl. SDS-PAGE of these two components is shown in Fig. 2. IgY consisted of two identical heavy chains at approximately 67 kDa and two identical light chains at approximately 25 kDa. Disulfide bridges among the chains were broken by mercaptoethanol from the loading buffer. IgY fractured into four chains due to the reduction of disulfide bonds at cysteine residues (C252, C340, C347) [20]. Compared with the native IgY (lane 2), the molecular weight of deglycosylated IgY (lane 3) was clearly reduced. N-linked carbohydrate chains were removed during the enzyme reaction. Galactosylated modification could affect the surface charge and isoelectric point of the protein [21]. The isoelectric point of native IgY was 5.2, and the ionic bonds between IgY and anion exchange packing was stronger because of the positively charged surfaces of IgY. Therefore, these characteristics helped us determine that peak 1 (lane 3 in SDSPAGE) was deglycosylated IgY and peak 2 (lane 2 in SDS-PAGE) was IgY.
132
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136
Fig. 3. FTIR spectrum characterization of IgY and deglycosylated IgY.
3.2. FT-IR spectrum The effect of the enzymatic deglycosylation on the intensity of the FT-IR spectra was investigated (Fig. 3). The spectra for IgY as well as that for deglycosylated IgY exhibited similar features. The amide I band (1700–1600 cm−1 ) and the amide II band (1580–1510 cm−1 ) were the most easily characterized structures of protein. Two strong peaks at approximately 1650 cm−1 and 1550 cm−1 were characteristic of Amide I and Amide II, respectively. Amide I was derived from C O stretching, while N H bending and C N stretching resulted in Amide II [22]. A broad band
at approximately 3400 cm−1 was attributed to O H stretching. A sharp band that appeared at 2900 cm−1 was due to C H stretching. Remarkable differences were observed in the intensity of the band at 1200–900 cm−1 . This band represented absorption of the protein-associated sugar chains. When compared to the absorption spectra of the protein before enzymatic treatment, deglycosylated IgY showed a drastic reduction in band intensity, which confirmed that the 1200–900 cm−1 band was due to the absorption of protein carbohydrates. These results were consistent with a previous report [23]. Natalello also found that reduced intensity of the band
Fig. 4. The far-UV CD of IgY and deglycosylated IgY. Protein solutions (0.2 mg/mL) were prepared in 50 mM phosphate buffer (pH 7.5) was measured with a 0.1 cm cell in the far-UV region. The wavelength was from 190 to 250 nm.
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136
133
Fig. 5. The intrinsic fluorescence of IgY and deglycosylated IgY. The excitation wavelength was 280 nm, and emission was from 300 to 450 nm. Protein solutions (0.1 mg/mL) were prepared in 20 mM phosphate buffer (pH 7.5).
at 1200–900 cm−1 was also observable in the FTIR spectrum of the PNGase F-treated protein [23].
3.3. Circular dichroism spectrum Fig. 4 shows the far-UV CD spectrum of IgY and deglycosylated IgY. A negative minimum at 217 nm and zero at 208 nm suggested the prominent -sheet content in IgY. The -sheet content of native IgY was approximately 42%, which was consistent with previous reports. Liu, et al. reported that native IgY contained approximately 45% -sheet content [24]. A positive band was also observed at195 nm, which suggested an ␣-helix structure in IgY. It was clear that IgY underwent a dramatic conformational change
Table 1 Calculation of secondary structures of IgY and deglycosylated IgY. Sample
␣-helix (%)
-sheet (%)
-turn (%)
Random (%)
IgY Deglycosylated IgY
30.9 23.2
41.8 23.3
22.3 25
5 28.5
after PNGase F-treatment. As shown in Table 1, the ␣-helix and -sheet content decreased to 23.2% and 23.3%, respectively. Meanwhile, the percentage of random coil increased from 5% to 28.5%. This phenomenon indicated that the IgY molecule became more flexible and disordered after removal of N-glycan. Hence, galactosylated modification was conducive to maintaining stability of IgY secondary structure. Jafari-Aghdam, et al. studied the effect of
Fig. 6. The SEC-HPLC analysis of aggregation and fragment of IgY and deglycosylated IgY.
134
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136
Fig. 7. IgY unfolding monitored by second derivative ultraviolet absorbance spectroscopy. (A) The UV-vis spectrum of IgY. (B) The 2nd derivative of UV-vis spectrum of IgY. (C) The 2nd derivative UV-vis spectrum monitoring GdnHCl-induced IgY unfolding.
deglycosylation on the structure of glucoamylase from Aspergillus niger. They found that the secondary structure of protein did not change after deglycosylation [25]. These differences might be due to different glycosylation sites on the proteins. 3.4. Fluorescence spectrum More information about protein folding at tertiary level can be obtained by studying the intrinsic fluorescence of protein. The intrinsic fluorescence of the protein came mainly from tryptophan (Trp) residues, which were very sensitive to changes in the microenvironment. The fluorescence emission spectra of IgY before and after deglycosylation were examined (Fig. 5). The intensity of deglycosylated IgY was lower than that of native IgY. The polarity of the environment surrounding the Trp residues affected the fluorescence intensity. This change indicated that the Trp residues were buried in a more hydrophobic environment after disposal of N-glycan.
3.5. SEC-HPLC Size exclusion chromatography (SEC) is a historical technique widely employed for the detailed characterization of therapeutic proteins. It can also be used as a reference and powerful technique for the qualitative and quantitative evaluation of aggregates and fragments [6]. The SEC-HPLC analysis of aggregation and fragmentation of native and deglycosylated IgY after a storage duration of 15 days at 4 ◦ C is shown in Fig. 6. The peak at 7.5–9.0 min represented monomeric IgY. The elution time of monomeric deglycosylated IgY occurred earlier than that of monomeric IgY. Low peaks at approximately 6.5–7.5 min represented the dimer and polymer of IgY. A series of peaks between 13.2 and 17.5 min represented the fragments of IgY. Apparently, both polymerization and fragmentation were enhanced after deglycosylation, especially the fragmentation. Therefore, N-glycan could help maintain the full and stable molecular structure of IgY.
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136
135
Fig. 8. The SDS-PAGE analysis of the pepsin hydrolysate of IgY and deglycosylated IgY. (A) the ratio of IgY to pepsin was 1:10 (w/w). (B) the ratio of deglycosylated IgY to pepsin was 1:10 (w/w). (C) the ratio of IgY to pepsin was 1:40 (w/w). M, protein marker; 1, incubating 15 min; 2, incubating 30 min; 3, incubating 1 h; 4, incubating 1.5 h; 5, incubating 2 h; 6, incubating 4 h; 7, incubating 6 h; 8, incubating 8 h; 9, incubating 16 h; 10, incubating 24 h.
3.6. GdnHCl induced denaturation Denaturant can cause protein unfolding and destroy the biologically active conformation of the protein. UV spectroscopy was used to monitor IgY unfolding induced by GdnHCl. After collecting high resolution UV absorbance spectrum of IgY (Fig. 7A), the second derivative of spectrum was calculated for resolution enhancement (Fig. 7B). Five negative peaks in the second derivative UV spectrum were from Phe (252, 258, and 267 nm), Tyr/Trp (283 nm), and Trp (290 nm) [26]. In this study, the negative peak approximately 290 nm (Fig. 7C) from the second derivative UV was chosen as a marker to monitor the changes in the microenvironment of Trp residues [27]. GdnHClinduced unfolding of glycosylated and deglycosylated IgY is shown in Fig. 7C. The midpoint of the IgY structural transition during the unfolding process was 291.7 nm, and the corresponding concentration was 2.6 M GdnHCl. Deglycosylated IgY was less resistant to GdnHCl-induced unfolding and the midpoint of the transition (291.5 nm) occurred at a concentration of GdnHCl that was lower by approximately 0.6 M. This result demonstrated that the N-glycan of IgY conferred stability during denaturation of the molecule.
also reported that the oligosaccharide chain at the Fc domain improved resistance to protease hydrolysis [32]. 4. Conclusions The results revealed that the removal of N-glycan changed the conformation, storage stability, and resistance to GdnHCl and pepsin digestion of the IgY molecule. It was obvious that Nglycosylation affected IgY stability because of the altered protein structure. It was concluded that a structural role for N-glycosylation in the IgY molecule and provide a basis for studying the role of glycoproteins in molecular stability. Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant No.31371810) and the Fundamental Research Funds for the Central Universities (Program No.2662015PY080; Program No.2662015BQ042). References
3.7. Anti-enzyme ability Pepsin is an enzyme expressed as a prototype of zymogen and pepsinogen and is released by the chief cells in the stomach. It digests food proteins by cleaving peptide bonds and was the first discovered animal enzyme [28]. Pepsin has a preference for cleaving between hydrophobic and aromatic amino acids such as phenylalanine or leucine [29]. Pepsin was commonly employed for limited proteolytic fragmentation of IgG, to generate a Fab fragment devoid of the Fc effector domain [30]. The SDS-PAGE results of the digestion of IgY is shown in Fig. 8. After 24 h of enzymatic hydrolysis with a ratio of protein to pepsin of 1:40 (w/w), a large amount of integrated IgY molecule was still present and the bands of hydrolyzed peptides were not obvious (Fig. 8A). With a ratio of protein to pepsin of 1:10 (w/w), IgY was gradually degraded. After the mixture was heated at 37 ◦ C following a 60 min incubation, a new 45 kDa band emerged (Fig. 8C). Compared with IgY, the band of deglycosylated IgY gradually disappeared when the ratio of protein to pepsin was 1:40 (w/w). Meanwhile, a new band of approximately 45 kDa appeared after a 6 h incubation (Fig. 8B). These new bands were attributed to the formation of the fragment Fab. Plenty of hydrophobic amino acid residues were located in C3 domain of IgY, which also contained N-glycosylation [31]. It seemed that the exposed hydrophobic amino acid residues were more easily attacked by pepsin. These results demonstrated that N-glycosylation protected IgY against pepsin hydrolysis. Raju et al.
[1] D. Carlander, H. Kollberg, P.E. Wejaker, A. Larsson, Peroral immunotherapy with yolk antibodies for the prevention and treatment of enteric infections, Immunol. Res. 21 (2000) 1–6. [2] A.K. Janson, C.I. Smith, L. Hammarstrom, Biological properties of yolk immunoglobulins, Adv. Exp. Med. Biol. 371 (1995) 685–690. [3] L. Jiang, W.S. Chang, H. Guo, P. Sun, X.M. Dai, Structure analysis of goose immunoglobulin Y Fc fragment, Cent. Eur. J. Immunol. 3 (2013) 299–304. [4] Y. Mine, J. Kovacs-Nolan, Chicken egg yolk antibodies as therapeutics in enteric infectious disease: a review, J. Med. Food 5 (2002) 159–169. [5] J. Hodoniczky, Y.Z. Zheng, D.C. James, Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro, Biotechnol. Progr. 21 (2005) 1644–1652. [6] S. Fekete, A. Beck, J.L. Veuthey, D. Guillarme, Theory and practice of size exclusion chromatography for the analysis of protein aggregates, J. Pharm. Biomed. Anal. 101 (2014) 161–173. [7] A. Diress, B. Lorbetskie, L. Larocque, X. Li, M. Alteen, R. Isbrucker, M. Girard, Study of aggregation, denaturation and reduction of interferon alpha-2 products by size-exclusion high-performance liquid chromatography with fluorescence detection and biological assays, J. Chromatogr. A 1217 (2010) 3297–3306. [8] V. Kayser, N. Chennamsetty, V. Voynov, K. Forrer, B. Helk, B.L. Trout, Glycosylation influences on the aggregation propensity of therapeutic monoclonal antibodies, Biotechnol. J. 6 (2011) 38–44. [9] S. Matsumiya, Y. Yamaguchi, J.I. Saito, M. Nagano, H. Sasakawa, S. Otaki, M. Satoh, K. Shitara, K. Kato, Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1, J. Mol. Biol. 368 (2007) 767–779. [10] P. Sondermann, A. Pincetic, J. Maamary, K. Lammens, J.V. Ravetch, General mechanism for modulating immunoglobulin effector function, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 9868–9872. [11] Y.A. Ioannou, K.M. Zeidner, M.E. Grace, R.J. Desnick, Human alpha-galactosidase A: glycosylation site 3 is essential for enzyme solubility, Biochem. J 332 (1998) 789–797. [12] B.D. Weintraub, B.S. Stannard, L. Meyers, Glycosylation of thyroid-stimulating hormone in pituitary tumor cells: influence of high mannose oligosaccharide
136
[13]
[14]
[15] [16]
[17]
[18]
[19]
[20]
[21]
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136 units on subunit aggregation, combination, and intracellular degradation, Endocrinology 112 (1983) 1331–1345. E.L. Smith, J.P. Giddens, A.T. Iavarone, G. Kamil, L.X. Wang, C.R. Bertozzi, Chemoenzymatic Fc glycosylation via engineered aldehyde tags, Bioconjugate Chem. 25 (2014) 788–795. M. Ohta, J. Hamako, S. Yamamoto, H. Hatta, M. Kim, T. Yamamoto, S. Oka, T. Mizuochi, F. Matsuura, Structures of asparagine-linked oligosaccharides from hen egg-yolk antibody (IgY). Occurrence of unusual glucosylated oligo-mannose type oligosaccharides in a mature glycoprotein, Glycoconj. J. 8 (1991) 400–413. N. Suzuki, Y.C. Lee, Site-specific N-glycosylation of chicken serum IgG, Glycobiology 14 (2004) 275–292. D. Shental-Bechor, Y. Levy, Effect of glycosylation on protein folding: a close look at thermodynamic stabilization Dalit, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 8256–8261. Y. Miyake, S. Tsuzuki, S. Mochida, T. Fushiki, K. Inouye, The role of asparagine-linked glycosylation site on the catalytic domain of matriptase in its zymogen activation, Biochim. et Biophys. Acta-Proteins Proteomics 1804 (2010) 156–165. F. Maley, R.B. Trimble, A.L. Tarentino, T.H. Plummer, Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases, Anal. Biochem. 180 (1989) 195–204. D. Pauly, P.A. Chacana, E.G. Calzado, B. Brembs, R. Schade, IgY technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation, J. Visualized Exp. 51 (2011), e3084-e3084. B. Haeduck, H. Honda, R. Murota, M. Kobayashi, F. Horio, A. Murai, Production of recombinant chicken IgY-Fc and evaluation of its transport ability into avian egg yolks, J. Poult. Sci. 47 (2010) 256–261. R.J. Sola, K. Griebenow, Effects of glycosylation on the stability of protein pharmaceuticals, J. Pharm. Sci. 98 (2009) 1223–1245.
[22] P.I. Haris, F. Severcan, FTIR spectroscopic characterization of protein structure in aqueous and non-aqueous media, J. Mol. Catal. B Enzym. 7 (1999) 207–221. [23] A. Natalello, D. Ami, S. Brocca, M. Lotti, S.M. Doglia, Secondary structure, conformational stability and glycosylation of a recombinant Candida rugosa lipase studied by Fourier-transform infrared spectroscopy, Biochem. J 385 (2005) 511–517. [24] J. Liu, J. Yang, H. Xu, J. Lu, Z. Cui, A new membrane based process to isolate immunoglobulin from chicken egg yolk, Food Chem. 122 (2010) 747–752. [25] J. Jafari-Aghdam, K. Khajeh, B. Ranjbar, M. Nemat-Gorgani, Deglycosylation of glucoamylase from Aspergillus niger: effects on structure activity and stability, Biochim. et Biophys. Acta-Proteins Proteomics 1750 (2005) 61–68. [26] K. Zheng, Biophysical Characterization of the EC5 Domain of E-cadherin: Conformation, Physical Stability, and Binding Properties. Ph.D. Thesis, Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence: The University of Kansas, 2007. [27] K. Zheng, C.R. Middaugh, T.J. Siahaan, Evaluation of the physical stability of the EC5 domain of E-cadherin: effects of pH, temperature, ionic strength and disulfide bonds, J. Pharm. Sci. 98 (2009) 63–73. [28] Aszmann, The life and work of theodore schwann, J. Reconstr. Microsurg. 16 (2000) 291–296. [29] D.W. Piper, B.H. Fenton, pH stability and activity curves of pepsin with special reference to their clinical importance, Gut 6 (1965) 506–508. [30] D. Yu, R. Ghosh, Method for studying immunoglobulin G binding on hydrophobic surfaces, Langmuir 26 (2010) 924–929. [31] A.I. Taylor, S.M. Fabiane, B.J. Sutton, R.A. Calvert, The crystal structure of an avian IgY-Fc fragment reveals conservation with both mammalian IgG and IgE, Biochemistry 48 (2008) 558–562. [32] T.S. Raju, B.J. Scallon, Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain, Biochem. Biophys. Res. Commun. 341 (2006) 797–803.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
The impact of N-glycosylation on conformation and stability of immunoglobulin Y from egg yolk Long Sheng, Zhenjiao He, Jiahui Chen, Yaofa Liu, Meihu Ma, Zhaoxia Cai ∗ National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China
a r t i c l e
i n f o
Article history: Received 5 October 2016 Received in revised form 8 December 2016 Accepted 9 December 2016 Available online 14 December 2016 Keywords: Immunoglobulin Y N-glycosylation Conformation
a b s t r a c t Immunoglobulin Y (IgY) is a new therapeutic antibody, and its applications in industry are very broad. To provide insight into the effects of N-glycosylation on IgY, its conformation and stability were studied. In this research, IgY was extracted from egg yolk and then digested by peptide-N4-(Nacetyl-beta-glucosaminyl) asparagine-amidase. SDS-PAGE and infrared absorption spectrum showed that carbohydrates were distinctly reduced after enzymolysis. The circular dichroism spectrum indicated that the IgY molecule became more flexible and disordered after removal of N-glycan. The fluorescence intensity revealed that Trp residues were buried in a more hydrophobic environment after disposal of N-glycan. Storage stability decreased with the removal of oligosaccharide chains based on size-exclusion chromatography analysis. Deglycosylated IgY exhibited less resistance to guanidine hydrochlorideinduced unfolding. After deglycosylation, IgY was more sensitive to pepsin. Therefore, N-glycosylation played an important role in the maintenance of the structure and stability of IgY. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Chicken egg yolk immunoglobulin, also known as immunoglobulin Y (IgY), has attracted considerable attention due to its ability to prevent and control disease. Compared with mammalian immunoglobulin G (IgG), IgY provides plenty of advantages, including cost-effectiveness, convenience and high yield [1]. In the formation of the chicken egg, serum IgY from the laying hen is deposited in large quantities in the egg yolk to protect the developing embryo from potential pathogens [2]. IgY/IgG can be divided into two functional parts, the antigen binding fragment (Fab) and crystalline fragment (Fc) [3]. Oral administration of the IgY antibody has been shown to be effective against a variety of intestinal pathogens, such as bovine and human rotaviruses, enterotoxigenic Escherichia coli (ETEC), Salmonella spp., Edwardsiella tarda, Yersinia ruckeri, Staphylococcus and Pseudomonas [4]. Glycosylation is one of the most important post-translational modifications, and has a significant effect on the structure and functions of proteins. Oligosaccharides, especially the N-glycan of glycoprotein play an important role in cell recognition, signal transduction and receptor modulation. Hodoniczky, et al. reported
∗ Corresponding author at: College of Food Science and Technology of Huazhong Agricultural University, Wuhan, Hubei Province, PR China. E-mail address: [email protected] (Z. Cai). http://dx.doi.org/10.1016/j.ijbiomac.2016.12.043 0141-8130/© 2016 Elsevier B.V. All rights reserved.
that IgGs mediated antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity only when N-glycan was involved [5]. Due to the unique physical and chemical properties of therapeutic proteins, they are prone to a number of changes such as oxidation, deamination, aggregation, misfolding, and adsorption during storage [6]. These modifications could result in the potential loss of therapeutic efficacy or unwanted immune reactions [7]. Glycosylation is a vital factor for protein folding and structural integrity [8]. The Fc portion of IgG possesses one conserved glycosylation site at Asn297 in each of the CH 2 domains, where complex biantennary type N-glycan is essential for the conformation of IgG [9]. Sondermann, et al. reported that N-acetylneuraminic acid induced significant structural changes in the IgG molecule [10]. Ioannou, et al. found that glycosylation at Asn215 could restrain the exposure of hydrophobic surface area and cause aggregation of ␣-galactosidase A [11]. Weintraub, et al. found that hormothyrin easily gathered after deglycosylation [12]. Presently, the research concerning N-linked glycosylation of mammalian IgG has focused on galactosylated modification at a specific site [13]. As a glycoprotein, the effects of N-glycosylation on conformation and stability of IgY were poorly understood. IgY contained monoglucosylated oligomannose-type oligosaccharides, oligomannose-type oligosaccharides, and biantennary complextype oligosaccharides [14]. Suzuki and Lee found that each heavy chain contained two potential N-glycosylation sites located on
130
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136
the CH 2 and CH 3 domains in chicken IgG [15]. However, there is still a lack of information about the effects of N-glycosylation on the structure and stability of IgY. The glycans, which are bulky hydrophilic polymers, contribute to the high solubility of the protein and increase its stability against proteolysis. Moreover, the covalent binding of the glycans to the protein surface may inherently enhance the thermal and kinetic stability of the protein [16]. To prepare deglycosylated protein, approaches including chemistry methods, loci mutation and in vitro enzymatic hydrolysis are used. In vitro enzymatic hydrolysis is a relatively easy and low-cost procedure [17]. Generally, peptide-N4-(N-acetylbeta-glucosaminyl) asparagine-amidase (PNGase F) is the most frequently used enzyme for N-linked carbohydrate release because of its specific cleavage capability under mild conditions between the innermost core N-acetylglucosamine in the carbohydrate structures and its holding asparagine residue [18]. Thus, a basic understanding of the effect of the structural features of proteins on their physicochemical stability can provide fundamental solutions to the formation of IgY. In this paper, the impact of N-glycosylation on the conformation and stability of IgY from egg yolk was investigated. Deglycosylated IgY was prepared from IgY by PNGase F treatment and purified by chromatography. The changes in protein conformation before and after deglycosylation were analyzed by spectrum technology. Meanwhile, the stability of the protein molecule such as storage stability, anti-enzyme ability and resistance of guanidine hydrochloride denaturation were also explored.
and diluted with two volumes of PBS (pH 7.5). Afterwards, 3.5% PEG 6000 (w/v) of the total volume was added to dilute yolk. The mixture was rolled for 20 min at 4 ◦ C and centrifuged at 10,000g for 20 min at 4 ◦ C. Hereafter, 8.5% PEG 6000 (w/v) of the total volume was added to the supernatant, rolled and centrifuged as the above way. The precipitate was collected and dissolved in 50 mM phosphate buffer (pH 7.5). A total of 12.5% PEG 6000 (w/v) was added to the dissolved solution followed by 20 min rolling at 4 ◦ C. The mixture was centrifuged at 10,000g for 20 min at 4 ◦ C, and the precipitate was collected for further purification by HiPREPTM 26/60 SEPHACRYL S-300 HR (GE, USA) gel permeation chromatography. The sample injection volume was 3–5% of the column volume. Twenty mM phosphate buffer (pH 7.5) was used as a mobile phase at a flow rate of 1 mL/min. Protein detection was at 280 nm. Samples were collected and freeze-dried (Model: FreeZone6L, Labconco, America). 2.2. Purification of deglycosylated IgY PNGase F (New England Biolabs, cat. P0705L) was used to remove the N-glycan by incubating 1 mg of IgY with 800 units of PNGase F at 37 ◦ C for 48 h. After digestion, DEAE Sepharose FF (GE, USA) chromatography was used for purification. The sample injection volume was 1 mL. A total of 50 mM phosphate buffer (pH 7.5) was used as a mobile phase at a flow rate of 2 mL/min. After the first eluting peak ended, 1 M NaCl dissolved in 50 mM PBS (pH 7.5) was used as a mobile phase at a flow rate of 2 mL/min. Protein detection was at 280 nm. Samples were collected and freeze-dried.
2. Materials and methods 2.3. SDS-PAGE 2.1. Purification of IgY from egg yolk Fresh eggs laid within 24 h were purchased from a local hennery (Jiufeng Farm, Wuhan, China). IgY was prepared from egg yolk according to a previously reported method [19]. The eggshell was carefully cracked, and the yolk was transferred to a “yolk spoon” to remove as much egg white as possible. The yolk was transferred to a filter paper and rolled to remove remaining egg white, and then the yolk membrane was cut with a lancet. The yolk was collected
SDS-PAGE was performed under both reducing and nonreducing conditions. SDS gel electrophoresis was performed in 1.0 M Tris-HCl buffer (pH 6.8) for stacking gel (4% acrylamide) and 1.5 M Tris-HCl buffer (pH 8.8) for separating gel (12% acryl-amide). Migration was performed at 80 V in the stacking gel and at 120 V in the separating gel. Ten g protein sample was loaded on the gel. After migration, the gel was stained with Coomassie brilliant blue R-250 composed of 25% ethanol and mixed with 8% acetic acid for 30 min.
Fig. 1. The anion exchange chromatography of isolation and enrichment of deglycosylated IgY.
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136
131
The destaining step was carried out after immersion of the gels in 7% acetic acid. 2.4. Fourier transform infrared spectrum FT-IR spectrum of representative sample was collected in KBr pellets on an infrared spectrometer (Perkin-Elmer 16 PC spectrometer, Boston, USA) over a wavelength range of 500–4000 cm−1 . 2.5. Circular dichroism spectrum Circular dichroism (CD) spectra were taken using a Jasco-810 CD spectropolarimeter (Easton, MD). Far-UV CD spectra of 0.2 mg/mL of protein in 50 mM phosphate buffer (pH 7.5) were recorded at 25 ◦ C in the range from 190 to 250 nm with a spectral resolution of 0.1 nm. The scan speed was 100 nm/s and the response time was 0.125 s with a bandwidth of 1 nm. Quartz cells with an optical path of 0.1 cm were used. Typically, 16 scans were accumulated and subsequently averaged. A total of three spectra were accumulated and averaged. 2.6. Fluorescence spectrum The fluorescence intensity was measured at an excitation wavelength of 295 nm and an emission wavelength from 300 to 450 nm using a RF-1501 Fluorescence spectrophotometer (Shimadzu, Kyoto, Japan). 2.7. Size-exclusion liquid chromatography (SEC-HPLC) Separation of proteins was carried out on a Tosoh TSKgel super SW3000 column on a Waters e2695 high-pressure liquid chromatograph. The column temperature was 22 ◦ C and 50 mM phosphate buffer (pH 7.5) was used as a mobile phase at a flow rate of 0.9 mL/min. The sample concentration and injection volume were 1.0 mg/mL and 5 L, respectively. Protein detection was at 280 nm. 2.8. Second derivative UV absorbance spectroscopy UV spectra of the antibodies were recorded using a NanoDrop 2000 UV–vis spectrophotometer during protein unfolding. An 80 L aliquot of a 0.5 mg/mL sample with 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 or 4.0 M of guanidine hydrochloride (GdnHCl) was loaded into a 1 cm pathlength quartz cuvette. Samples were equilibrated in GdnHCl solutions for 5 min before the UV measurement. Spectra were collected from 240 to 320 nm at room temperature and a buffer blank spectrum was subtracted. Origin 7.0 software was used to calculate second derivative spectra. 2.9. Anti-pepsin activity The protein sample was digested by pepsin digestion in 20 mM sodium acetate, pH 4.0 buffer at 37 ◦ C; the protein concentration during digestion was 1 mg/mL. The ratio of protein to pepsin (Sigma) was 40:1 (w/w) and 10:1 (w/w). Digested samples were collected at 15 and 30 min, and at 1, 1.5, 2, 4, 6, 8, 16, and 24 h. The digestion was quenched by adding 2 M tris base with 1/20 of the total volume. Native SDS-PAGE was used to measure the digestion of protein. 3. Results and discussion 3.1. Preparation of deglycosylation IgY Due to incomplete enzymolysis, the products still had a few IgY remaining. Anion exchange chromatography was used to separated
Fig. 2. The SDS-PAGE analysis of IgY and deglycosylated IgY. Lane 1, protein marker; lane 2, IgY; lane 3, deglycosylated IgY.
deglycosylated IgY from residual native IgY. As shown in Fig. 1, two elution peaks appeared with increased NaCl concentration. The component of peak 1 belonged to protein incapable of binding with the weak anion-exchange packing. Peak 2 only appeared under a high concentration of NaCl. SDS-PAGE of these two components is shown in Fig. 2. IgY consisted of two identical heavy chains at approximately 67 kDa and two identical light chains at approximately 25 kDa. Disulfide bridges among the chains were broken by mercaptoethanol from the loading buffer. IgY fractured into four chains due to the reduction of disulfide bonds at cysteine residues (C252, C340, C347) [20]. Compared with the native IgY (lane 2), the molecular weight of deglycosylated IgY (lane 3) was clearly reduced. N-linked carbohydrate chains were removed during the enzyme reaction. Galactosylated modification could affect the surface charge and isoelectric point of the protein [21]. The isoelectric point of native IgY was 5.2, and the ionic bonds between IgY and anion exchange packing was stronger because of the positively charged surfaces of IgY. Therefore, these characteristics helped us determine that peak 1 (lane 3 in SDSPAGE) was deglycosylated IgY and peak 2 (lane 2 in SDS-PAGE) was IgY.
132
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136
Fig. 3. FTIR spectrum characterization of IgY and deglycosylated IgY.
3.2. FT-IR spectrum The effect of the enzymatic deglycosylation on the intensity of the FT-IR spectra was investigated (Fig. 3). The spectra for IgY as well as that for deglycosylated IgY exhibited similar features. The amide I band (1700–1600 cm−1 ) and the amide II band (1580–1510 cm−1 ) were the most easily characterized structures of protein. Two strong peaks at approximately 1650 cm−1 and 1550 cm−1 were characteristic of Amide I and Amide II, respectively. Amide I was derived from C O stretching, while N H bending and C N stretching resulted in Amide II [22]. A broad band
at approximately 3400 cm−1 was attributed to O H stretching. A sharp band that appeared at 2900 cm−1 was due to C H stretching. Remarkable differences were observed in the intensity of the band at 1200–900 cm−1 . This band represented absorption of the protein-associated sugar chains. When compared to the absorption spectra of the protein before enzymatic treatment, deglycosylated IgY showed a drastic reduction in band intensity, which confirmed that the 1200–900 cm−1 band was due to the absorption of protein carbohydrates. These results were consistent with a previous report [23]. Natalello also found that reduced intensity of the band
Fig. 4. The far-UV CD of IgY and deglycosylated IgY. Protein solutions (0.2 mg/mL) were prepared in 50 mM phosphate buffer (pH 7.5) was measured with a 0.1 cm cell in the far-UV region. The wavelength was from 190 to 250 nm.
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136
133
Fig. 5. The intrinsic fluorescence of IgY and deglycosylated IgY. The excitation wavelength was 280 nm, and emission was from 300 to 450 nm. Protein solutions (0.1 mg/mL) were prepared in 20 mM phosphate buffer (pH 7.5).
at 1200–900 cm−1 was also observable in the FTIR spectrum of the PNGase F-treated protein [23].
3.3. Circular dichroism spectrum Fig. 4 shows the far-UV CD spectrum of IgY and deglycosylated IgY. A negative minimum at 217 nm and zero at 208 nm suggested the prominent -sheet content in IgY. The -sheet content of native IgY was approximately 42%, which was consistent with previous reports. Liu, et al. reported that native IgY contained approximately 45% -sheet content [24]. A positive band was also observed at195 nm, which suggested an ␣-helix structure in IgY. It was clear that IgY underwent a dramatic conformational change
Table 1 Calculation of secondary structures of IgY and deglycosylated IgY. Sample
␣-helix (%)
-sheet (%)
-turn (%)
Random (%)
IgY Deglycosylated IgY
30.9 23.2
41.8 23.3
22.3 25
5 28.5
after PNGase F-treatment. As shown in Table 1, the ␣-helix and -sheet content decreased to 23.2% and 23.3%, respectively. Meanwhile, the percentage of random coil increased from 5% to 28.5%. This phenomenon indicated that the IgY molecule became more flexible and disordered after removal of N-glycan. Hence, galactosylated modification was conducive to maintaining stability of IgY secondary structure. Jafari-Aghdam, et al. studied the effect of
Fig. 6. The SEC-HPLC analysis of aggregation and fragment of IgY and deglycosylated IgY.
134
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136
Fig. 7. IgY unfolding monitored by second derivative ultraviolet absorbance spectroscopy. (A) The UV-vis spectrum of IgY. (B) The 2nd derivative of UV-vis spectrum of IgY. (C) The 2nd derivative UV-vis spectrum monitoring GdnHCl-induced IgY unfolding.
deglycosylation on the structure of glucoamylase from Aspergillus niger. They found that the secondary structure of protein did not change after deglycosylation [25]. These differences might be due to different glycosylation sites on the proteins. 3.4. Fluorescence spectrum More information about protein folding at tertiary level can be obtained by studying the intrinsic fluorescence of protein. The intrinsic fluorescence of the protein came mainly from tryptophan (Trp) residues, which were very sensitive to changes in the microenvironment. The fluorescence emission spectra of IgY before and after deglycosylation were examined (Fig. 5). The intensity of deglycosylated IgY was lower than that of native IgY. The polarity of the environment surrounding the Trp residues affected the fluorescence intensity. This change indicated that the Trp residues were buried in a more hydrophobic environment after disposal of N-glycan.
3.5. SEC-HPLC Size exclusion chromatography (SEC) is a historical technique widely employed for the detailed characterization of therapeutic proteins. It can also be used as a reference and powerful technique for the qualitative and quantitative evaluation of aggregates and fragments [6]. The SEC-HPLC analysis of aggregation and fragmentation of native and deglycosylated IgY after a storage duration of 15 days at 4 ◦ C is shown in Fig. 6. The peak at 7.5–9.0 min represented monomeric IgY. The elution time of monomeric deglycosylated IgY occurred earlier than that of monomeric IgY. Low peaks at approximately 6.5–7.5 min represented the dimer and polymer of IgY. A series of peaks between 13.2 and 17.5 min represented the fragments of IgY. Apparently, both polymerization and fragmentation were enhanced after deglycosylation, especially the fragmentation. Therefore, N-glycan could help maintain the full and stable molecular structure of IgY.
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136
135
Fig. 8. The SDS-PAGE analysis of the pepsin hydrolysate of IgY and deglycosylated IgY. (A) the ratio of IgY to pepsin was 1:10 (w/w). (B) the ratio of deglycosylated IgY to pepsin was 1:10 (w/w). (C) the ratio of IgY to pepsin was 1:40 (w/w). M, protein marker; 1, incubating 15 min; 2, incubating 30 min; 3, incubating 1 h; 4, incubating 1.5 h; 5, incubating 2 h; 6, incubating 4 h; 7, incubating 6 h; 8, incubating 8 h; 9, incubating 16 h; 10, incubating 24 h.
3.6. GdnHCl induced denaturation Denaturant can cause protein unfolding and destroy the biologically active conformation of the protein. UV spectroscopy was used to monitor IgY unfolding induced by GdnHCl. After collecting high resolution UV absorbance spectrum of IgY (Fig. 7A), the second derivative of spectrum was calculated for resolution enhancement (Fig. 7B). Five negative peaks in the second derivative UV spectrum were from Phe (252, 258, and 267 nm), Tyr/Trp (283 nm), and Trp (290 nm) [26]. In this study, the negative peak approximately 290 nm (Fig. 7C) from the second derivative UV was chosen as a marker to monitor the changes in the microenvironment of Trp residues [27]. GdnHClinduced unfolding of glycosylated and deglycosylated IgY is shown in Fig. 7C. The midpoint of the IgY structural transition during the unfolding process was 291.7 nm, and the corresponding concentration was 2.6 M GdnHCl. Deglycosylated IgY was less resistant to GdnHCl-induced unfolding and the midpoint of the transition (291.5 nm) occurred at a concentration of GdnHCl that was lower by approximately 0.6 M. This result demonstrated that the N-glycan of IgY conferred stability during denaturation of the molecule.
also reported that the oligosaccharide chain at the Fc domain improved resistance to protease hydrolysis [32]. 4. Conclusions The results revealed that the removal of N-glycan changed the conformation, storage stability, and resistance to GdnHCl and pepsin digestion of the IgY molecule. It was obvious that Nglycosylation affected IgY stability because of the altered protein structure. It was concluded that a structural role for N-glycosylation in the IgY molecule and provide a basis for studying the role of glycoproteins in molecular stability. Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant No.31371810) and the Fundamental Research Funds for the Central Universities (Program No.2662015PY080; Program No.2662015BQ042). References
3.7. Anti-enzyme ability Pepsin is an enzyme expressed as a prototype of zymogen and pepsinogen and is released by the chief cells in the stomach. It digests food proteins by cleaving peptide bonds and was the first discovered animal enzyme [28]. Pepsin has a preference for cleaving between hydrophobic and aromatic amino acids such as phenylalanine or leucine [29]. Pepsin was commonly employed for limited proteolytic fragmentation of IgG, to generate a Fab fragment devoid of the Fc effector domain [30]. The SDS-PAGE results of the digestion of IgY is shown in Fig. 8. After 24 h of enzymatic hydrolysis with a ratio of protein to pepsin of 1:40 (w/w), a large amount of integrated IgY molecule was still present and the bands of hydrolyzed peptides were not obvious (Fig. 8A). With a ratio of protein to pepsin of 1:10 (w/w), IgY was gradually degraded. After the mixture was heated at 37 ◦ C following a 60 min incubation, a new 45 kDa band emerged (Fig. 8C). Compared with IgY, the band of deglycosylated IgY gradually disappeared when the ratio of protein to pepsin was 1:40 (w/w). Meanwhile, a new band of approximately 45 kDa appeared after a 6 h incubation (Fig. 8B). These new bands were attributed to the formation of the fragment Fab. Plenty of hydrophobic amino acid residues were located in C3 domain of IgY, which also contained N-glycosylation [31]. It seemed that the exposed hydrophobic amino acid residues were more easily attacked by pepsin. These results demonstrated that N-glycosylation protected IgY against pepsin hydrolysis. Raju et al.
[1] D. Carlander, H. Kollberg, P.E. Wejaker, A. Larsson, Peroral immunotherapy with yolk antibodies for the prevention and treatment of enteric infections, Immunol. Res. 21 (2000) 1–6. [2] A.K. Janson, C.I. Smith, L. Hammarstrom, Biological properties of yolk immunoglobulins, Adv. Exp. Med. Biol. 371 (1995) 685–690. [3] L. Jiang, W.S. Chang, H. Guo, P. Sun, X.M. Dai, Structure analysis of goose immunoglobulin Y Fc fragment, Cent. Eur. J. Immunol. 3 (2013) 299–304. [4] Y. Mine, J. Kovacs-Nolan, Chicken egg yolk antibodies as therapeutics in enteric infectious disease: a review, J. Med. Food 5 (2002) 159–169. [5] J. Hodoniczky, Y.Z. Zheng, D.C. James, Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro, Biotechnol. Progr. 21 (2005) 1644–1652. [6] S. Fekete, A. Beck, J.L. Veuthey, D. Guillarme, Theory and practice of size exclusion chromatography for the analysis of protein aggregates, J. Pharm. Biomed. Anal. 101 (2014) 161–173. [7] A. Diress, B. Lorbetskie, L. Larocque, X. Li, M. Alteen, R. Isbrucker, M. Girard, Study of aggregation, denaturation and reduction of interferon alpha-2 products by size-exclusion high-performance liquid chromatography with fluorescence detection and biological assays, J. Chromatogr. A 1217 (2010) 3297–3306. [8] V. Kayser, N. Chennamsetty, V. Voynov, K. Forrer, B. Helk, B.L. Trout, Glycosylation influences on the aggregation propensity of therapeutic monoclonal antibodies, Biotechnol. J. 6 (2011) 38–44. [9] S. Matsumiya, Y. Yamaguchi, J.I. Saito, M. Nagano, H. Sasakawa, S. Otaki, M. Satoh, K. Shitara, K. Kato, Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1, J. Mol. Biol. 368 (2007) 767–779. [10] P. Sondermann, A. Pincetic, J. Maamary, K. Lammens, J.V. Ravetch, General mechanism for modulating immunoglobulin effector function, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 9868–9872. [11] Y.A. Ioannou, K.M. Zeidner, M.E. Grace, R.J. Desnick, Human alpha-galactosidase A: glycosylation site 3 is essential for enzyme solubility, Biochem. J 332 (1998) 789–797. [12] B.D. Weintraub, B.S. Stannard, L. Meyers, Glycosylation of thyroid-stimulating hormone in pituitary tumor cells: influence of high mannose oligosaccharide
136
[13]
[14]
[15] [16]
[17]
[18]
[19]
[20]
[21]
L. Sheng et al. / International Journal of Biological Macromolecules 96 (2017) 129–136 units on subunit aggregation, combination, and intracellular degradation, Endocrinology 112 (1983) 1331–1345. E.L. Smith, J.P. Giddens, A.T. Iavarone, G. Kamil, L.X. Wang, C.R. Bertozzi, Chemoenzymatic Fc glycosylation via engineered aldehyde tags, Bioconjugate Chem. 25 (2014) 788–795. M. Ohta, J. Hamako, S. Yamamoto, H. Hatta, M. Kim, T. Yamamoto, S. Oka, T. Mizuochi, F. Matsuura, Structures of asparagine-linked oligosaccharides from hen egg-yolk antibody (IgY). Occurrence of unusual glucosylated oligo-mannose type oligosaccharides in a mature glycoprotein, Glycoconj. J. 8 (1991) 400–413. N. Suzuki, Y.C. Lee, Site-specific N-glycosylation of chicken serum IgG, Glycobiology 14 (2004) 275–292. D. Shental-Bechor, Y. Levy, Effect of glycosylation on protein folding: a close look at thermodynamic stabilization Dalit, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 8256–8261. Y. Miyake, S. Tsuzuki, S. Mochida, T. Fushiki, K. Inouye, The role of asparagine-linked glycosylation site on the catalytic domain of matriptase in its zymogen activation, Biochim. et Biophys. Acta-Proteins Proteomics 1804 (2010) 156–165. F. Maley, R.B. Trimble, A.L. Tarentino, T.H. Plummer, Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases, Anal. Biochem. 180 (1989) 195–204. D. Pauly, P.A. Chacana, E.G. Calzado, B. Brembs, R. Schade, IgY technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation, J. Visualized Exp. 51 (2011), e3084-e3084. B. Haeduck, H. Honda, R. Murota, M. Kobayashi, F. Horio, A. Murai, Production of recombinant chicken IgY-Fc and evaluation of its transport ability into avian egg yolks, J. Poult. Sci. 47 (2010) 256–261. R.J. Sola, K. Griebenow, Effects of glycosylation on the stability of protein pharmaceuticals, J. Pharm. Sci. 98 (2009) 1223–1245.
[22] P.I. Haris, F. Severcan, FTIR spectroscopic characterization of protein structure in aqueous and non-aqueous media, J. Mol. Catal. B Enzym. 7 (1999) 207–221. [23] A. Natalello, D. Ami, S. Brocca, M. Lotti, S.M. Doglia, Secondary structure, conformational stability and glycosylation of a recombinant Candida rugosa lipase studied by Fourier-transform infrared spectroscopy, Biochem. J 385 (2005) 511–517. [24] J. Liu, J. Yang, H. Xu, J. Lu, Z. Cui, A new membrane based process to isolate immunoglobulin from chicken egg yolk, Food Chem. 122 (2010) 747–752. [25] J. Jafari-Aghdam, K. Khajeh, B. Ranjbar, M. Nemat-Gorgani, Deglycosylation of glucoamylase from Aspergillus niger: effects on structure activity and stability, Biochim. et Biophys. Acta-Proteins Proteomics 1750 (2005) 61–68. [26] K. Zheng, Biophysical Characterization of the EC5 Domain of E-cadherin: Conformation, Physical Stability, and Binding Properties. Ph.D. Thesis, Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence: The University of Kansas, 2007. [27] K. Zheng, C.R. Middaugh, T.J. Siahaan, Evaluation of the physical stability of the EC5 domain of E-cadherin: effects of pH, temperature, ionic strength and disulfide bonds, J. Pharm. Sci. 98 (2009) 63–73. [28] Aszmann, The life and work of theodore schwann, J. Reconstr. Microsurg. 16 (2000) 291–296. [29] D.W. Piper, B.H. Fenton, pH stability and activity curves of pepsin with special reference to their clinical importance, Gut 6 (1965) 506–508. [30] D. Yu, R. Ghosh, Method for studying immunoglobulin G binding on hydrophobic surfaces, Langmuir 26 (2010) 924–929. [31] A.I. Taylor, S.M. Fabiane, B.J. Sutton, R.A. Calvert, The crystal structure of an avian IgY-Fc fragment reveals conservation with both mammalian IgG and IgE, Biochemistry 48 (2008) 558–562. [32] T.S. Raju, B.J. Scallon, Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain, Biochem. Biophys. Res. Commun. 341 (2006) 797–803.