Isolation and analysation of soybean agglutinin-specific binding proteins for erythrocyte membrane in different animal species

Abstract

Soybean agglutinin (SBA) represents the anti-nutritional molecule in soybean. This molecule agglutinates erythrocytes of several animal species. The specific binding of SBA to glycoprotein of the cellular plasma membrane is necessary for SBA to exert its toxic action. The sensitivity of erythrocyte to SBA for various species is different, however, the pathway is unclear. We aimed to optimise the method of extracting erythrocyte membrane (EM) and to analyse the pathway of the different sensitivity of erythrocyte to SBA in three animals species (rabbit as a rodent, pig as an omnivore, and bovine as a ruminant). The method of low osmotic haemolysis was used to extract the EM. To precipitate the EM, we evaluated the purification effects of three lysis bufferes, including NP-40, RIPA (medium) and RIPA (weak). All SBA-specific binding proteins (SSBP) on erythrocyte membranes were conducted using co-immunoprecipitation (CO-IP) and analysed using liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS (Q-E)). The results showed that NP-40 lysis buffer was the most effective than the other two kinds of lysates to extract EM proteins. Functionally, the SSBP of EM could be divided into three categories in each animal, including cytoskeleton proteins, catalytic enzyme proteins and the functional regulatory proteins. The cytoskeleton proteins were the main different expressed proteins among the EM proteins in pig, rabbit than that in bovine, which may be one important reason for the difference of the SBA sensitivity. This research provides basis to reveal the pathway of SBA-induced hemagglutination differences in erythrocytes in different animal species.

Highlights

• We optimised the method of extracting EM to isolate and identify SSBP in different animal species.

• NP-40 lysis buffer was the most effective than the other two kinds of lysates to extract EM proteins.

• Cytoskeleton proteins were the main different expressed proteins among the EM proteins in pig, rabbit, and cow.

Keywords:

• Anti-nutritional factor
• agglutination activity
• sensitive difference
• pathway
• LC-MS-MS (Q-E)

Introduction

Soybean agglutinin (SBA) is a kind of glycoprotein with a specific binding to N-acetyl-d-galactosamine/d-galactose. Such glycoprotein possesses a molecular weight of 120 kDa. SBA represents a main anti-nutritional factor in soybean (Lotan et al. 1974). This anti-nutritional factor plays a unique biological function as a bioactive protein through direct or indirect contact with human blood cells (Pereira et al. 1974). The biological functions of SBA can be shown in two ways. First, anti-nutritional functions that appear in animal digestion and absorption of nutrients and influencing multiple biological functions of cells (Pan et al. 2013; Halder et al. 2016). The other way is the specificity of sugar binding. Therefore, the specific binding of SBA to glycoprotein is a necessary condition for SBA to show its toxic action.

The understanding of SBA has begun with the discovery of its agglutination activity. SBA can agglutinate the erythrocyte (red blood cells, RBCs), therefore, it was initially called haemagglutinin (Goldstein et al. 1980; Tremblay et al. 2011). Lotan et al. (1973) found that SBA possesses species-specific coagulation activity and sensitive to the agglutination of rabbit and human erythrocytes. In many researches, SBA showed that the rabbit blood was the most sensitive convenient among the animal species for this coagulation activity (Tateno et al. 1998; Kakiuchi et al. 2002). Liu (2006) also confirmed that the rabbit is the most sensitive species to SBA agglutination, while pig had less responsive than rabbits, and ruminant erythrocytes can be only agglutinated at a specific concentration, for example the weak agglutinin in sheep erythrocytes only occurred when SBA was diluted between 8 and 64 times. Hughes-Jones et al. (1963) indicated that the immune agglutination of erythrocyte comes from a dynamic process which can be divided into two stages. The first immune agglutination stage starts immediately following the mixing antibody molecules with antigens on the surface of the cells. The second stage begins directly after the first stage and results from fruitful collisions occurring between erythrocyte so that antibody molecules bound to one erythrocyte to another one. Guesdon et al. (1979) proved that SBA can be polymerised due to chemical or physical factors, the monomers can cross-linked with each other, and the sites of reaction with erythrocyte also can be increased, and consequently the agglutination activity of erythrocyte enhances.

Erythrocyte are the main components of the blood circulation system in the body. The main functions of erythrocyte are carrying oxygen, transporting carbon dioxide and maintaining electrolytes balance (Tomova et al. 2008). The erythrocyte membrane (EM) components are complex compound, including bilayer of phospholipid, membrane protein and membrane skeleton (Tse and Lux 1999; Gordon-Smith 2009). The membrane protein of erythrocyte mainly includes peripheral protein and integrin protein, that represents the basics for the functions of the erythrocytes. Because the structure and function of EM are overlapped, the normal structure and function are directly related to the normal physiological activities of erythrocyte.

The sensitivity of erythrocytes from different animal species to SBA is different (Lotan et al. 1973; Tateno et al. 1998; Kakiuchi et al. 2002; Liu 2006). However, no clear reasons indicating why SBA causes the difference of haemagglutinin activity in different animal species. Therefore, this study aims to optimise the method of extracting erythrocyte membrane (EM), and to analyse the pathway of the different sensitivity of different animals’ erythrocyte (rodents, rabbit; omnivore, pig; and ruminant, bovine) to SBA. This research can provide a major basis to reveal the pathway of SBA-induced hemagglutination differences in erythrocytes among different animal species.

Materials and methods

Animal management

To conform to national and international research standards, this study was conducted according to ethical principles governing the usage of animal samples in research studies of this nature. Permission to use samples was also acquired from the Ethical Clearance Committee of Jilin Agricultural University. The management and care of animals were performed according to the guidelines and recommendations of the Jilin Agricultural University Guide (JNDZ [2019] No.19). This study was conducted under the ethical approval no. kt2015010 of Animal Experimental Ethics of Key Laboratory of Animal Production and Product Quality and Safety.

Collection of blood samples

Before blood collection, the animals were fasted for 24 h (Niu et al. 2013). Ten millilitre (10 mL) of blood were withdrawn from the jugular vein of pig (2-month old Landrace boar), from the heart of rabbits (2-month old experimental rabbit Japanese big-eared rabbits). Blood samples were withdrawn from the jugular vein of bovine (6-month old Holstein bull) (Yuan et al. 2018). In order to prevent blood clotting, anticoagulant (5% sodium citrate, 1 mL) was added and mixed evenly for the preparation of EM.

Preparation and purification of EM

The erythrocytes possess the highest weight compared to serum and the other formed elements. The blood samples were leuko-reduced by first centrifuging at 3000 rpm for 10 min to decant plasma. The packed cells were then centrifuged at 700 xg for 30 min with no brakes to remove the leukocytes. Because there is no nucleus and other organelles in mammalian erythrocyte, the preparation of the membrane was only through simple low permeability solution treatment and centrifugation. To improve the extraction efficiency of EM protein, we compared several common lysis buffers that had no effect on subsequent co-immunoprecipitation assays (CO-IP). All lysis buffers were obtained from Gene Star, Beijing, China. We evaluated the purification effects of three lysis buffers, including NP-40 lysis buffer, RIPA lysis buffer (weak), and RIPA lysis buffer (middle). A sample of 1 mL from each lysis buffer was used to precipitate the EM. After standing for 30 min, the supernatant was separated and then was centrifuged for 15 min at 4 °C and 10,000 xg, for SDS-PAGE electrophoresis. Different lysis buffers were compared through the analysis of electrophoretogram, and the most effective lysis buffer was selected to be used in the next experiment.

The obtained opalescent precipitate was dissolved in 1 mL of lysis buffer (PMSF contained), and ultrasonic breaked for 1 min (5 s per 10 s interval) at 80 Hz and 4 °C. After 40 min of standing at 4 °C, the proteins centrifugated at 4 °C and 10,000 xg for 15 minutes, then the supernatant was used as a protein solution for EM.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

The extracted EM proteins were isolated by SDS-PAGE. EM proteins were re-suspended in a loading buffer (CWBIO, Beijing, China) with the ratio of 4:1 (sample weight to buffer weight). Then the samples were centrifuged and the supernatant was heated to 95 °C for 3 min. The boiled samples were kept in iced condition before loading into a gel.

Both 5% stacking gel and 10% separation gel were prepared for separating the proteins. 10 μL of 1.0 mg/mL protein samples, as well as protein marker (BioLab, Beijing, China) was applied on the gel. The SDS-PAGE was performed at 80 V through the stacking gel and 150 V through the separation gel. After fixation, these proteins were stained with coomassie brilliant blue at room temperature for 2 h, followed by washing with double-distilled water for 3 times.

CO-IP

CO-IP experiment was conducted to separate all specific proteins that specially combined with SBA on EM from pig, rabbit or as previously described in bovine (Yuan et al. 2018). CO-IP were carried out using dynabeads CO-IP kiit (Invitrogen, Carlsbad, CA). The experiment was strictly conducted in accordance with the manufacturer’s instructions. Prior to immunoprecipitation, 50 μg of SBA (Sigma, St. Louis, MO) was mixed with beads, and incubated on a roller at 37 °C overnight.

After washed 3 times with 0.01% Tween-20 HB solution, we transferred 1.5 mg SBA-coupled beads to a fresh tube. Then the SBA-coupled beads were re-suspended in 1.0 g EMs and incubated on a roller at 4 °C for 30 min. Then the Protein A beads were finally re-suspended in ethidium bromide (EB) solution and incubated on a roller at room temperature for 5 min. The immunoprecipitate was centrifuged and the supernatant which contained SBA-binding protein complexes was transferred to a clean tube.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS (Q-E)) detection

After separation by CO-IP, all SSBP were detected by Beijing Protein Innovation CO., Ltd. (Beijing, China) and analysed using an electro-spray ionisation tandem mass spectrometry LC-MS-MS (Q-E). MASCOT protein scores (combined MS and MS/MS spectra) >22 in rabbit, MASCOT protein scores > 23 in pig, and >24 in bovine (Yuan et al. 2018), were considered statistically significant (p < .05). The individual MS/MS spectrum, with a statistical significant (confidence interval >95%) iron score was accepted.

Retrieval of database

The database search was performed with Mascot Daemon (Version 2.3.0, Matrix Science). Bos taurus database was used for data analysis and was downloaded from UniProt. The database link is http://www.uniprot.org/taxonomy/9913?Sort = score.

Results

Comparison of purification effects of different lysis buffers on EM protein extraction

In this experiment, the time of haemolysis (overnight haemolysis) was prolonged on the basis of traditional hypotonic haemolysis method, and the method of ultrasonic fragmentation was used to accelerate the fragmentation of erythrocyte, so the breakage of erythrocyte and the cleavage of proteins from the membrane were more thorough.

The SDS-PAGE electrophoretogram results are shown in Figure 1. The results showed that NP-40 was the most effective lysis buffer compared to RIPA lysis buffer (medium) and RIPA lysis buffer (weak). In addition, the mild NP-40 lysis buffer had no effect on subsequent CO-IP. Such effective buffer was selected as the lysis buffer for the extraction of EM proteins.

Figure 1. Comparison of extraction effect of different lysis buffers on erythrocyte membrane in sodium salt (sds)-polyacrylamide gel electrophoresis (SDS-PAGE). M. Standard protein maker 1. Octylphenoxypolyethoxyethanol (NP-40) lysis buffer 2. Radio immunoprecipitation assay (RIPA) lysis buffer (medium) 3. RIPA lysis buffer (weak).

Comparison among EM proteins in different animal species

The protein compositions of EM of pig and rabbit were similar (Figure 2). Most of these compositions were distributed between 25 kDa and 250 kDa, and the proportion of high molecular weight protein was significantly higher than that of low molecular weight protein. The difference was the protein abundance of 180 kDa, 65 kDa and 35 kDa in rabbit EM which was higher than that in EM of pig.

Figure 2. SDS-PAGE of erythrocyte membrane (EM) proteins in rabbit and pig. M. Standard protein maker 1. EM proteins of pig 2. EM proteins of rabbit.

Identification of SSBP of EMs in different animal species

In herein experiment, SSBP on the surface of EM of pig and rabbit were detected and analysed using (LC-MS-MS (Q-E)). The Mascot software showed that the proteins with score higher than 23 in pig and with score higher than 22 in rabbit were all positive results. Then, we compared and analysed the SSBP of EMs in different animal species (pig, rabbit and that in bovine). Functionally, SSBP proteins could be divided into three protein categories including cytoskeleton, catalytic enzymes, and functional regulatory proteins. The cytoskeleton proteins were the main different expressed proteins among the EM proteins of pig, rabbit and that in bovine, as shown in Tables 1–3.

Table 1. The soybean agglutinin specific binding cytoskeleton protein of EMs in three animal species.

		Rabbit					Swine					Bovine
No.	Protein name	AN	S	MW (kDa)	CP (%)	PI	AN	S	MW (kDa)	CP (%)	PI	AN	S	MW/kDa	CP (%)	PI
1	Keratin 10	G1T1V0	884	58,260	25.8	4.98	I3LDS3	597	58243	17.1	4.93	A6QNZ7	649	54987	16.2	5.01
2	Keratin 1	G1U9I8	609	64,422	14.4	8.6	F1SGG3	456	65381	12.2	8.18	G3N0V2	549	63341	13	8.41
3	Spectrin beta	G1SD01	48	268,766	1	5.38	F1SA60	530	271142	20.1	5.23	F1MKE9	591	269692	54.2	5.16
4	Spectrin alpha	G1STR7	48	281,789	3.1	4.94	F1RNB8	550	27767	26.6	4.92	F1MMQ8	650	282352	79.3	4.84
5	Keratin 5	G1TDN6	493	62,414	17.2	7.62	F1SGG6	250	63,668	18.7	7.74	M0QVZ6	608	60,800	25	7.63
6	Actin	P29751	166	42,071	21.1	5.3	I3LVD5	240	42,108	11.5	5.31	F1MRD0	105	42,167	16.5	5.16
7	Keratin 75	G1SHY2	335	61,390	12.5	8.43	F1SGI7	183	65,354	9	9.27	Q08D91	276	59,399	10.3	7.56
8	Keratin 14	G1T1Y7	324	48,662	15.1	4.87	F1S0L1	352	51,811	19.8	4.94	F1MC11	466	52,164	28.7	5.08
9	Keratin 3	G1SY72	296	64,858	10.2	7.6	I3LDM6	137	65,497	11.4	8.03	G3MXL3	459	63,082	14	5.78
10	Ankyrin 1	G1TG85	267	203,233	8	6.11	F1SE30	351	207,769	16.3	6	F1MY81	581	123,754	15.9	6.92
11	Keratin 8	G1SWS0	59	51,483	3.6	4.9	F1SGG2	63	54,394	7.4	5.7	P05786	141	53,594	11.7	5.6
12	Keratin 18	G1TMP1	137	48,347	5.8	5.33	F1SGG1	100	49,430	3.6	5.39	A6H7D3	68	49,218	3.6	5.33
13	Keratin 13	G1T4S1	454	48,712	19	4.86	F1S0K1	225	49,505	9.7	4.88	_	_	_	_	_
14	Ankyrin 2	G1SZP4	55	42,7545	0.6	4.98	F1S146	141	428,715	4.4	4.93	_	_	_	_	_
15	Keratin 35	G1T4P5	65	51,439	6.2	4.87	F1S0K7	99	51,637	3.5	4.95	_	_	_	_	_
16	Keratin 16	G1T4R6	332	49,878	12	49878	_	_	_	_	_	_	_	_	_	_
17	Keratin 20	G1TE74	147	84,376	4.6	4.89	_	_	_	_	_	_	_	_	_	_
18	Tubulin alpha chain	G1SL10	97	50,834	18.2	4.94	_	_	_	_	_	_	_	_	_	_
19	Keratin 222	G1U0K9	68	34,270	5.8	5.83	_	_	_	_	_	_	_	_	_	_
20	Keratin 7 C	G1TP66	53	55,448	7.8	5.8	_	_	_	_	_	_	_	_	_	_
21	Keratin 2	G1SUH8	618	63,646	19.1	8.98	_	_	_	_	_	E1B991	246	64,570	6.8	8.92
22	Keratin 7	G1SHZ4	239	40,889	11.5	4.88	_	_	_	_	_	Q29S21	288	51,603	10.3	5.79
23	Keratin 85	G1SHW8	40	56,850	5.1	7.03	_	_	_	_	_	Q0VD04	61	57,171	8.7	6.15
24	Keratin 15	_	_	_	_	_	F1S0K2	246	49,501	12	4.87	Q17QL7	295	49,083	12.6	4.83
25	Keratin 28	_	_	_	_	_	F1RXG2	180	50,560	5.4	5.08	Q148H6	160	51,314	7.3	5.19
26	Keratin 6 A	_	_	_	_	_	LOC100737483	172	60,375	15	8.53	M0QVY0	525	61,166	19.6	8.28
27	Keratin 19	_	_	_	_	_	F1S0J8	159	44,186	11.4	5.05	P08728	160	43,858	13.5	4.92
28	Keratin 34	_	_	_	_	_	F1S0K4	98	47,282	5.7	4.94	F1MSA6	197	45,782	19.4	4.86
29	Keratin 2 A	_	_	_	_	_	A5A759	259	66,510	8.3	7.47	_	_	_	_	_
30	Keratin 77	_	_	_	_	_	I3LNT6	202	62,707	8.3	5.89	_	_	_	_	_
31	Plakoglobin	_	_	_	_	_	Q8WNW3	108	82,539	2.4	5.75	_	_	_	_	_
32	Protein 4.2	_	_	_	_	_	F1SI48	106	77,185	3.6	6.93	_	_	_	_	_
33	Keratin 4	_	_	_	_	_	I3LQN8	62	57,648	6.7	7.45	_	_	_	_	_
34	Keratin 6 C	_	_	_	_	_	_	_	_	_	_	F1MUY2	460	61,275	19.4	8.58
35	Band 3 protein	_	_	_	_	_	_	_	_	_	_	Q9TUQ0	346	95,867	18.7	6
36	Keratin 17	_	_	_	_	_	_	_	_	_	_	A1L595	280	48,967	18.8	5.08
37	Keratin 24	_	_	_	_	_	_	_	_	_	_	F1MFW9	232	50,904	6.9	4.68
38	Keratin 28	_	_	_	_	_	_	_	_	_	_	Q148H6	183	51,314	7.3	5.19
39	Keratin 31	_	_	_	_	_	_	_	_	_	_	Q148I8	169	48,526	12.3	4.79
40	Protein 4.1	_	_	_	_	_	_	_	_	_	_	F1MCU7	158	58,782	13.3	6.45
41	Keratin 12	_	_	_	_	_	_	_	_	_	_	F1MJ66	152	50,303	7.9	4.55
42	Keratin 33B	_	_	_	_	_	_	_	_	_	_	A3KN26	90	44,440	10.7	4.81
43	Keratin 73	_	_	_	_	_	_	_	_	_	_	A7YWK3	88	59,540	3.5	6.96
44	Keratin 71	_	_	_	_	_	_	_	_	_	_	Q148H5	70	57,885	4	6.27
45	Keratin 86	_	_	_	_	_	_	_	_	_	_	E1B898	62	54,951	8.6	5.49
46	Keratin 83	_	_	_	_	_	_	_	_	_	_	A4FUZ0	62	55,665	8.5	5.43
47	Keratin 81	_	–	_	_	_	_	_	_	_	_	Q148H4	62	56,403	8.4	5.62
48	Vimentin	_	_	_	_	_	_	_	_	_	_	P48616	29	53,752	3.4	5.06

Accession number in the NCBInr database.

AC: accession number; MW: molecular weight; IP: isoelectric points; CP: coverage percentage; S: score.

Peptide score is shown as −10log(p), where p is the probability that the observed match is a random event. MASCOT protein scores (combined MS and MS/MS spectra) > 22 in rabbit, protein score >23 in pig, and >24 in bovine, were considered statistically significant (p < .05).

Table 2. The soybean agglutinin specific binding catalytic enzyme proteins in the EMs of three different animal species.

		Rabbit					Swine					Bovine
No.	Protein name	AN	S	MW (kDa)	CP (%)	PI	AN	S	MW (kDa)	CP (%)	PI	AN	S	MW/kDa	CP (%)	PI
1	Protein arginine methyltransferase 5	G1SGM2	72	73286	7.4	5.95	C3RZ98	26	73,252	4.4	5.88	F1N443	84	73,283	7.4	5.88
2	Methyltransferase like 3	G1SD33	28	65,012	2.9	6.05	F1S8J8	28	65,501	4.8	6.12		_	_	_	_
3	Pyruvate kinase	P11974	90	58,524	6	7.62	_	_	_	_	_	_	_	_	_	_
4	Enolase 2	G1SK04	26	47,536	12.7	4.95	_	_	_	_	_	_	_	_	_	_
5	Glutathione S-transferase theta-4	G1SN46	23	28,140	2.5	6.76	_	_	_	_	_	_	_	_	_	_
6	Trypsinogen	_	_	_	_	_	F1SRS2	306	26,563	7.3	6.85		_	_	_	_
7	Phosphodiesterase 4 D interacting protein	_	_	_	_	_	F1SB10	22	96,486	0.7	5.19		_	_	_	_
8	Adenosine deaminases, RNA specific B1	_	_	_	_	_	_	_	_	_	_	F1N0T4	113	79,070	4	9.08
9	Receptor for activated C kinase 1	_	_	_	_	_	_	_	_	_	_	P63243	100	35,511	13.9	7.6
10	Serine/threonine-protein kinase 38	_	_	_	_	_	_	_	_	_	_	F1MBL1	65	54,533	11.8	6.67

Accession number in the NCBInr database.

AN: accession number; MW: molecular weight; IP: isoelectric points; CP: coverage percentage; S: score.

Peptide score is shown as −10log(p), where p is the probability that the observed match is a random event. MASCOT protein scores (combined MS and MS/MS spectra) > 22 in rabbit, protein score >23 in pig, and >24 in bovine, were considered statistically significant (p < .05).

Table 3. Differences of soybean agglutinin specific binding functional regulatory proteins in EMs of different animal species.

		Rabbit					Swine					Bovine
No.	Protein name	AN	S	MW (kDa)	CP (%)	PI	AN	S	MW (kDa)	CP (%)	PI	AN	S	MW/kDa	CP (%)	PI
1	Solute carrier family 4 member 1	G1SLY0	275	102,346	9.8	5.13	F1RQY6	85	104,116	5.9	5.49	_	_	_	_	_
2	Anion exchange protein	G1SLY0	65	102,346	2.5	5.13	_	_	_	_	_	_	_	_	_	_
3	Sodium channel protein	G1U7U1	35	229,912	1.5	5.51	_	_	_	_	_	_	_	_	_	_
4	Cullin 9	G1SIL5	25	291,709	0.6	5.34	_	_	_	_	_	_	_	_	_	_
5	Rap guanine nucleotide exchange factor like 1	G1TJY0	25	71,524	1.9	5.91	_	_	_	_	_	_	_	_	_	_
6	Family with sequence similarity 71 member B	G1SHD8	24	59,557	3.1	9.64	_	_	_	_	_	_	_	_	_	_
7	Calmodulin regulated spectrin associated protein 1	_	_	_	_	_	F1S018	23	168,082	1	6.67	_	_	_	_	_
8	Heat shock 70 kDa protein 1 A	_	_	_	_	_	_	_	_	_	_	Q27975	169	70,500	16.1	5.68
9	T-complex protein 1 subunit beta	_	_	_	_	_	_	_	_	_	_	Q3ZBH0	97	57,781	2.8	6.18
10	78 kDa glucose-regulated protein	_	_	_	_	_	_	_	_	_	_	Q0VCX2	69	72,470	4.4	5.07

Accession number in the NCBInr database.

AN: accession number; MW: molecular weight; IP: isoelectric points; CP: coverage percentage; S: score.

Peptide score is shown as −10log(p), where p is the probability that the observed match is a random event. MASCOT protein scores (combined MS and MS/MS spectra) > 22 in rabbit, protein score >23 in pig, and >24 in bovine, were considered statistically significant (p < .05).

The SSBP on bovine EM were the most abundant than the other two animal species (23 proteins in rabbit, 25 proteins in pig and 35 proteins in bovine). The cytoskeleton proteins that specifically bound with SBA on the EMs of these three species were mainly family members of the keratin, as depicted in Table 1 and Figure 3. We found 12 overlapping SBA-specific binding cytoskeleton proteins among pig, rabbit, and bovine EM. There were mainly some family members of keratin, spectrin, actin and ankyrin 1. There were three overlapping SBA-specific binding cytoskeleton proteins in both pig and rabbit (some family members of keratin and ankyrin 2), three overlapping SSBP in both rabbit and bovine (family members of keratin), and five overlapping SSBP in both pig and bovine (family members of keratin). To expect the difference of cytoskeleton protein keratin subunits among these three animal species, the difference was detected for tubulin alpha chain specially in rabbit, while plakoglobin and protein 4.2 were specially expressed in pig, whereas band 3 protein, protein 4.1 and vimentin were specially expressed in bovine.

Figure 3. The overlapping and differential soybean agglutinin specific binding cytoskeleton proteins in three animal species.

Regarding the SBA binding catalytic enzyme proteins, only 1 overlapping protein, (arginine methyltransferase 5) was expressed in the EM of these three animal species (Table 2 and Figure 4). The methyltransferase like 3, pyruvate kinase, enolase 2 and glutathione S-transferase theta-4 specially have expressed in rabbit, while trypsinogen and phosphodiesterase 4D interacting proteins were specially expressed in pig. Yet, adenosine deaminases, RNA-specific B1, receptor for activated C kinase 1, serine/threonine-protein kinase 38 were specially expressed in the bovine.

Figure 4. The overlapping and differential soybean agglutinin specific binding catalytic enzyme proteins in three animal species.

The highest difference was detected in the functional regulatory proteins expression (Table 3 and Figure 5). We found 1 overlapping SSBP, called solute carrier family 4 member 1 in both rabbit and pig. The calmodulin regulated spectrin associated protein 1 was specially expressed in pig. While three proteins were specially expressed in the bovine including heat shock 70 kDa protein 1A, T-complex protein 1 subunit beta, and 78 kDa glucose-regulated protein, while other five proteins were expressed in rabbit, including anion exchange protein, sodium channel protein, cullin 9, rap guanine nucleotide exchange factor like 1, family with sequence similarity 71member B.

Figure 5. The overlapping and differential soybean agglutinin functional regulatory proteins in three animal species.

Discussion

In this experiment, the extraction method of EM protein was optimised. The time of haemolysis (overnight haemolysis) was prolonged on the basis of traditional hypotonic haemolysis method. The method of ultrasonic fragmentation was used to accelerate the fragmentation of erythrocyte. Therefore, the breakage of erythrocyte and the cleavage of proteins from the membrane were more thorough than the original method.

The active ingredients in RIPA lysis buffer (Medium) were 50 mM Tris HCl (pH7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS with some other components like sodium pyrophosphate, sodium fluoride, EDTA. The difference in RIPA lysis buffer (low) and RIPA lysis buffer (medium) was noticed in sodium deoxycholate which was decreased by 0.25%. This difference was noticed in NP-40 lysis buffer in another component (sodium orthovanadate). Sodium deoxycholate, an anionic surfactant, can destroy the membranes of the damaged or non-viable cells to enhance permeability of cells (Ling et al. 2020). A related research indicated that sodium deoxycholate-based extraction buffer with heat denaturation was the most effective approach for highly efficient extraction of proteins from complex tissues such as the zebrafish liver tumour (Wang et al. 2015). In the present research, the increased content of sodium deoxycholate could improve the extraction effect of cell membrane protein. In addition, the sodium orthovanadate could effectively inhibit protein degradation and maintain the original protein–protein interaction. Therefore, the NP-40 lysis buffer was more effective than the other two kinds of lysates to extract of cell membrane protein.

The sensitivity of animal species to SBA is different (Kakiuchi et al. 2002), and the premise of SBA hemagglutination or anti-nutritional functions are related to species-specific effect with cell membrane protein (Dam and Brewer 2010). Hasan et al. (2018) also reported that the decreased hemagglutination activity may be related to the effective irreversibly binding proteins of human kidney lectins to the erythrocyte membrane, lectins oligomerization or both actions. Moreover, chitosan nanoparticles can promote agglutination of rabbit blood cells by binding to the EM (Fan et al. 2012). Therefore, we speculated that the different sensitivity of erythrocyte to SBA among animal species may be ascribed to the variations of the SSBP on EM in such species.

Functionally, the results of SSBP in rabbit, pig and the precious report of bovine (Yuan et al. 2018). EM proteins were divided into three categories including cytoskeleton proteins, catalytic enzyme proteins, and the functional regulatory proteins. According to the current experimental results, the cytoskeleton proteins were the main different expressed proteins among the EM proteins in pig and rabbit than that in bovine. In these proteins, overlapping and differential proteins are mainly family members of keratin. Thakur et al. (2016) reported that several herbal secondary bioactive compounds such as flavonoids and polyphenols possess anti-oxidant properties to stabilise EM by scavenging free radicals, and thereby protecting host cells from oxidative damage, which can be caused by haemolysis and hemagglutination. Therefore, the stability of EM is of great significance for erythrocyte agglutination. Previous studies have shown that there is a significant difference in SBA sensitivity among erythrocyte of different species (Contreras and Tagle 1974; Liu 2006). The most sensitive animal species for erythrocyte agglutination is rabbit, followed by pig, and bovine. The recent species shows agglutinate at a specific concentration (since there was a weak agglutination reaction in 8–64 times dilution of SBA for ruminant). From our results, the SSBP on bovine EM were the most abundant than the other two species (23 proteins in rabbit, 25 proteins in pig and 35 proteins in bovine). Additionally, these proteins were mainly family members of keratin and the functions of keratin, that can maintain the integrity and continuity of the cells. Band 3 protein and protein 4.1 were only detected in SSBP of the bovine. EM mechanical function is regulated by the spectrin-based membrane cytoskeleton, which composed of α-and β-spectrin, actin, protein 4.1R and adducing (Manno et al. 2005). One of EM major organising centres is the cytoplasmic domain of band 3, which links multiple proteins to the membrane (Zhang et al. 2000). Because the effect of SBA-specific binding cytoskeleton proteins on bovine EM is more abundant than the other to animals, the stability of bovine erythrocytes was higher than that of pig and rabbit. However, there is no research report on the relationship between keratin/band 3 protein/protein 4.1 and erythrocyte agglutination, these cytoskeleton proteins play an important role in maintaining EM stability. When SBA binds to these proteins, it can inhibit changes in cell structure and stability. Therefore, this inhibition can lead to decrease the sensitivity of bovine's erythrocytes to SBA than those of rabbit and pig.

We analysed other SBA-specific binding cytoskeleton proteins that were related to erythrocyte agglutination on EMs of these three animal species. Except some members of keratin family, tubulin alpha chains are SSBP that could only be expressed on the EM of rabbit. Tubulin, the subunit protein of microtubules, is found in all eukaryotic cells. Microtubules play an important in various cellular activities such as mitosis, cell shaping, secretion, and motility. Previous investigations reported that tubulin plays an important role in hemagglutination, 100 μg of tubulin per 1 mL of packed formalinized cells during the coating procedure can cause a significant agglutination (Gozes et al. 1977; Kim et al. 2001). Therefore, when the tubulin alpha protein interacts with SBA, may be leading to significant agglutination sensitivity to erythrocyte of rabbit. In addition to ankyrin1, the overlapped proteins of rabbit and pig also contained ankyrin 2, which was lack in bovine EM. Minion and Goguen (1985) showed that the attachment of EM proteins to a semi-rigid cytoskeletal network prevents or greatly reduces their lateral diffusion. The erythrocyte from ankyrin-deficient mice in which the linkage between glycophorin and cytoskeleton is disrupted and can be agglutinated by M. pulmonis. Thus, ankyrin 2 may declare the sensitive agglutination potential for SBA in the erythrocytes of rabbit and pig than bovine. After the binding of SBA to microproteins, the expression or activity of ankyrin may cause erythrocyte agglutination. These proteins may be some pathways among SBA-induced-hemagglutination in different animal species; however, these conjectures still need to be confirmed by further studies.

Protein kinases and transferases are the most important enzymes in cell membrane signal transduction (Pajaud et al. 2012). Once these enzymes have activated, they can induce many intracellular reactions. Most catalytic enzymes detected in our results belong to these two enzymes. Among the results of SBA-specific binding catalytic enzymes on EMs of different animal species, the major enzyme related to erythrocyte agglutination was erythrocyte pyruvate kinase (PK), which detected only in rabbit. Erythrocyte PK represents one of the rate limiting key enzymes in the Embden–Meyerhof pathway, which the erythrocyte depends to get the energy and viability (Nakashima 1974). The deficiency of this enzyme leads to decrease of energy supply. Pollack and Reckel (1977) reported that the immune agglutination of erythrocyte also needs two major opposing energy sources. First, the attraction energy which mostly comes from interfacial energy, the kinetic or thermally driven Brownian motion. Such attraction energy is opposed by the second energy source, the repulsion energy, due to ζ-potential. Therefore, when SBA combined with erythrocyte PK, this may affect the energy supply, and finally leading to exhibit the agglutination sensitivity of rabbit's erythrocyte to SBA.

The functional regulatory proteins in the erythrocyte are necessary for the cell metabolism. These functions involve transportation for oxygen, carbon dioxide, electrolytes, glucose, amino acids, etc. The results from the SBA-binding functional regulatory proteins only showed a typical trans-membrane protein, solute carrier family 4 member 1 in rabbits and pig. Such functional proteins play an important role in maintaining the stability of EM structure and regulating energy metabolism. Other proteins are responsible for the transport of amino acids, sugars, nucleotides and inorganic ions through the cell membranes. Therefore, we speculated that functional regulatory proteins may be a marginal reason for the sensitivity difference of EM proteins to SBA in different animal species.

In this study, we just compared the differences of SSBP on EM proteins from three representative animal species, as the agglutination activity of SBA on erythrocytes of these three animals is quite different. Although we have detected the binding between the proteins, the current study has a limitation in evaluating the strength of the various proteins. Therefore, further studies should be conducted to evaluate the binding strength of SSBP on the EM of different animal species, and to perform a systematic analysis of these differences. In addition, we could also verify the function of these different SSBP, and indicate the reason and pathway for the difference of SBA agglutination activity of erythrocyte of different animal species. This study is of great theoretical significance for further understanding and analysing the toxic mechanism of SBA. Such study is of great practical value to the diversity and precision feeding of different animal groups, as well as to optimise the feed resource allocation and feed processing and utilisation.

Conclusions

In conclusion, NP-40 lysis buffer was the most effective lysates in the extraction of EM proteins. The specific binding cytoskeleton proteins on erythrocyte membrane of different animal species may be one important reason for the difference of the sensitivity of rabbit, pig, and bovine erythrocyte to SBA. The protein kinases (erythrocyte PK) and the functional regulatory proteins (solute carrier family 4 member 1) may be another reason for the most sensitive agglutination in rabbit erythrocyte than that of pig and bovine to SBA. This research provides an important experimental basis for revealing the pathway of the SBA-induced hemagglutination variations among erythrocytes of different animal species.

Disclosure statement

We certify that there is no conflict of interest with any financial organisation regarding the material discussed in the manuscript. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [No. 31802093].