Research On The Glycosylation Patterns And Sensitivity To Soybean Agglutinin In The Intestinal Tracts Of Different Animal Species

This research is composed of4parts, the depiction of intestinal glycosylation patterns in different animal species, isolation and identification of SBA receptors, establishment of a kind of affinity chromatography method with high efficiency for SBA purification and detection of the effects of SBA on the intestinal glycosylation of different animal species.In this research, biotin labeled SBA, WGA, Con A, RCA, and UEA were used as probes in Lectin-histochemical staining to depict the glycosylation patterns in duodenum, pre-jejunum, mid-jejunum, post-jejunum, ileum in pig, chicken and rabbit at1/4sexual maturity stage. The comparison of glycosylation patterns was performed between different intestinal tracts and cell types. Results:biotin labeled SBA, WGA, ConA, RCA and UEA can bind to all tracts of intestine in pig and rabbit. In chicken, all biotin labeled lectins except UEA can bind to all tracts of intestine. The main cell types that lectins can bind to are goblet cells, Columnar epithelial cells and some lymphocytes. Meanwhile, significant differences can be found between the positive signals of different lectins in one cell type. Our results also show that had differences in the quality of positive signals of one lectin between different intestinal tracts in one kind of animal and between the same intestinal tracts in different kinds of animals. It showed that in all intestinal tracts in pigs, rabbits and chickens, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, galactose, mannose can be detected, and fucose can be found in pigs and rabbits but not in chickens. N-acetyl-D-galactosamine residues that can be recognized by SBA are distributed in the free surface of goblet cells and columnar epithelial cells. Differences can be found between the main glycol-residues in different kinds of animals, in pig intestine, the main residues are fucoses and mannoses, in rabbits, are fucose and N-acetyl-D-glucosamine. and in chicken are N-acetyl-D-glucosamine and galactose, respectively. In chikens, there are more N-acetyl-D-glucosamine, N-Acetyl-D-galactosamine, galactose, mannose than other two kinds of animals.Then new born and not breast-feeding pigs, rabbits and chickens were chosen in this research, and crude membrane proteins were isolated from intestinal tissue and then SBA receptors in the intestine in these animals were purified by SBA affinity chromatography;2-D electrophoresis was performed and MW and pI of SBA receptors that purified by glycol-capture method were obtained. At last, glycoproteins were identified through MS-TOF/TOF. Results:the expression map of SBA receptors was obtained by the means of SBA affinity chromatography and2-D electrophoresis. pI of SBA receptors in piglets are distributed in the range of5-8, and MV30kD-97.2kD. Among those glycoproteins13proteins were chosen for MS-TOF/TOF, after identification and blast in IPI base,11known protein were identified, which were named as Intelectin-1β,Keratin, pattern Ⅱ cytoskeletal1, Keratin, pattern Ⅰ cytoskeletal10,Annexin A4, Protein SET,78kD glucose-regulated protein, Heat shock cognate71kD protein, Calcium-activated chloride channel regulator1. pI of SBA receptors in chickens is located in the range of5-8, and MW29.0kD-64.2kD. Among those proteins18dots were cut, after MS-TOF/TOF identification and blast in IPI base,12known proteins were identified, and among which9proteins were enzyme proteins with catalytic activity such as L-lactate dehydrogenase A chain. Protein disulfide-isomerase A3, Triosephosphate isomerase, Folic acid synthesis protein FOL1, Creatine kinase U-pattern, mitochondrial, ATP synthase subunit β,mitochondrial, Malate dehydrogenase, Annexin A2. A cell structure and function of protein, Keratin, pattern Ⅱ cytoskeletal1, and one HSP. PI of SBA receptors in rabbits intestine was located in the range of5-8, and MV in the range of26.0kD-90.0kD, after MS-TOF/TOF and blast in IPI base,3protein were identified with the names of helix-destabilizing protein,actin, type Ⅰ cytoskeletal9.In order to obtain enough SBA for animal experiment, D-galactose-FF-sepharose4B was synthesized as an affinity adsorbent for affinity chromatography. SBA was purified in this system, and the purity, immunogenicity, hemagglutinational activity were detected and compared with standard SBA. Results:the affinity activity of GalN-FF-sepharose-4B is lOmgSBA per ml. The MV of the SBA we purified is30kD, purity is98%. Western blot shows the purified SBA is active. hemagglutinational activity value is1/210. There are no difference of the purity, immunogenicity and hemagglutinational activity between theSBA that we obtained and the standard SBA.In this research,1/4sexual maturity piglets, chickens and rabbits were, and divided into five groups and then were feed with diet containing SBA with the concentration of0,0.025%,0.25%,0.50%and1.00%respectively. Lectin-histochemical staining was performed as described in experiment Ⅰ. the change of N-acetyl-D-glucosamine was detected through the biotin labeled SBA as probe. Results:N-acetyl-D-glucosamine in the intestine can be changed by feed diet containing SBA. N-acetyl-D-glucosamine in all the animals intestine can be increased by SBA, and in1%group, all intestinal tracts in all kind of animals, the expression of N-acetyl-D-glucosamine glycosylation is significantly higher than that of control group(P<0.05). The trend of N-Acetyl-D-galactosamine in piglet intestine is decreased from duodenum to ileum, and the sensitivities in duodenum and pre-jejunum, were higher than that of mid-jejunum, post-jejunum and ileum. In the intestine of chickens, the trend of N-Acetyl-D-galactosamine was increased from duodenum to ileum, and the sensitivities of pre-jejunum and mid-jejunum were higher than other tracts. In rabbits, N-Acetyl-D-galactosamine was increased by the raise SBA contention.This study showed that there are many differences in the glycol-patterns, quantity of glycol-residues, and dominant glycol-residues between different animal species. It showed that in intestine in piglets and rabbits which stand for mammalian, all5kinds of glycol-residues can be detected and in chickens which stand for poultry only4kinds of glycol-residues can be detected. The distribution of glycol-residues is located in the free surface of goblet cells, Columnar epithelial cells in all animals that were used in this study According to intestinal glycol-pattern in these three kinds of animals, N-Acetyl-D-galactosamine residues are not the dominant glycol-residues. According to the structural and functional information, the proteins can be divided into3classes, cell membrane structural proteins, functional regulating proteins, and enzyme proteins with catalytic activities. These results elucidated that the anti-nutrition activities of SBA may be mediated by their binding proteins. The increase of N-Acetyl-D-galactosamine in intestine of different animals illustrate that SBA can regulate the level of the expression of N-Acetyl-D-galactosamine, that may associated with the anti-nutrition of SBA, and the sensitivity to SBA is dependent on the kind of animals and the tracts of the intestine.