Effects Of Site-specific Mutant For ?4GalT1 Gene On Physiological Function,Glycosylation Of Protein And Intestinal Microorganism In Mice

?4GalT1 is ?1,4 galactosyltransferase,There are seven members in the family,?4Gal T1-?34GalT7.Every ?4GalT has different functions in the biosynthesis of different glycoco njugates and glycan structures.So far,the most studied is ?4GalT1 in ?4GalT family,whic h is also the most widely distributed and very important glycosyltransferase in the body.?4 GalT1 is involved in the construction of cytoskeleton,cell-matrix interaction,embryonic de velopment,nervous system development,immune and inflammatory response,colorectal ca ncer and other tumors.Although there are many studies on the function of ?4GalT1,they ar e all carried out by knocking out the ?4GalT1 gene in mice.It has been proved that the kno ckout of ?4GalT1 gene has a great influence on the normal physiological function of mice.In addition,little research has been done on the effect of the mutation of the ?4GalT1 gene i nto GalNAcT on the glycosylation of proteins in mice,especially on the structure of intesti nal microorganisms in mice.Therefore,the homozygotes,heterozygotes and wild-type mic e with point mutations of the ?4GalT1 gene were obtained through breeding and gene identi fication.On this basis,the effects of point mutations of the ?4GalT1 gene on the growth,ne rve development,reproductive capacity and glycosylation of protein in mice were investiga ted.The effects of point mutations of the ?4GalT1 gene on intestinal microorganisms in mi ce were also studied.The possible ameliorative effect of N-glycans in human milk and cow milk on this effect was also discussed.1.Effect of point mutation of ?4GalT1 gene on physiological function of miceThe homozygous,heterozygous and wild-type mice of the point mutation of the ?4Gal T1 gene were successfully obtained by genotype identification of offspring of heterozygous self-bred mice.By observing the daily state of homozygous and wild-type mice,it was fou nd that homozygous mice were less responsive to external stimuli than wild-type mice.The tail suspension test showed that the resting time of homozygous mice was significantly lon ger than that of wild-type mice(P<0.01),indicating that homozygous mice were in a depre ssive state for a longer time,which further proved that ?4GalT1 played an important role in the process of neurodevelopment.Homozygous female and homozygous male mice,homozygous female and wild type m ale mice,heterozygous female and heterozygous male mice,wild type female mice and wild t ype male mice were fed in cages.It was found that homozygous female mice were difficult to conceive,or could not normally produce mice after conception,or even died of dystocia.Stat istical analysis of the average per-fetal algebraic number of homozygous,heterozygous and w ild-type mice showed that the average per-fetal algebraic number of homozygous,heterozygo us and wild-type mice were 0,6±2 and 9±2 respectively.The average number of homozygou s per-fetal mice was significantly less than that of heterozygous and wild-type mice(P<0.01).which indicated that ?4GalT1 played an important role in the reproduction,It not only affect s the fertility rate of mice,but also closely related to embryonic development.2.Effect of point mutation of ?4GalT1 gene on glycosylation of protein in serum and s mall intestline of miceSerum and small intestinal N-glycans were extracted from homozygous mice and wild-type mice with point mutation of ?4GalT1 gene for structural analysis.UPLC and MALDI-TOF-MS were used to detect the N-glycans released by enzymatic interpretation.The struc ture of the N-glycans in the serum of mice with point mutation of ?4GalT1 gene was analyz ed and compared.The structure of serum N-glycans in mice with point mutation of ?4GalT 1 gene only contains a small amount of galactose,but contains a certain amount of N-acetyl galactosamine,while the structure of serum N-glycans in wild-type mice contains more gal actose and does not contain N-acetylgalactosamine.It was proved that galactose in serum N-glycans of mice was mainly linked to N-glycans catalyzed by ?4GalT1.After point mutati on of ?4GalT1 gene in mice,the activity of ?4GalT1 in mice is lost,Although this process can be completed by other members of the ?4GalT family,due to the specificity of the distr ibution of substrates,the process is mainly catalyzed by ?4GalT1.Therefore,compared wit h wild type mice,the structure of N-glycans in serum of homozygous mice significantly de creased.At the same time,due to the introduction of N-acetylgalactosaminotransferase gen e,the structure of N-acetylgalactosamine increased.Based on the important biological funct ion of N-glycans,the changes of N-glycosylation of protein in blood of homozygous mice will inevitably change the physiological function of mice.By analyzing the structure of N-glycans in the small intestine of homozygous and wild-type mice,the N-glycans containing galactose were not found in the small intestine of hom ozygous mice,which may be the structure containing galactose was low and undetected.Th e structure of galactose in the small intestine of wild type mice is similar to that of N-chain oligosaccharides in the serum of homozygous and wild type mice.These results suggest tha t ?4GalT1 also alters the structure of N-glycans in the small intestine of mice.Because the s ugar chain structure on the surface of intestinal epithelial cells is the recognition receptor of microorganisms,the change of N-glycans structure in intestinal tract also indicates that it may affect the structure of intestinal microorganisms.The structure of N-glycans in serum and small intestine of homozygous and wild-type mice showed that the point mutation of ?4GalT1 gene had an important effect on glycosylat ion of protein in mice.3.The effect of point mutation of ?4GalT1 gene on intestinal microorganisms and the improvement treated with milk N-glycans in miceThe feces of homozygous and wild-type mice were analyzed by high-throughput sequ encing.The results showed that there were about 10%unique OTUs in each group,principa 1 component analysis,cluster tree analysis and differential analysis showed that the point m utation of ?4GalT1 gene had a significant effect on the structure of intestinal micro flora in t he feces of mice.In addition,high throughput sequencing results showed that the intake of N-glycans from human milk and bovine milk also significantly affected the fecal intestinal microbial abundance and flora composition of wild-type mice after feeding a certain amoun t of N-glycans from human milk and bovine milk.Specifically,the point mutation of ?4GalT1 gene significantly increased the abundanc e of Ruminococcaceae_UCG₀09,Ruminococcaceae_UCG₀10,Parabacteroides and Tan nerellaceae in feces of mice(p<0.05),while the N-glycans intake of human milk and bovi ne milk significantly decreased the abundance of Ruminococceae,Tanneraceae and Paraba cteroides in feces of wild-type mice(p<0.05).Point mutation of the ?4GalT1 gene signific antly decreased the abundance of Ruminococcaceae UCG 014 and other bacteria in the fec es of mice(p<0.05),while the N-glycans intake of bovine milk significantly increased the abundance of Ruminococcaceae_UCG₀14 and other bacteria in the feces of wild-type mic e(p<0.05).Therefore,it can be inferred that the intestinal flora imbalance of the homozyg ous mice with point mutation of ?4GalT1 gene can be regulated by feeding a certain amoun t of N-glycans in human milk or bovine milk.