Structural And Functional Study Of Mouse And Human Leg1

Healthy growth of livestock animals and efficient absorbtion and utilization of feed nutrients is one of the crucial questions to livestock production.Salivary glands can secrete growth factors,digestive enzymes and digestive cofactors,and genes encoding target proteins beneficial for animal growth and nutritional utilization can be expressed specifically in the salivary glands via transgenic technology to improve livestock production,therefore,the study of the regulation of expression and fucntion of salivary gland proteins is expected to be applied in animal husbandary.Leg1(Liver-enriched gene 1)is a secreted protein which is conserved in vertebrate.Previous studies in our laboratory firstly investigated the function of MLEG1.In mice,MLEG1 may act as a new regulator of fat metabolism.MLEG1 is expressed in salivary glands and then secreted into saliva,it can withstand degradation by digestive system such as gastric and intestinal fluids and reach the liver through blood circulation.In liver,MLEG1 maily regulates the expression of some lipogenesis transfactor genes.However,the post-transcriptional modifications,structure and functional domains of Leg1 protein have been poorly studied,and there is still a gap in the study of Leg1 protein(HLEG1)in human,which poses a great challenge for us to study the function and application of Leg1.In this thesis,we carried out an in-depth study on the function of Leg1 in mice.Firstly,we investigated the chemical modifications of MLEG1 and its function in mice,and secondly,we investigated the sturcture of MLEG1 by Cryo-EM.Finally,we probed the expression of the HLEG1 and its asscoaition with human physiology.We found in MLEG1,three Asn(117th,196th,and 257th)are attached to N-linked glycans while two Ser(119th and 123th)are attached to O-linked glycans.The N-and O-linked glycosylation modifications mutually affect each other.In addition,we found an unknown type of modification at the 117th Asn.This modification can be recognized and cleaved by PNGaseF and also rely on the existence of S119.We carried out preliminary structure analysis of MLEG1 protein with glycosylation modification by cryoelectron microscopy with the purpose to establish the relationship between its structure and function.We also conducted a study on the modification and function of HLEG1 in human.A total of 621 human saliva samples were collected to determine the abundance of HLEG1,and the correlation between HLEG1 abundance in saliva samples and various physiological parameters was analyzed in an attempt to develop a new marker factor from saliva for monitoring human health.Our results will help to reveal the function of the human salivary protein HLEG1 and its potential signaling pathways in the future.In summary,the results presented in this thesis provide an in-depth extension of the chemical modifications and functions of Leg1 in mice and human by identifying two types of glycosylation modification,intiating a cryoelectron microscopy study of MLEG1 protein,and pioneering the human population survey of HLEG1 abundance in saliva.These studies will provide a solid foundation for future studies of MLEG1 and HLEG1 function.In addition,given the relatively specific high expression of the leg1 gene in the salivary gland,the promoter of the leg1 gene can be used in the livestock breeding process to construct transgenic animals with saliva-specific expression of the target gene according to production needs,which may help us to improve the germplasm of agricultural animals and thus achieve the goals of improving production efficiency and reducing environmental pollution.