The Structure Of The Outer Membrane Amuc＿1100 Protein Of Akkermansia Muciniphila And Its Effect On Serotonin In The Intestine

Macromolecular compounds are compounds with a relative molecular mass of several thousand to several million.According to their sources,they can be divided into two categories:natural polymers and synthetic polymers.They have been widely used in people’s clothing,food,housing,and transportation.Various sectors of the economy and cutting-edge technology.As a natural macromolecule,protein is widely used in the development of medicine and drugs in addition to its wide application in materials.Understanding the structure of proteins provides a good research foundation for the development of protein drugs.Akkermansia muciniphila is a probiotic inhabiting host intestinal mucus layers and displays evident easing or therapeutic effects on host enteritis and metabolic disorders such as obesity and diabetes.The outer membrane protein Amuc＿1100 of A.muciniphila is likely to play a crucial role during the interaction with the host.Amuc＿1100 can reduce fat,insulin resistance and dyslipidemia function in mice,and can also activate the toll-like receptor 2（TLR2）the NF-κB signaling pathway to producing host immune cytokines such as IL-1β,IL-6,IL-8,IL-10 and TNFαto adjusting the host’s immune response,and it can also improve the transepithelial electrical resistance of the intestinal epithelial cells by activation of TLR2 or increase the expression of the TJ-proteins of the intestinal epithelial to regulate the host intestinal barrier function,but the molecular mechanism is unclear.5-hydroxytryptamine（5-HT）is not only a neurotransmitter,but also a key signal molecule regulating the functions of gastrointestinal tract and other organs.It is related to a variety of physiological and pathological processes.TLR2 as a microbial recognition receptor,in addition to help maintain the intestinal homeostasis,it can also improve the inflammatory bowel injury by regulating Tryptophan hydroxylase 1,Tph1 and 5-HT transporters（serotonin from,SERT）expression to regulate the intestinal 5-HT levels and play a role in intestinal physiology and pathology,whether Amuc＿1100 regulates the intestinal 5-HT system by activating TLR2 remains unclear.In this study,we obtained the extracellular domain protein of Amuc＿1100（residues,31-317 aa）was obtained by Escherichia coli heterologous expression system and obtained the high purity and good homogeneity recombinant proteins by nickel column purification and gel filtration chromatography,finally solved the crystal structure of Amuc＿1100 by X-ray diffraction.Amuc＿1100 consists of a four-stranded antiparallelβ-sheet and fourα-helices.Two C-terminal helices and the four-strandedantiparallelβ-sheet formed two“αββ”motifs and constituted the core domain,which shared a similar fold with type IV pili and type II Secretion system protein.The coiled-coilα1 helix at the N end shows a new topological structure.Amuc＿1100 presents trimer structure in crystal,but exists as monomer in solution.Analysis of its structure shows that theα1 helix of N-terminal plays an important role in the aggregation state of protein.In order to study the effect ofα1helix on the state of aggregation,we generated theα1 helix deletion mutant Amuc＿1100^Δ80 and heterologous expression in Escherichia coli,after nickel column purification and molecular sieve purification to obtain high purity and good uniformity protein,using X-ray diffraction technology to solve the Amuc＿1100^Δ80 crystal structure,Amuc＿1100^Δ80adopts to a dimers in the crystal structure,and share a similar fold the with the reported type IV pili protein or type II secretion system proteins and Amuc＿1100^Δ80 also adopted to a dimer in solution.All these results indicate that theα1 helix of Amuc＿1100 plays an important role in the aggregation state of Amuc＿1100.Although Amuc＿1100 can activate TLR2,the exact mechanism of action is not clear.In this study,we use biofilm interference technology and enzyme-linked immunosorbent assay experiments to confirm that Amuc＿1100 is a new ligand of TLR2,which can directly interact with TLR2.Moreover,Amuc＿1100^Δ80 has higher binding affinity with TLR2,suggesting that Amuc＿1100^Δ80 may be a more favorable candidate gene for the development of A.muciniphila related drugs.After clarifying the direct interaction between Amuc＿1100 and TLR2,this study demonstrated that Amuc＿1100 can increase the expression of Tph1 in RIN-14B cells through direct interaction with TLR2,and reduce the expression of SERT in Caco-2 cells,thereby promoting 5-HT biosynthesis and improves the extracellular availability of 5-HT.In animal experiment,we used the antibiotic-treated mice as animal models.After gavage with A.muciniphila or Amuc＿1100,the expression of Tph1 in the colon tissue of the mouse is increased,the expression of SERT is decreased,and the 5-HT level in the colon and blood is significantly increased.Changes in 5-HT in the intestine will affect the function of intestinal motility.We also tested the effects of Amuc＿1100 and A.muciniphila on intestinal motility and found that Amuc＿1100 and A.muciniphila improved the production of 5-HT in the intestine of antibiotic mice.Changes in 5-HT levels in the intestine not only directly affect various physiological functions,but also affect the composition and abundance of intestinal microbes,which in turn affect various physiological functions of the host.After administration of A.muciniphila or Amuc＿1100 to antibiotic treated mice,the amount and type of intestinal microbes in antibiotic-treated mice were restored.This clarifies the specific mechanisms by which A.muciniphila and host interact with each other and explains the action pathways through which A.muciniphila and Amuc＿1100 improve host intestinal function,inflammation,and metabolic disorders.This study reveals the molecular mechanism and pathways whereby A.muciniphila interacts with hosts,and it provides the foundation for new treatment approaches targeting the intestine and developing therapeutic drugs for related human diseases.