Cloning And Functional Analysis Of HISerpin-a And HISerpin-b From Serpin Family Of Haemaphysalis Iongicornis

Ticks are one of the blood-feeding arthropods that transmit numerous pathogens,imposing a serious threat to human health.During their long-term blood feeding period,ticks secrete saliva to transmit pathogens into the host.Tick saliva contain critical biological active components that evade host defenses and facilitate the blood feeding.These proteins could regulate inflammatory response,complement activation,blood coagulation,platelet aggregation,and also affect host adaptive immune response.Serpins are important inhibitors of Serine proteases,which are closely related to the immune regulation and disease.Serpins are evolutionarily conserved,and widely distributed in arthropods.In this paper,we reported that cloning and functional analysis of two novel Serpin genes,HISerpin-a(SA)and HISerpin-b(SB),from the hard tick Haemaphysalis longicornis in China.SA gene encodes a 399 amino acids protein with a signal peptide from residues 1 to 21.SB gene encodes a 379 amino acids protein without a signal peptide.The inhibitory activities of SA and SB against a series of mammalian proteases were tested.SA inhibits protease acitivies of Cathepsin B,Cathepsin G,F X a,and papin,while SB inhibits Cathepsin G,F ? a,and papin activities.SA and SB could delay the plasma clotting in the blood coagulation assay suggesting that SA and SB may facilitate blood feeding.Inflammatory response is an important process in vertebrate defense during a skin injury.SA and SB impair the expression of inflammatory cytokines such as TNF-?,IL-6,IL-1? from mouse bone marrow-derived macrophages(BMDMs)or mouse bone marrow-derived dendritic cells(BMDCs)that stimulated with LPS.The sequence of SA and SB were compared with reported Serpin IRS-2 using the method of homology modeling.Both SA and SB have a reaction center loop(RCL),which is reported to bind to the target enzymes and contains 20 amino acids.We synthesized a minimum active region of Serpin SA,named SA-RCL.SA-RCL shows similar biological activities to SA in the protease inhibition assay and immune suppression assay in terms of detecting the expression of inflammatory cytokines from LPS-induced BMDMs.Since Cathepsins play important roles in immune responses,we constructed mouse macrophage cell lines that deficient in Cathepsin G,Cathepsin B Cathepsin L,respective,by the CRISPR/Cas9 technology.The cytokine expression assay suggested that the immunosuppressive function of SA and SA-RCL are depended on their protease inhibitory activity.Finally,SA and SA-RCL relive the joint inflammation in collagen-induced mouse arthritis models.Futhermore,SA-RCL can reduce the mRNA expression of TNF-?,IFN-? and IL-? from Babesia microti infected mice.The above results showed that two novel Serpins--SA and SB derived from the Haemaphysalis longicornis display immunosuppressive effects and may play a role in blood-sucking and pathogen transmission processes of arthropod vectors.The minimum active region SA-RCL has similar immunosuppressive activity to full length SA,and the later can be a good targetin the development of pharmaceutical applications.