Liver-expressed antimicrobial peptide 2(LEAP-2)is a cationic peptide that plays an important role in a host’s innate immune system.We previously demonstrated that mudskipper(Boleophthalmus pectinirostris)LEAP-2(BpLEA...Liver-expressed antimicrobial peptide 2(LEAP-2)is a cationic peptide that plays an important role in a host’s innate immune system.We previously demonstrated that mudskipper(Boleophthalmus pectinirostris)LEAP-2(BpLEAP-2)induces chemotaxis and activation of monocytes/macrophages(MO/MΦ).However,the molecular mechanism by which BpLEAP-2 regulates MO/MΦ remains unclear.In this study,we used yeast twohybrid cDNA library screening to identify mudskipper protein(s)that interacted with BpLEAP-2,and characterized a sequence encoding motile sperm domain-containing protein 2(BpMOSPD2).The interaction between BpLEAP-2 and BpMOSPD2 was subsequently confirmed by co-immunoprecipitation(Co-IP).Sequence analyses revealed that the predicted BpMOSPD2 contained an N-terminal extracellular portion composed of a CRAL-TRIO domain and a motile sperm domain,a C-terminal transmembrane domain,and a short cytoplasmic tail.Phylogenetic tree analysis indicated that BpMOSPD2 grouped tightly with fish MOSPD2 homologs and was most closely related to that of the Nile tilapia(Oreochromis niloticus).The recombinant BpMOSPD2 was produced by prokaryotic expression and the corresponding antibody was prepared for protein concentration determination.RNA interference was used to knockdown BpMOSPD2 expression in the mudskipper MO/MΦ,and the knockdown efficiency was confirmed by quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting.Knockdown of BpMOSPD2 significantly inhibited BpLEAP-2-induced chemotaxis of mudskipper MO/MΦand BpLEAP-2-induced bacterial killing activity.Furthermore,knockdown of BpMOSPD2 inhibited the effect of BpLEAP-2 on mRNA expression levels of BpIL-10,BpTNFα,BpIL-1β,and BpTGFβ in MO/MΦ.In general,BpMOSPD2 directly interacted with BpLEAP-2,and mediated the effects of BpLEAP-2 on chemotaxis and activation of mudskipper MO/MΦ.This is the first identification of MOSPD2 as a receptor for LEAP-2.展开更多
Liver-expressed antimicrobial peptide 2(LEAP2)is a key regulator of innate immune defense in teleosts,yet the molecular basis of its chemotactic function remains largely unidentified.Boleophthalmus pectinirostris MOSP...Liver-expressed antimicrobial peptide 2(LEAP2)is a key regulator of innate immune defense in teleosts,yet the molecular basis of its chemotactic function remains largely unidentified.Boleophthalmus pectinirostris MOSPD2(BpMOSPD2)was previously identified as a candidate receptor for BpLEAP2 in monocytes/macrophages(MO/MΦ).In the present study,BpLEAP2 stimulation was found to trigger a retromer-dependent intracellular trafficking program essential for BpMOSPD2-mediated chemotaxis.Exposure to BpLEAP2 significantly enhanced BpMO/MΦmigration and promoted the accumulation of BpMOSPD2 at the plasma membrane.Subcellular fractionation and immunofluorescence analyses revealed that BpMOSPD2 translocated from the endoplasmic reticulum(ER)to early endosomes upon BpLEAP2 stimulation,followed by redistribution to the cell surface.Blockade of ER export or knockdown of core retromer subunits(BpVPS35,BpVPS26,or BpVPS29)abolished membrane localization and attenuated BpLEAP2-induced migration.Co-immunoprecipitation combined with mass spectrometry confirmed direct binding between BpMOSPD2 and BpVPS35,while domain-mapping indicated that this interaction was not exclusively dependent on MSP or CRAL-TRIO domains.Depletion of individual retromer components led to retention of BpMOSPD2 in early endosomes,establishing the necessity of the retromer complex for receptor recycling.Functionally,disruption of this complex eliminated the pro-migratory activity of BpLEAP2 on BpMO/MΦ.These findings identify the retromer complex as a critical regulator of BpMOSPD2 trafficking and uncover a previously unrecognized mechanism through which BpLEAP2 promotes MO/MΦmigration in teleosts.展开更多
基金supported by the National Natural Science Foundation of China(31972821,31772876)Zhejiang Provincial Natural Science Foundation of China(LZ18C190001)+1 种基金Scientific Innovation Team Project of Ningbo(2015C110018)K.C.Wong Magna Fund in Ningbo University。
文摘Liver-expressed antimicrobial peptide 2(LEAP-2)is a cationic peptide that plays an important role in a host’s innate immune system.We previously demonstrated that mudskipper(Boleophthalmus pectinirostris)LEAP-2(BpLEAP-2)induces chemotaxis and activation of monocytes/macrophages(MO/MΦ).However,the molecular mechanism by which BpLEAP-2 regulates MO/MΦ remains unclear.In this study,we used yeast twohybrid cDNA library screening to identify mudskipper protein(s)that interacted with BpLEAP-2,and characterized a sequence encoding motile sperm domain-containing protein 2(BpMOSPD2).The interaction between BpLEAP-2 and BpMOSPD2 was subsequently confirmed by co-immunoprecipitation(Co-IP).Sequence analyses revealed that the predicted BpMOSPD2 contained an N-terminal extracellular portion composed of a CRAL-TRIO domain and a motile sperm domain,a C-terminal transmembrane domain,and a short cytoplasmic tail.Phylogenetic tree analysis indicated that BpMOSPD2 grouped tightly with fish MOSPD2 homologs and was most closely related to that of the Nile tilapia(Oreochromis niloticus).The recombinant BpMOSPD2 was produced by prokaryotic expression and the corresponding antibody was prepared for protein concentration determination.RNA interference was used to knockdown BpMOSPD2 expression in the mudskipper MO/MΦ,and the knockdown efficiency was confirmed by quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting.Knockdown of BpMOSPD2 significantly inhibited BpLEAP-2-induced chemotaxis of mudskipper MO/MΦand BpLEAP-2-induced bacterial killing activity.Furthermore,knockdown of BpMOSPD2 inhibited the effect of BpLEAP-2 on mRNA expression levels of BpIL-10,BpTNFα,BpIL-1β,and BpTGFβ in MO/MΦ.In general,BpMOSPD2 directly interacted with BpLEAP-2,and mediated the effects of BpLEAP-2 on chemotaxis and activation of mudskipper MO/MΦ.This is the first identification of MOSPD2 as a receptor for LEAP-2.
基金supported by the National Natural Science Foundation of China(32173004)Natural Science Foundation of Zhejiang Province(LZ23C190001)。
文摘Liver-expressed antimicrobial peptide 2(LEAP2)is a key regulator of innate immune defense in teleosts,yet the molecular basis of its chemotactic function remains largely unidentified.Boleophthalmus pectinirostris MOSPD2(BpMOSPD2)was previously identified as a candidate receptor for BpLEAP2 in monocytes/macrophages(MO/MΦ).In the present study,BpLEAP2 stimulation was found to trigger a retromer-dependent intracellular trafficking program essential for BpMOSPD2-mediated chemotaxis.Exposure to BpLEAP2 significantly enhanced BpMO/MΦmigration and promoted the accumulation of BpMOSPD2 at the plasma membrane.Subcellular fractionation and immunofluorescence analyses revealed that BpMOSPD2 translocated from the endoplasmic reticulum(ER)to early endosomes upon BpLEAP2 stimulation,followed by redistribution to the cell surface.Blockade of ER export or knockdown of core retromer subunits(BpVPS35,BpVPS26,or BpVPS29)abolished membrane localization and attenuated BpLEAP2-induced migration.Co-immunoprecipitation combined with mass spectrometry confirmed direct binding between BpMOSPD2 and BpVPS35,while domain-mapping indicated that this interaction was not exclusively dependent on MSP or CRAL-TRIO domains.Depletion of individual retromer components led to retention of BpMOSPD2 in early endosomes,establishing the necessity of the retromer complex for receptor recycling.Functionally,disruption of this complex eliminated the pro-migratory activity of BpLEAP2 on BpMO/MΦ.These findings identify the retromer complex as a critical regulator of BpMOSPD2 trafficking and uncover a previously unrecognized mechanism through which BpLEAP2 promotes MO/MΦmigration in teleosts.
