Mechanistic Insight Into The Action Of Vibrio Extracellular Metalloproteases On Collagen

Collagen is the major protein of the extracellular matrix(ECM)of animal,providing structural framework for skin,bones,tendons,and other connective tissues.Collagen is assembled by triple-helical tropocollagen molecules through various intermolecular interactions and covalent cross-links,and its function depends on the supramolecular assembly process.Collagenases,capable of hydrolyzing native collagen under physiological conditions,are found in animals and microbes.Many Vibrio species are pathogens of humans and certain aquatic animals.Extracellular metalloproteases produced by pathogenic Vibrio species are closely related to their pathogenesis.According to the substrate specificity,amino acid sequence similarity and domain organization,Vibrio extracellular metalloproteases are classified into three classes.Class ? proteases belong to the M4 family,and both Classes ? and ? proteases are from the M9A subfamily.Class ? proteases participate in Vibrio pathogenesis mainly via digestion of various protein substrates with important physiological functions or activation of other toxins.In addition,Class ? proteases possess promising potential in industrial applications due to their thermostability or organic solvent-resistance.Class? and Class ? proteases exhibit collagenolytic activity and are one of the important members of bacterial collagenases.They have been regarded as important virulence factors,which digest collagen-containing components in the host ECM to accelerate bacterial dissemination and assist the Vibrio colonization and the diffusion of other toxins.Although many Vibrio collagenases have been identified and characterized,knowledge concerning the collagen degradation mechanism of Vibrio collagenase is still limited.Moreover,little is known about the mechanism of Vibrio collagenase for collagen recognition and degradation due to the lack of structural information.In this study,the mechanisms of three classes of Vibrio extracellular metalloproteases acting on collagen were studied,including the collagen swelling mechanism of Class ? protease in the M4 family and the collagen degradation mechanism of Class ? and Class ? collagenases in the M9A subfamily.(1)The collagen swelling mechanism of Class ? protease VP9 from the M4 family.The protease VP9 secreted by Vibrio pomeroyi strain 12613 was identified as a Class ? protease of the M4 family in previous study.In this study,VP9 was heterologously overexpressed and purified.We successfully obtained the mature recombinant VP9 and characterized it.VP9 can hydrolyze casein and gelatin but not elastin or collagen.It has good thermostability,is stable in a range of pH 5.0-11.0,and has good tolerance to NaCl,non-ionic detergents,and some organic solvents.These properties indicate its potential for industrial application.In addition,although VP9 cannot degrade collagen,it has obvious collagen swelling ability.The relative expansion volume of collagen increased by approximately eightfold after treatment with 10 ?M VP9 at 37? for 12 h.Atomic force microscopy(AFM)observation and biochemical analyses showed that VP9 can degrade proteoglycans in collagen fibers,resulting in the release of collagen fibrils from collagen fibers and the swelling of the latter.In addition,VP9 can degrade glycoproteins,a non-collagenous constituent interacting with collagen.The degradation of proteoglycans and glycoproteins by VP9 results in structural destruction of collagen and ECM,suggesting that VP9 has pathological effects on the physiological functions of the host ECM.In addition,the characteristics of VP9,such as sufficient specificity toward proteoglycans and glycoproteins but no activity toward collagen,suggest its promising potential in the unhairing and fiber-opening processing in leather industry.(2)The collagen degradation pattern of Class ? collagenase VP397 from the M9A subfamily.Class ? collagenase VP397 derived from V.pomeroyi strain 12613 is composed of a collagenase module(CM)containing an activator domain and a peptidase domain,and two accessory bacterial prepeptidase C-terminal domains(PPC domains).VP397 has broad specificity to various collagenous substrates such as fish collagen,mammalian collagens of types ?-?.We further studied its degradation pattern on insoluble type ? collagen fibers by AFM observation and biochemical analyses.Our results revealed that VP397 first assaults the C-telopeptide region to destroy the compact structure of collagen and dissociate tropocollagen fragments.Subsequently,the dissociated tropocollagen fragments are further digested into peptides and amino acids by VP397 mainly at the Y-Gly bonds in the repeating Gly-X-Y triplets.In addition,domain deletion mutagenesis analysis showed that the collagenase module of VP397 alone is capable of hydrolyzing type ? collagen fibers.The intermediate PPC1 domain that has no collagen binding or swelling ability may function as a linker between the upstream and downstream domains in VP397 collagenolysis.The C-terminal PPC2 domain that has collagen binding but no swelling ability may function as a collagen binding domain(CBD)during collagen degradation.Based on our results,a model for the collagenolytic pattern of VP397 was proposed.Altogether,the degradation pattern of Class ? collagenase VP397 in the M9A subfamily on insoluble type ? collagen fibers was revealed,which offers a new insight into the role and mechanism of Vibrio collagenases in the pathogenesis of Vibrio.(3)Characterization and structural analysis of Class ? collagenase VhaC from the M9A subfamily.Vibrio collagenase of the M9A subfamily is closely related to the pathogenesis of several pathogenic Vibrio species through the degradation of collagenous component in the ECM.However,the structural basis and molecular mechanism of collagen degradation by Vibrio collagenase have not been clarified.Here,the gene encoding a Class ? collagenase,VhaC,from pathogenic Vibrio harveyi VHJR7 was synthesized and overexpressed in E.coli BL21(DE3),and the recombinant VhaC was purified and characterized.VhaC is a Class ? collagenase composed of a collagenase module(CM)featuring an activator domain and a peptidase domain,a polycystic kidney disease-like(PKD-like)domain and a PPC domain.VhaC presents as a monomer in solution and has activity toward fish collagen,bovine collagens of types ?-?,human collagens of types ?-?,and gelatin.The crystal structure of the CM was solved to 1.8 A,which adopts a more contracted saddle-shaped architecture and lacks the catalytic helper subdomain,compared to that of Clostridium collagenase ColG,the only CM structure in the M9 family.Small-angle X-ray scattering(SAXS)analysis showed that the sideby-side arrangement of the domains of VhaC results in a long and flat conformation of the enzyme in solution,and the PPC domain connected by a flexible linker extends outside the core region.The crystal structure of the collagenase module of Vibrio collagenase VhaC and its solution conformation revealed here represent the first structural information of Vibrio collagenase,which lays a structural foundation for further study on the mechanism for collagen recognition and degradation by Vibrio collagenase and helps in structure-based drug design targeting Vibrio collagenase.(4)Mechanism for triple helical collagen recognition and catalysis by the collagenase module of VhaC.Biochemical analyses showed that the CM of VhaC is the minimal unit capable of breaking down triple helical collagen.In this study,the mechanism of CM for the recognition and catalysis of triple helical collagen was illustrated based on biochemical and mutagenesis analyses,and molecular dynamics simulation(MDS).Our results indicated that the activator domain is responsible for substrate binding in the degradation of triple helical collagen and collagen fibers,and Phe107,Arg153 and Tyr157 in the activator domain are the key residues involved in triple helical collagen binding.In addition,our results also showed that CM adopts different strategies for the recognition of peptide substrate and triple helical collagen.Peptide substrate is directly recognized and degraded by the peptidase domain,whereas triple helical collagen is initially recognized by the activator domain.We found that CM shows continuous opening and closing changes in MDS.The MDS process of the CM in complex with triple helical collagen further showed that,as the system gradually reached equilibrium,the triple helical molecule initially bound to the activator domain is close to the catalytic center of the peptidase domain with the conformation closing of CM.Combining with the biochemical verification of the key residues in the catalytic center of the peptidase domain,we proposed that triple helical collagen is initially recognized by the activator domain of VhaC,followed by subsequent cleavage driven by the closing movement of CM.Our results revealed the mechanism for triple helical collagen recognition and catalysis of Vibrio collagenase in the M9A subfamily for the first time,which is helpful to further study the mechanism of native collagen degradation by Vibrio collagenase.(5)Functional analysis of the PKD-like and the PPC domain and collagen degradation pattern of VhaC.Class ? collagenase VhaC contains two accessory domains,the PKD-like domain and the PPC domain,in addition to the collagenase module.Domain deletion mutagenesis analysis showed that the absence of either accessory domain significantly affected the activity of VhaC towards collagen fibers.Functional analysis showed that the PKD-like domain of VhaC did not exhibit any collagen binding or swelling effect.As an intermediate domain,the PKD domain is likely to function as a linker between the upstream and downstream domains.The PPC domain of VhaC showed noticeable collagen binding ability,but no collagen swelling ability,suggesting that the PPC domain likely functions as a CBD.Structural analysis showed that the PPC domain is connected to the core region by a flexible linker,suitable to exert the collagen binding function.We further studied the degradation pattern of VhaC on insoluble type ?collagen fibers by AFM observation and biochemical analyses.The results showed that Class ? collagenase VhaC adopts a collagen degradation pattern similar to Class ?collagenase VP397.Both enzymes disintegrate collagen fibrils and dissociate tropocollagen fragments by initially degrading the C-telopeptide,and then hydrolyze the dissociated tropocollagen fragments into peptides and amino acids in a manner of cleaving the Y-Gly bonds in the repeating Gly-X-Y triplets.Finally,combined with the triple helical collagen recognition and catalytic mechanism of the collagenase module,the functions of the accessory domains,and the collagen degradation pattern,a model for the integrated collagenolytic mechanism of Class ? collagenase VhaC was proposed.In summary,the collagen swelling mechanism of Vibrio Class ? protease in the M4 family and the collagen degradation mechanism of Vibrio Class ? and Class ?collagenases in the M9A subfamily were studied in this dissertation,which is helpful in better understanding the pathogenesis of Vibrio extracellular metalloproteases and in developing their application potentials.