RESUMO
Extracellular proTGF-ß is covalently linked to "milieu" molecules in the matrix or on cell surfaces and is latent until TGF-ß is released by integrins. Here, we show that LRRC33 on the surface of microglia functions as a milieu molecule and enables highly localized, integrin-αVß8-dependent TGF-ß activation. Lrrc33-/- mice lack CNS vascular abnormalities associated with deficiency in TGF-ß-activating integrins but have microglia with a reactive phenotype and after 2 months develop ascending paraparesis with loss of myelinated axons and death by 5 months. Whole bone marrow transplantation results in selective repopulation of Lrrc33-/- brains with WT microglia and halts disease progression. The phenotypes of WT and Lrrc33-/- microglia in the same brain suggest that there is little spreading of TGF-ß activated from one microglial cell to neighboring microglia. Our results suggest that interactions between integrin-bearing cells and cells bearing milieu molecule-associated TGF-ß provide localized and selective activation of TGF-ß.
Assuntos
Proteínas de Transporte/metabolismo , Microglia/metabolismo , Sistema Nervoso/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Axônios/metabolismo , Transplante de Medula Óssea , Encéfalo/metabolismo , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Células Cultivadas , Integrinas/metabolismo , Estimativa de Kaplan-Meier , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Mutagênese Sítio-Dirigida , Doenças Neurodegenerativas/mortalidade , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/terapia , Filogenia , Ligação Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
Integrins are adhesion receptors that transmit force across the plasma membrane between extracellular ligands and the actin cytoskeleton. In activation of the transforming growth factor-ß1 precursor (pro-TGF-ß1), integrins bind to the prodomain, apply force, and release the TGF-ß growth factor. However, we know little about how integrins bind macromolecular ligands in the extracellular matrix or transmit force to them. Here we show how integrin αVß6 binds pro-TGF-ß1 in an orientation biologically relevant for force-dependent release of TGF-ß from latency. The conformation of the prodomain integrin-binding motif differs in the presence and absence of integrin binding; differences extend well outside the interface and illustrate how integrins can remodel extracellular matrix. Remodelled residues outside the interface stabilize the integrin-bound conformation, adopt a conformation similar to earlier-evolving family members, and show how macromolecular components outside the binding motif contribute to integrin recognition. Regions in and outside the highly interdigitated interface stabilize a specific integrin/pro-TGF-ß orientation that defines the pathway through these macromolecules which actin-cytoskeleton-generated tensile force takes when applied through the integrin ß-subunit. Simulations of force-dependent activation of TGF-ß demonstrate evolutionary specializations for force application through the TGF-ß prodomain and through the ß- and not α-subunit of the integrin.
Assuntos
Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Integrinas/química , Integrinas/metabolismo , Fator de Crescimento Transformador beta1/agonistas , Fator de Crescimento Transformador beta1/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Evolução Molecular , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Fator de Crescimento Transformador beta1/metabolismoRESUMO
We show that the three conformational states of integrin α5ß1 have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000-fold higher affinity of the extended-open state than the bent-closed and extended-closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α5ß1 On the surface of K562 cells, α5ß1 is 99.8% bent-closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N-glycans and leg domains in each subunit that connect the ligand-binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single-span transmembrane domains.
Assuntos
Integrina alfa5beta1/química , Integrina alfa5beta1/metabolismo , Linhagem Celular , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
The role of the hybrid domain in integrin affinity regulation is unknown, as is whether the kinetics of ligand binding is modulated by integrin affinity state. Here, we compare cell surface and soluble integrin αVß6 truncation mutants for ligand-binding affinity, kinetics, and thermodynamics. Removal of the integrin transmembrane/cytoplasmic domains or lower legs has little effect on αVß6 affinity, in contrast to ß1 integrins. In integrin opening, rearrangement at the interface between the ßI and hybrid domains is linked to remodeling at the ligand-binding site at the opposite end of the ßI domain, which greatly increases in affinity in the open conformation. The larger size of the ßI-hybrid interface in the closed state suggests that the hybrid domain stabilizes closing. In agreement, deletion of the hybrid domain raised affinity by 50-fold. Surface plasmon resonance and isothermal titration calorimetry gave similar results and the latter revealed tradeoffs between enthalpy and entropy not apparent from affinity. At extremely high affinity reached in Mn2+ with hybrid domain truncation, αVß6 on-rate for both pro-TGF-ß1 and fibronectin declined. The results suggest that the open conformation of αVß6 has lower on-rate than the closed conformation, correlate with constriction of the ligand-binding pocket in open αVß6 structures, and suggest that the extended-closed conformation is kinetically selected for ligand binding. Subsequent transition to the extended-open conformation is stabilized by its much higher affinity for ligand and would also be stabilized by force exerted across ligand-bound integrins by the actin cytoskeleton.
Assuntos
Antígenos de Neoplasias/metabolismo , Citoesqueleto/metabolismo , Integrinas/metabolismo , Conformação Proteica , Fator de Crescimento Transformador beta1/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Sítios de Ligação , Humanos , Integrinas/química , Integrinas/genética , Ligantes , Manganês/metabolismo , Modelos Moleculares , Ligação Proteica , Deleção de Sequência , Fator de Crescimento Transformador beta1/química , Fator de Crescimento Transformador beta1/genéticaRESUMO
Integrins αVß6 and αVß8 are specialized for recognizing pro-TGF-ß and activating its growth factor by releasing it from the latency imposed by its surrounding prodomain. The integrin αVß8 is atypical among integrins in lacking sites in its cytoplasmic domain for binding to actin cytoskeleton adaptors. Here, we examine αVß8 for atypical binding to pro-TGF-ß1. In contrast to αVß6, αVß8 has a constitutive extended-closed conformation, and binding to pro-TGF-ß1 does not stabilize the open conformation of its headpiece. Although Mn2+ potently activates other integrins and increases affinity of αVß6 for pro-TGF-ß1 25- to 55-fold, it increases αVß8 affinity only 2- to 3-fold. This minimal effect correlates with the inability of Mn2+ and pro-TGF-ß1 to stabilize the open conformation of the αVß8 headpiece. Moreover, αVß8 was inhibited by high concentrations of Mn2+ and was stimulated and inhibited at markedly different Ca2+ concentrations than αVß6 These unusual characteristics are likely to be important in the still incompletely understood physiologic mechanisms that regulate αVß8 binding to and activation of pro-TGF-ß.
Assuntos
Integrinas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Cálcio/metabolismo , Humanos , Manganês/metabolismoRESUMO
TGF-ß is synthesized as a proprotein that dimerizes in the endoplasmic reticulum. After processing in the Golgi to cleave the N-terminal prodomain from the C-terminal growth factor (GF) domain in each monomer, pro-TGF-ß is secreted and stored in latent complexes. It is unclear which prodomain and GF monomer are linked before proprotein convertase cleavage and how much conformational change occurs following cleavage. We have determined a structure of pro-TGF-ß1 with the proprotein convertase cleavage site mutated to mimic the structure of the TGF-ß1 proprotein. Structure, mutation, and model building demonstrate that the prodomain arm domain in one monomer is linked to the GF that interacts with the arm domain in the other monomer in the dimeric structure (i.e. the prodomain arm domain and GF domain in each monomer are swapped). Swapping has important implications for the mechanism of biosynthesis in the TGF-ß family and is relevant to the mechanism for preferential formation of heterodimers over homodimers for some members of the TGF-ß family. Our structure, together with two previous ones, also provides insights into which regions of the prodomain-GF complex are highly structurally conserved and which are perturbed by crystal lattice contacts.
Assuntos
Modelos Moleculares , Precursores de Proteínas/química , Fator de Crescimento Transformador beta1/química , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Mutação , Domínios Proteicos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Whether ß1 integrin ectodomains visit conformational states similarly to ß2 and ß3 integrins has not been characterized. Furthermore, despite a wealth of activating and inhibitory antibodies to ß1 integrins, the conformational states that these antibodies stabilize, and the relation of these conformations to function, remain incompletely characterized. Using negative-stain electron microscopy, we show that the integrin α5ß1 ectodomain adopts extended-closed and extended-open conformations as well as a bent conformation. Antibodies SNAKA51, 8E3, N29, and 9EG7 bind to different domains in the α5 or ß1 legs, activate, and stabilize extended ectodomain conformations. Antibodies 12G10 and HUTS-4 bind to the ß1 ßI domain and hybrid domains, respectively, activate, and stabilize the open headpiece conformation. Antibody TS2/16 binds a similar epitope as 12G10, activates, and appears to stabilize an open ßI domain conformation without requiring extension or hybrid domain swing-out. mAb13 and SG/19 bind to the ßI domain and ßI-hybrid domain interface, respectively, inhibit, and stabilize the closed conformation of the headpiece. The effects of the antibodies on cell adhesion to fibronectin substrates suggest that the extended-open conformation of α5ß1 is adhesive and that the extended-closed and bent-closed conformations are nonadhesive. The functional effects and binding sites of antibodies and fibronectin were consistent with their ability in binding to α5ß1 on cell surfaces to cross-enhance or inhibit one another by competitive or noncompetitive (allosteric) mechanisms.
Assuntos
Integrina alfa5beta1/química , Escherichia coli , Fibronectinas/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
At the acidic pH of the trans-Golgi and Weibel-Palade bodies (WPBs), but not at the alkaline pH of secretion, the C-terminal â¼1350 residues of von Willebrand factor (VWF) zip up into an elongated, dimeric bouquet. Six small domains visualized here for the first time between the D4 and cystine-knot domains form a stem. The A2, A3, and D4 domains form a raceme with three pairs of opposed, large, flower-like domains. N-terminal VWF domains mediate helical tubule formation in WPBs and template N-terminal disulphide linkage between VWF dimers, to form ultralong VWF concatamers. The dimensions we measure in VWF at pH 6.2 and 7.4, and the distance between tubules in nascent WPB, suggest that dimeric bouquets are essential for correct VWF dimer incorporation into growing tubules and to prevent crosslinking between neighbouring tubules. Further insights into the structure of the domains and flexible segments in VWF provide an overall view of VWF structure important for understanding both the biogenesis of ultralong concatamers at acidic pH and flow-regulated changes in concatamer conformation in plasma at alkaline pH that trigger hemostasis.
Assuntos
Fator de von Willebrand/metabolismo , Velocidade do Fluxo Sanguíneo , Plaquetas/metabolismo , Dimerização , Dissulfetos/química , Endotélio Vascular/metabolismo , Células HEK293 , Hemostasia , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica/métodos , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Corpos de Weibel-Palade/metabolismoRESUMO
Sporozoite gliding motility and invasion of mosquito and vertebrate host cells in malaria is mediated by thrombospondin repeat anonymous protein (TRAP). Tandem von Willebrand factor A (VWA) and thrombospondin type I repeat (TSR) domains in TRAP connect through proline-rich stalk, transmembrane, and cytoplasmic domains to the parasite actin-dependent motility apparatus. We crystallized fragments containing the VWA and TSR domains from Plasmodium vivax and Plasmodium falciparum in different crystal lattices. TRAP VWA domains adopt closed and open conformations, and bind a Mg(2+) ion at a metal ion-dependent adhesion site implicated in ligand binding. Metal ion coordination in the open state is identical to that seen in the open high-affinity state of integrin I domains. The closed VWA conformation associates with a disordered TSR domain. In contrast, the open VWA conformation crystallizes with an extensible ß ribbon and ordered TSR domain. The extensible ß ribbon is composed of disulfide-bonded segments N- and C-terminal to the VWA domain that are largely drawn out of the closed VWA domain in a 15 Å movement to the open conformation. The extensible ß ribbon and TSR domain overlap at a conserved interface. The VWA, extensible ß ribbon, and TSR domains adopt a highly elongated overall orientation that would be stabilized by tensile force exerted across a ligand-receptor complex by the actin motility apparatus of the sporozoite. Our results provide insights into regulation of "stick-and-slip" parasite motility and for development of sporozoite subunit vaccines.
Assuntos
Movimento/fisiologia , Plasmodium/fisiologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Esporozoítos/fisiologia , Sequência de Aminoácidos , Animais , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Eletricidade EstáticaRESUMO
Circumsporozoite (CS) protein is the major surface component of Plasmodium falciparum sporozoites and is essential for host cell invasion. A vaccine containing tandem repeats, region III, and thrombospondin type-I repeat (TSR) of CS is efficacious in phase III trials but gives only a 35% reduction in severe malaria in the first year postimmunization. We solved crystal structures showing that region III and TSR fold into a single unit, an "αTSR" domain. The αTSR domain possesses a hydrophobic pocket and core, missing in TSR domains. CS binds heparin, but αTSR does not. Interestingly, polymorphic T-cell epitopes map to specialized αTSR regions. The N and C termini are unexpectedly close, providing clues for sporozoite sheath organization. Elucidation of a unique structure of a domain within CS enables rational design of next-generation subunit vaccines and functional and medicinal chemical investigation of the conserved hydrophobic pocket.
Assuntos
Vacinas Antimaláricas/química , Malária Falciparum/prevenção & controle , Modelos Moleculares , Plasmodium falciparum , Dobramento de Proteína , Proteínas de Protozoários/química , Esporozoítos/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cristalografia , Células HEK293 , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Espalhamento a Baixo Ângulo , Alinhamento de SequênciaRESUMO
In the present study, we re-annotated von Willebrand factor (VWF), assigned its entire sequence to specific modules, and related these modules to structure using electron microscopy (EM). The D domains are assemblies of smaller modules visible as lobes in EM. Modules in the D-domain assemblies include von Willebrand D, 8-cysteine, trypsin inhibitor-like, E or fibronectin type 1-like domains, and a unique D4N module in D4. The D1-D2 prodomain shows 2 large connected assemblies, each containing smaller lobes. The previous B and C regions of VWF are re-annotated as 6 tandem von Willebrand C (VWC) and VWC-like domains. These 6 VWC domains correspond to 6 elongated domains that associate in pairs at acidic pH in the stem region of VWF dimeric bouquets. This correspondence is demonstrated by binding of integrin α(IIb)ß(3) to the fourth module seen in EM, VWC4, which bears the VWF Arg-Gly-Asp motif. The C-terminal cystine knot domain dimerizes end-to-end in a manner predicted by homology to TGF-ß and orients approximately perpendicular to the VWC domains in dimeric bouquets. Homologies of domains in VWF to domains in other proteins allow many disulfide bonds to be tentatively assigned, which may have functional implications.
Assuntos
Fator de von Willebrand/química , Fator de von Willebrand/genética , Sequência de Aminoácidos , Sítios de Ligação , Dimerização , Humanos , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Fator de von Willebrand/ultraestruturaRESUMO
We study a mechanism by which dimerization of the EGF receptor (EGFR) cytoplasmic domain is transmitted to the ectodomain. Therapeutic and other small molecule antagonists to the kinase domain that stabilize its active conformation, but not those that stabilize an inactive conformation, stabilize ectodomain dimerization. Inhibitor-induced dimerization requires an asymmetric kinase domain interface associated with activation. EGF and kinase inhibitors stimulate formation of identical dimer interfaces in the EGFR transmembrane domain, as shown by disulfide cross-linking. Disulfide cross-linking at an interface in domain IV in the ectodomain was also stimulated similarly; however, EGF but not inhibitors stimulated cross-linking in domain II. Inhibitors similarly induced noncovalent dimerization in nearly full-length, detergent-solubilized EGFR as shown by gel filtration. EGFR ectodomain deletion resulted in spontaneous dimerization, whereas deletion of exons 2-7, in which extracellular domains III and IV are retained, did not. In EM, kinase inhibitor-induced dimers lacked any well defined orientation between the ectodomain monomers. Fab of the therapeutic antibody cetuximab to domain III confirmed a variable position and orientation of this domain in inhibitor-induced dimers but suggested that the C termini of domain IV of the two monomers were in close proximity, consistent with dimerization in the transmembrane domains. The results provide insights into the relative energetics of intracellular and extracellular dimerization in EGFR and have significance for physiologic dimerization through the asymmetric kinase interface, bidirectional signal transmission in EGFR, and mechanism of action of therapeutics.
Assuntos
Receptores ErbB/química , Fosfotransferases/química , Multimerização Proteica , Estrutura Terciária de Proteína , Animais , Sítios de Ligação/genética , Western Blotting , Linhagem Celular , Reagentes de Ligações Cruzadas/química , Dissulfetos/química , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gefitinibe , Humanos , Microscopia Eletrônica , Mutação , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/metabolismo , Conformação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologiaRESUMO
The lengths of von Willebrand factor (VWF) concatamers correlate with hemostatic potency. After secretion in plasma, length is regulated by hydrodynamic shear force-dependent unfolding of the A2 domain, which is then cleaved by a specific protease. The 1.9-A crystal structure of the A2 domain demonstrates evolutionary adaptations to this shear sensor function. Unique among VWF A (VWA) domains, A2 contains a loop in place of the alpha4 helix, and a cis-proline. The central beta4-strand is poorly packed, with multiple side-chain rotamers. The Tyr-Met cleavage site is buried in the beta4-strand in the central hydrophobic core, and the Tyr structurally links to the C-terminal alpha6-helix. The alpha6-helix ends in 2 Cys residues that are linked by an unusual vicinal disulfide bond that is buried in a hydrophobic pocket. These features may narrow the force range over which unfolding occurs and may also slow refolding. Von Willebrand disease mutations, which presumably lower the force at which A2 unfolds, are illuminated by the structure.
Assuntos
Fator de von Willebrand/química , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Doenças de von Willebrand/genética , Fator de von Willebrand/genéticaRESUMO
Here, we study the gamete fusogen HAP2 from Cyanidioschyzon merolae (Cyani), an extremophile red algae that grows at acidic pH at 45 °C. HAP2 has a trimeric postfusion structure with similarity to viral class II fusion proteins, but its prefusion structure has been elusive. The crystal structure of a monomeric prefusion state of Cyani HAP2 shows it is highly extended with three domains in the order D2, D1, and D3. Three hydrophobic fusion loops at the tip of D2 are each required for postfusion state formation. We followed by negative stain electron microscopy steps in the process of detergent micelle-stimulated postfusion state formation. In an intermediate state, two or three linear HAP2 monomers associate at the end of D2 bearing its fusion loops. Subsequently, D2 and D1 line the core of a trimer and D3 folds back over the exterior of D1 and D2. D3 is not required for formation of intermediate or postfusion-like states.
Assuntos
Extremófilos , Proteínas do Envelope Viral , Extremófilos/metabolismo , Células Germinativas/metabolismo , Conformação Proteica , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/metabolismoRESUMO
Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker.
Assuntos
Plaquetas , Fator de von Willebrand , Plaquetas/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Ureia/metabolismo , Fator de von Willebrand/metabolismoRESUMO
HAP2 is a transmembrane gamete fusogen found in multiple eukaryotic kingdoms and is structurally homologous to viral class II fusogens. Studies in Plasmodium have suggested that HAP2 is an attractive target for vaccines that block transmission of malaria. HAP2 has three extracellular domains, arranged in the order D2, D1, and D3. Here, we report monoclonal antibodies against the D3 fragment of Plasmodium berghei HAP2 and crystal structures of D3 in complex with Fab fragments of two of these antibodies, one of which blocks fertilization of Plasmodium berghei in vitro and transmission of malaria in mosquitoes. We also show how this Fab binds the complete HAP2 ectodomain with electron microscopy. The two antibodies cross-react with HAP2 among multiple plasmodial species. Our characterization of the Plasmodium D3 structure, HAP2 ectodomain architecture, and mechanism of inhibition provide insights for the development of a vaccine to block malaria transmission.
Assuntos
Anticorpos Monoclonais/metabolismo , Células Germinativas/imunologia , Malária/prevenção & controle , Malária/transmissão , Plasmodium berghei/imunologia , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Animais , Sítios de Ligação de Anticorpos , Fenômenos Biofísicos , Culicidae/parasitologia , Células Germinativas/fisiologia , Malária/imunologia , Fusão de Membrana , Ligação Proteica , Proteínas de Protozoários/químicaRESUMO
Inserted (I) domains function as ligand-binding domains in adhesins that support cell adhesion and migration in many eukaryotic phyla. These adhesins include integrin αß heterodimers in metazoans and single subunit transmembrane proteins in apicomplexans such as TRAP in Plasmodium and MIC2 in Toxoplasma. Here we show that the I domain of TRAP is essential for sporozoite gliding motility, mosquito salivary gland invasion and mouse infection. Its replacement with the I domain from Toxoplasma MIC2 fully restores tissue invasion and parasite transmission, while replacement with the aX I domain from human integrins still partially restores liver infection. Mutations around the ligand binding site allowed salivary gland invasion but led to inefficient transmission to the rodent host. These results suggest that apicomplexan parasites appropriated polyspecific I domains in part for their ability to engage with multiple ligands and to provide traction for emigration into diverse organs in distant phyla.
Assuntos
Anopheles/parasitologia , Malária/parasitologia , Plasmodium berghei/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Evolução Molecular , Feminino , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Plasmodium berghei/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Glândulas Salivares/parasitologia , Alinhamento de Sequência , Esporozoítos/fisiologiaRESUMO
The circumsporozoite protein (CSP) and thrombospondin-related adhesion protein (TRAP) are major targets for pre-erythrocytic malaria vaccine development. However, the CSP-based vaccine RTS,S provides only marginal protection, highlighting the need for innovative vaccine design and development. Here we design and characterize expression and folding of P. berghei (Pb) and P. falciparum (Pf) TRAP-CSP fusion proteins, and evaluate immunogenicity and sterilizing immunity in mice. TRAP N-terminal domains were fused to the CSP C-terminal αTSR domain with or without the CSP repeat region, expressed in mammalian cells, and evaluated with or without N-glycan shaving. Pb and Pf fusions were each expressed substantially better than the TRAP or CSP components alone; furthermore, the fusions but not the CSP component could be purified to homogeneity and were well folded and monomeric. As yields of TRAP and CSP fragments were insufficient, we immunized BALB/c mice with Pb TRAP-CSP fusions in AddaVax adjuvant and tested the effects of absence or presence of the CSP repeats and absence or presence of high mannose N-glycans on total antibody titer and protection from infection by mosquito bite both 2.5 months and 6 months after the last immunization. Fusions containing the repeats were completely protective against challenge and re-challenge, while those lacking repeats were significantly less effective. These results correlated with higher total antibody titers when repeats were present. Our results show that TRAP-CSP fusions increase protein antigen production, have the potential to yield effective vaccines, and also guide design of effective proteins that can be encoded by nucleic acid-based and virally vectored vaccines.
Assuntos
Vacinas Antimaláricas/farmacologia , Malária/tratamento farmacológico , Proteínas de Protozoários/genética , Animais , Anticorpos/genética , Anticorpos/imunologia , Antígenos/genética , Antígenos/imunologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/imunologia , Humanos , Imunização , Malária/imunologia , Malária/parasitologia , Vacinas Antimaláricas/imunologia , Camundongos , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/imunologia , Plasmodium berghei/patogenicidade , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/imunologia , Plasmodium falciparum/patogenicidade , Polissacarídeos/genética , Polissacarídeos/imunologia , Dobramento de Proteína , Proteínas de Protozoários/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Transforming growth factorß1 (TGF-ß1) is a versatile cytokine. It has context-dependent pro- and anti-cell proliferation functions. Activation of latent TGF-ß1 requires release of the growth factor from pro-complexes and is regulated through TGF-ß binding proteins. Two types of TGF-ß binding partners, latent TGF-ß-binding proteins (LTBPs) and leucine-rich-repeat-containing protein 32 (LRRC32), have been identified and their expression are cell specific. TGF-ß1 also plays important roles in acute myeloid leukemia (AML) cells. However, the expression of LTBPs and LRRC32 are lacking in myeloid lineage cells and the binding protein of TGF-ß1 in these cells are unknown. Here we show that a novel leucine-rich-repeat-containing protein family member, LRRC33, with high mRNA level in AML cells, to be the binding and regulating protein of TGF-ß1 in AML cells. Using two representative cell lines MV4-11 and AML193, we demonstrate that the protein expression of LRRC33 and TGF-ß1 are correlated. LRRC33 co-localizes and forms complex with latent TGF-ß1 protein on the cell surface and intracellularly in these cells. Similar as in other cell types, the activation of TGF-ß1 in MV4-11 and AML193 cells are also integrin dependent. We anticipate our study to be a starting point of more comprehensive research on LRRC33 as novel TGF-ß regulating protein and potential non-genomic based drug target for AML and other myeloid malignancy.