RESUMEN
Here, we describe molecular engineering of monovalent ultra-long acting two-chain insulin-Fc conjugates. Insulin-Fc conjugates were synthesized using trifunctional linkers with one amino reactive group for reaction with a lysine residue of insulin and two thiol reactive groups used for re-bridging of a disulfide bond within the Fc molecule. The ultra-long pharmacokinetic profile of the insulin-Fc conjugates was the result of concertedly slowing insulin receptor-mediated clearance by (1) introduction of amino acid substitutions that lowered the insulin receptor affinity and (2) conjugating insulin to the Fc element. Fc conjugation leads to recycling by the neonatal Fc receptor and increase in the molecular size, both contributing to the ultra-long pharmacokinetic and pharmacodynamic profiles.
Asunto(s)
Hipoglucemiantes/síntesis química , Inmunoconjugados/química , Fragmentos Fc de Inmunoglobulinas/química , Insulina de Acción Prolongada/síntesis química , Secuencia de Aminoácidos , Animales , Línea Celular , Diabetes Mellitus Experimental/tratamiento farmacológico , Humanos , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/uso terapéutico , Inmunoconjugados/farmacocinética , Inmunoconjugados/uso terapéutico , Fragmentos Fc de Inmunoglobulinas/farmacología , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Insulina de Acción Prolongada/farmacocinética , Insulina de Acción Prolongada/uso terapéutico , Masculino , Mesocricetus , Ingeniería de Proteínas , Ratas Sprague-DawleyRESUMEN
Birch-allergic patients often experience oral allergy syndrome upon ingestion of vegetables and fruits, most prominently apple, that is caused by antibody cross-reactivity of the IgE antibodies in patients to proteins sharing molecular surface structures with the major birch pollen group 1 allergen from Betula verrucosa (Bet v 1). Still, to what extent two molecular surfaces need to be similar for clinically relevant antibody cross-reactivity to occur is unknown. Here, we describe the grafting of a defined conformational antibody epitope from Bet v 1 onto the surface of the homologous apple allergen Malus domestica group 1 (Mal d 1). Engineering of the epitope was accomplished by genetic engineering substituting amino acid residues in Mal d 1 differing between Bet v 1 and Mal d 1 within the epitope defined by the mAb BV16. The kinetic parameters characterizing the antibody binding interaction to Bet v 1 and to the mutated Mal d 1 variant, respectively, were assessed by Biacore experiments demonstrating indistinguishable binding kinetics. This demonstrates that a conformational epitope defined by a high affinity antibody-allergen interaction can successfully be grafted onto a homologous scaffold molecule without loss of epitope functionality. Furthermore, we show that increasing surface similarity to Bet v 1 of Mal d 1 variants by substitution of 6-8 residues increased the ability to trigger basophil histamine release with blood from birch-allergic patients not responding to natural Mal d 1. Conversely, reducing surface similarity to Bet v 1 of a Mal d 1 variant by substitution of three residues abolished histamine release in one patient reacting to Mal d 1.
Asunto(s)
Alérgenos/química , Antígenos de Plantas/química , Epítopos/química , Inmunoglobulina E/química , Proteínas de Plantas/química , Alérgenos/genética , Alérgenos/inmunología , Alérgenos/metabolismo , Antígenos de Plantas/genética , Antígenos de Plantas/inmunología , Antígenos de Plantas/metabolismo , Epítopos/genética , Epítopos/inmunología , Epítopos/metabolismo , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/genética , Inmunoglobulina E/inmunología , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Rinitis Alérgica Estacional/sangre , Rinitis Alérgica Estacional/genética , Rinitis Alérgica Estacional/inmunologíaRESUMEN
The 159 residue Bet v 1 is the major allergen from birch tree pollen. Its natural function is unknown although it is capable of binding several types of physiologically relevant ligands in a centrally placed cavity in the protein structure. Here we use circular dichroism and fluorescence spectroscopy to show that Bet v 1 binds to DOPC and DOPG phospholipid vesicles in a pH-dependent manner. Binding is facilitated by low pH, negatively charged phospholipids, and high vesicle curvature, indicating that electrostatic interactions and vesicle surface defects are important parameters for binding. Binding is accompanied by major structural rearrangements, involving an increase in alpha-helical structure and a decrease in beta-structure. A bilayer structure per se is not a prerequisite for these rearrangements, since they also occur in the presence of the micelle-forming lysophospholipids lysoMPC and lysoMPG. Two major bound states (A and B) with distinct secondary structure compositions were identified, which predominate in the pH ranges approximately 9.5-6.5 and approximately 5-2.5, respectively. Despite the high content of secondary structure, the A- and B-states are partially unfolded as they unfold noncooperatively in CD thermal scans, in contrast to the native state. In addition, the B-state (but not the A-state) shows intermediate proteolysis-resistance and is able to induce complete leakage of calcein from the vesicles, indicating that this state is partially inserted into and significantly perturbs the bilayer structure. We conclude that Bet v 1 is a membrane binding protein, highlighting a possible biological function of this protein.
Asunto(s)
Alérgenos/metabolismo , Liposomas/metabolismo , Alérgenos/efectos de los fármacos , Antígenos de Plantas , Betula/química , Proteínas Portadoras/química , Dicroismo Circular , Detergentes/farmacología , Glucósidos/farmacología , Proteínas de la Membrana/química , Micelas , Permeabilidad/efectos de los fármacos , Fosfatidilcolinas/química , Proteínas de Plantas/metabolismo , Liposomas Unilamelares/metabolismoRESUMEN
Staphylococcal gamma-hemolysins are bicomponent toxins forming a protein family with leucocidins and alpha-toxin. Two active toxins (AB and CB) can be formed combining one of the class-S components, HlgA or HlgC, with the class-F component HlgB. These two gamma-hemolysins form pores with marked similarities to alpha-toxin in terms of conductance, nonlinearity of the current-voltage curve, and channel stability in the open state. AB and CB pores, however, are cation-selective, whereas alpha-toxin is anion-selective. gamma-Hemolysins' pores are hetero-oligomers formed by three or four copies of each component (indicated as 3A3B and 3C3B or 4A4B and 4C4B). Point mutants located on a beta-strand of the class-S component that forms part of the protomer-protomer contact region can prevent oligomer assembly. Interestingly, these mutants inhibit growth of pores formed not only by their natural components but also by nonstandard components. This lead to the hypothesis that mixed ABC pores could also be formed. By studying the conductance of pores, assembled in the presence of all three components (in different ratios), it was observed that the magnitudes expected for mixed pores were, indeed, present. We conclude that the gamma-hemolysin/leucocidin bicomponent toxin family may form a larger than expected number of active toxins by cross-combining various S and F components.
Asunto(s)
Proteínas Hemolisinas/química , Staphylococcus aureus/química , Eritrocitos/fisiología , Proteínas Hemolisinas/genética , Hemólisis , Humanos , Membrana Dobles de Lípidos , Lípidos/química , Membranas Artificiales , Mutagénesis , Permeabilidad , Mutación Puntual , Staphylococcus aureus/genéticaRESUMEN
Human type 1 immediate allergic response symptoms are caused by mediator release from basophils and mast cells. This event is triggered by allergens aggregating preformed IgE Abs bound to the high-affinity receptor (FcepsilonRI) on these cells. Thus, the allergen/IgE interaction is crucial for the cascade leading to the allergic and anaphylactic response. Two genetically engineered forms of the white birch pollen major allergen Bet v 1 with point mutations directed at molecular surfaces have been characterized. Four and nine point mutations led to a significant reduction of the binding to human serum IgE, suggesting a mutation-induced distortion of IgE-binding B cell epitopes. In addition, the mutated allergens showed a decrease in anaphylactic potential, because histamine release from human basophils was significantly reduced. Retained alpha-carbon backbone folding pattern of the mutated allergens was indicated by x-ray diffraction analysis and circular dichroism spectroscopy. The rBet v 1 mutants were able to induce proliferation of T cell lines derived from birch pollen allergic patients. The stimulation indices were similar to the indices of nonmutated rBet v 1 and natural Bet v 1 purified from birch pollen. The ability of anti-rBet v 1 mutant specific mouse IgG serum to block binding of human serum IgE to rBet v 1 demonstrates that the engineered rBet v 1 mutants are able to induce Abs reactive with nonmodified Bet v 1. rBet v 1 mutants may constitute vaccine candidates with improved efficacy/safety profiles for safer allergy vaccination.
Asunto(s)
Alérgenos/inmunología , Inmunoglobulina E/metabolismo , Pliegue de Proteína , Vacunas Sintéticas/inmunología , Alérgenos/química , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Antígenos de Plantas , Basófilos/metabolismo , Liberación de Histamina , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , Linfocitos T/inmunologíaRESUMEN
Osteoclasts require matrix metalloproteinase (MMP) activity and cathepsin K to resorb bone, but the critical MMP has not been identified. Osteoclasts express MMP-9 and MMP-14, which do not appear limiting for resorption, and the expression of additional MMPs is not clear. MMP-12, also called metalloelastase, is reported only in a few cells, including tissue macrophages and hypertrophic chondrocytes. MMP-12 is critical for invasion and destruction in pathologies such as aneurysm and emphysema. In the present study, we demonstrate that osteoclasts express MMP-12, although only in some situations. Northern blots show that highly purified rabbit osteoclasts in culture express MMP-12 at the same level as macrophages, whereas in situ hybridizations performed on rabbit bone do not show any MMP-12 expression in osteoclasts whatever the bone type. In contrast, in situ hybridizations performed on mouse bone show MMP-12 expression in osteoclasts in calvariae and long bones. We also demonstrate that recombinant MMP-12 cleaves the putative functional domains of osteopontin and bone sialoprotein, two bone matrix proteins that strongly influence osteoclast activities, such as attachment, spreading and resorption. Furthermore, we investigated the role of MMP-12 in bone resorption and osteoclast recruitment by comparing MMP-12 knockout and wild-type mice in specialized culture models known to depend on MMP activity, as well as in the ovariectomy model, and we did not find any indication for a limiting role of MMP-12 in these processes. In conclusion, we found that osteoclasts are able to express MMP-12, but MMP-12 did not appear critical for osteoclast recruitment or resorption. The fact that none of the MMPs identified so far in osteoclasts appears limiting for resorption, gives strength to the hypothesis that the critical MMP for bone solubilization is produced by non-osteoclastic cells.
Asunto(s)
Resorción Ósea/enzimología , Metaloendopeptidasas/metabolismo , Osteoclastos/enzimología , Secuencia de Aminoácidos , Animales , Northern Blotting , Matriz Ósea/metabolismo , Resorción Ósea/metabolismo , Células Cultivadas , Clonación Molecular , ADN Complementario/genética , Femenino , Metaloproteinasa 12 de la Matriz , Metaloendopeptidasas/deficiencia , Metaloendopeptidasas/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Osteoclastos/citología , Osteoclastos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Upon termination of bone matrix synthesis, osteoblasts either undergo apoptosis or differentiate into osteocytes or bone lining cells. In this study, we investigated the role of matrix metalloproteinases (MMPs) and growth factors in the differentiation of osteoblasts into osteocytes and in osteoblast apoptosis. The mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts were either grown on two-dimensional (2-D) collagen-coated surfaces, where they morphologically resemble flattened, cuboidal bone lining cells, or embedded in three-dimensional (3-D) collagen gels, where they resemble dendritic osteocytes constituting a network of cells. When MC3T3-E1 osteoblasts were grown in a 3-D matrix in the presence of an MMP inhibitor (GM6001), the cell number was dose-dependently reduced by approximately 50%, whereas no effect was observed on a 2-D substratum. In contrast, the murine mature osteocyte cell line, MLO-Y4, was unaffected by GM6001 under all culture conditions. According to TUNEL assay, the osteoblast apoptosis was increased 2.5-fold by 10 microm GM6001. To investigate the mechanism by which MMPs mediate the survival of osteoblasts, we examined the effect of GM6001 on MC3T3-E1 osteoblasts in the presence of extracellular matrix components and growth factors, including tenascin, fibronectin, laminin, collagenase-cleaved collagen, gelatin, parathyroid hormone, basic fibroblast growth factor, vascular epidermal growth factor, insulin-like growth factor, interleukin-1, and latent and active transforming growth factor-beta (TGF-beta). Only active TGF-beta, but not latent TGF-beta or other agents tested, restored cell number and apoptosis to control levels. Furthermore, we found that the membrane type MMP, MT1-MMP, which is produced by osteoblasts, could activate latent TGF-beta and that antibodies neutralizing endogenous TGF-beta led to a similar decrease in cell number as GM6001. Whereas inhibitors of other protease families did not induce osteoblast apoptosis, an inhibitor of the p44/42 mitogen-activated protein kinase showed the same but non-synergetic effect as GM6001. These findings suggest that MMP-activated TGF-beta maintains osteoblast survival during trans-differentiation into osteocytes by a p44/42-dependent pathway.