RESUMEN
Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered ß-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the ß-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation.
Asunto(s)
Serpinas/metabolismo , Regulación Alostérica , Anticuerpos Monoclonales/química , Espectroscopía de Resonancia por Spin del Electrón , Ensayo de Inmunoadsorción Enzimática , Transferencia Resonante de Energía de Fluorescencia , Mutagénesis Sitio-Dirigida , Polimerizacion , Temperatura , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genéticaRESUMEN
Misfolding, polymerization, and defective secretion of functional alpha-1 antitrypsin underlies the predisposition to severe liver and lung disease in alpha-1 antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterized it relative to the wild-type (M) and Glu342Lys (Z) alleles. The index case is a homozygous individual of consanguineous parentage, with levels of circulating alpha-1 antitrypsin in the moderate deficiency range, but is a biochemical phenotype that could not be classified by standard methods. The majority of the protein was present as functionally inactive polymer, and the remaining monomer was 37% active relative to the wild-type protein. These factors combined indicate an 85 to 95% functional deficiency, similar to that seen with ZZ homozygotes. Biochemical, biophysical, and computational studies further defined the molecular basis of this deficiency. These studies demonstrated that native Ala336Pro alpha-1 antitrypsin could populate the polymerogenic intermediate-and therefore polymerize-more readily than either wild-type alpha-1 antitrypsin or the Z variant. In contrast, folding was far less impaired in Ala336Pro alpha-1 antitrypsin than in the Z variant. The data are consistent with a disparate contribution by the "breach" region and "shutter" region of strand 5A to folding and polymerization mechanisms. Moreover, the findings demonstrate that, in these variants, folding efficiency does not correlate directly with the tendency to polymerize in vitro or in vivo. They therefore differentiate generalized misfolding from polymerization tendencies in missense variants of alpha-1 antitrypsin. Clinically, they further support the need to quantify loss-of-function in alpha-1 antitrypsin deficiency to individualize patient care.
Asunto(s)
Mutación , Deficiencia de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , Adulto , Análisis Mutacional de ADN , Estabilidad de Enzimas , Femenino , Predisposición Genética a la Enfermedad , Homocigoto , Humanos , Cinética , Modelos Moleculares , Fenotipo , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Multimerización de Proteína , alfa 1-Antitripsina/química , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/enzimologíaRESUMEN
Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.
Asunto(s)
Polimerizacion/efectos de los fármacos , Inhibidores de Proteasas/metabolismo , Anticuerpos de Cadena Única/farmacología , alfa 1-Antitripsina/metabolismo , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Humanos , Immunoblotting , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Cinética , Elastasa de Leucocito/metabolismo , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación , Inhibidores de Proteasas/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/inmunologíaRESUMEN
A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen-whose native state is susceptible to the formation of a proto-oligomeric intermediate-we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/inmunología , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/inmunología , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales de Origen Murino/química , Especificidad de Anticuerpos , Epítopos/química , Epítopos/genética , Variación Genética , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Mutación , Conformación Proteica , Multimerización de Proteína/inmunología , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Termodinámica , alfa 1-Antitripsina/químicaRESUMEN
A number of human diseases are associated with the conversion of proteins from their native state into well defined fibrillar aggregates, depositing in the extracellular space and generally termed amyloid fibrils. Heparan sulfate (HS), a glycosaminoglycan normally present in the extracellular matrix, has been found to be universally associated with amyloid deposits and to promote amyloid fibril formation by all studied protein systems. We have studied the impact of HS on the amyloidogenesis of human muscle acylphosphatase, monitoring the process with an array of techniques, such as normal and stopped-flow far-UV circular dichroism, thioflavin T fluorescence, static and dynamic light scattering, and atomic force microscopy. The results show that HS accelerates the conversion of the studied protein from the native state into the amyloidogenic, yet monomeric, partially folded state. They also indicate that HS does not simply accelerate the conversion of the resulting partially folded state into amyloid species but splits the process into two distinct pathways occurring in parallel: a very fast phase in which HS interacts with a fraction of protein molecules, causing their rapid aggregation into ThT-positive and beta-sheet containing oligomers, and a slow phase resulting from the normal aggregation of partially folded molecules that cannot interact with HS. The HS-mediated aggregation pathway is severalfold faster than that observed in the absence of HS. Two aggregation phases are generally observed when proteins aggregate in the presence of HS, underlying the importance of a detailed kinetic analysis to fully understand the effect of this glycosaminoglycan on amyloidogenesis.
Asunto(s)
Ácido Anhídrido Hidrolasas/química , Amiloide/química , Matriz Extracelular/química , Heparitina Sulfato/química , Proteínas Musculares/química , Tiazoles/química , Ácido Anhídrido Hidrolasas/metabolismo , Amiloide/metabolismo , Benzotiazoles , Dicroismo Circular , Matriz Extracelular/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Cinética , Proteínas Musculares/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , AcilfosfatasaRESUMEN
Many human diseases are caused by the conversion of proteins from their native state into amyloid fibrils that deposit in the extracellular space. Heparan sulfate, a component of the extracellular matrix, is universally associated with amyloid deposits and promotes fibril formation. The formation of cytotoxic prefibrillar oligomers is challenging to study because of its rapidity, transient appearance and the heterogeneity of species generated. The process is even more complex with agents such as heparan sulfate. Here we have used a stopped-flow device coupled to turbidometry detection to monitor the rapid conversion of human muscle acylphosphatase into oligomers with varying heparan sulfate and protein concentrations. We also analyzed mutants of the 15 basic amino acids of acylphosphatase, identifying the residues primarily involved in heparan sulfate-induced oligomerization of this protein and tracing the process with unprecedented molecular detail.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Heparitina Sulfato/metabolismo , Músculos/enzimología , Ácido Anhídrido Hidrolasas/química , Ácido Anhídrido Hidrolasas/genética , Amiloide/química , Amiloide/genética , Amiloide/metabolismo , Humanos , Músculos/metabolismo , Mutación , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , AcilfosfatasaRESUMEN
Amyloid formation is the hallmark of many diseases. The propensity of a protein to aggregate depends on a number of biological factors like the presence of sulphated polysaccharides termed as glycosaminoglycans (GAGs). Here we assessed whether the polymeric nature of GAGs is responsible for their protein aggregation-promoting effect. We studied the effect of different monosaccharide derivatives, featuring the main characteristics of heparin and heparan sulphate (HS) building blocks, on the aggregation kinetics of human muscle acylphosphatase (mAcP), a useful model protein for these studies. We observed that while heparin and HS changed the mAcP aggregation kinetic profile, the monosaccharide derivatives had no effect, whatever their concentration could be and both when they are studied separately or in combination. In contrast, heparin fragments with six or more monosaccharides reproduced the effects of HS and in part those of heparin. We conclude that the effect of heparin and HS on protein aggregation arises from the clustering and regular distribution of their composing units on a polymeric structure. We propose a model in which heparin and HS promote mAcP aggregation through a scaffolding-based mechanism, in which the regularly spaced sulphate moieties of the polymer interact with mAcP molecules increasing their local concentration and facilitating their orientation.