A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

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

An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behavior

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 β-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 β-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

Amyloid Formation by the Model Protein Muscle Acylphosphatase is Accelerated by Heparin and Heparan Sulphate Through a Scaffolding-based Mechanism

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

Deficiency mutations of α1-antitrypsin differentially affect folding, function and polymerization

Misfolding, polymerisation and defective secretion of functional α1-antitrypsin underlies the predisposition to severe liver and lung disease in α1-antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterised 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 α1-antitrypsin in the moderate deficiency range, but 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 a 85-95% functional deficiency, similar to that seen with ZZ homozygotes. Biochemical, biophysical and computational studies further defined the molecular basis of this deficiency. These demonstrated that native Ala336Pro α1-antitrypsin could populate the polymerogenic intermediate – and therefore polymerise – more readily than either wild-type α1-antitrypsin or the Z variant. In contrast, folding was far less impaired in Ala336Pro α1-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 to polymerisation mechanisms. Moreover, the findings demonstrate that in these variants, folding efficiency does not correlate directly with the tendency to polymerise in vitro or in vivo. They therefore differentiate generalised misfolding from polymerisation tendencies in missense variants of α1-antitrypsin. Clinically they further support the need to quantify loss-of-function in α1-antitrypsin deficiency to individualise patient care

Kinetic Analysis of Amyloid Formation in the Presence of Heparan Sulfate: FASTER UNFOLDING AND CHANGE OF PATHWAY*

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 β-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

Rapid oligomer formation of human muscle acylphosphatase induced by heparan sulfate

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