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Extreme sensitivity of botulinum neurotoxin domains towards mild agitation

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Abstract

Botulinum neurotoxins (BoNTs) and their fragments are targets of therapeutic developments and are increasingly used as therapeutic, prophylactic, and research reagents. However, published data on their properties vary widely. In order to gain a better understanding of these variations, we initiated a systematic investigation of the stability parameters of catalytic light chains (Lc) as well as of cell surface binding domains (Hc) of the neurotoxin. When followed by CD spectroscopy, we noticed that the recombinant light chains of serotypes A (LcA), B, D, E, and G rapidly lost their secondary structures by mild stirring. Denaturation of LcA increased with stirring speed and temperature resulting in a catalytically inactive precipitate. Reducing agents or an anaerobic environment were ineffective in the denaturation. Under identical conditions, bovine serum albumin, ovalbumin, carboxypeptidase B, and of thermolysin, a structural and functional analogue of LcA, remained unchanged. Hc domains of serotype A, B, C, E, and F were also denatured by mild stirring. Adding the nonionic detergent Tween-20 to LcA completely prevented the denaturation. We speculate that the BoNT domains undergo surface denaturation due to rapid exposure of hydrophobic residues by mechanical agitation. This study has important implications for handling BoNT proteins used in therapeutic development. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3302–3311, 2009

Section snippets

INTRODUCTION

Understanding and monitoring the stability of proteins are important factors in their therapeutic applications. Botulinum neurotoxins (BoNTs) are most lethal of all toxins.1,2 The neurotoxins and their nontoxic fragments are also finding increasing application as therapeutic and prophylactic agents,3, 4, 5, 6, 7, 8, 9 and are targets of drug development.10, 11, 12, 13 These neurotoxins are initially expressed by strains of Clostridium botulinum as 150-kDa single polypeptides along with other

BoNT/A LC, Chemicals, Buffers, and Reagents

The 449-residue recombinant BoNT/A LC (LcA) with an extra valine residue at position 218 was expressed and purified as described.17 The homogeneous preparation was stored at −20°C in 50 mM Na-phosphate, pH 6.5 containing 150–250 mM NaCl and 2 mM EDTA. Recombinant LcB was purified as published,23 those of LcD and LcG will be published elsewhere. Recombinant LcE was purchased from Bibitech (Dartmouth, MA). Recombinant Hc proteins were purified in Dr. Smith's laboratory. Before use, each 1 mL of

Denaturation of LcA Induced by Mechanical Stirring

Agitation by stirring of 2.7 mL solution in all experiments was set at a speed that did not produce any vortex or visible turbulence at the surface, which was 13 mm above the “Star Head” magnetic stirrer (or 18 mm above the “Micro-Flea” stirrer bar) and 5 mm above the center of light path. The stirring therefore will be considered as mild. Figure 2 (curve 1) shows that mild stirring of LcA with the “Star Head” bar led to rapid loss of its CD signal related to α-helical secondary structure as a

Extreme Instability

Protein denaturation by shaking or mechanical agitation is an old observation. As early as 1927, Wu30 (and the references therein) reported shaking a solution of protein destabilizes or denatures protein, forming aggregates and precipitates which are often catalytically inactive. Yet, destabilization leading to aggregation still remains a common problem with protein pharmaceuticals.31,32 These destabilizations are usually attributed to exposure of kinetically stable unfolded33 protein molecules

SCHEME I

The CD technique employed in these studies could not detect any intermediate such as TS* or its aggregate-competent D1 (Fig. 2B) but agitation might have simply enhanced the formation of D1 that rapidly formed the inactive species Dm and Dm+1. In the absence of significant agitation, the catalytically inactive or poorly active D1 may have accumulated in a solution yielding suboptimal catalytic activity of a LC preparation. Indeed, large variations in the catalytic activity of the same LcA are

Acknowledgements

The research described herein was sponsored by JSTO-CBD (RIID 3.10011_06_RD_B (to SAA)). We thank Dr. John Carra, Dr. Wieslaw Swietnicki, and Dr. Frank Lebeda for helpful comments and critical reading of the manuscript.

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