Characterization of SNARE Cleavage Products Generated by Formulated Botulinum Neurotoxin Type-A Drug Products

被引：11

作者：

Hunt, Terrence ^{[1
]}

Rupp, David ^{[1
]}

Shimizu, Gary ^{[1
]}

Tam, Karen ^{[1
]}

Weidler, Julia ^{[1
]}

Xie, Jack ^{[1
]}

机构：

[1] Allergan Pharmaceut Inc, Irvine, CA 92612 USA

来源：

TOXINS | 2010年 / 2卷 / 08期

关键词：

abotulinumtoxinA; botulinum toxin; BOTOX (R); Dysport (R); incobotulinumtoxinA; onabotulinumtoxin A; SNAP-25; SNARE; syntaxin; Xeomin (R);

D O I：

10.3390/toxins2082198

中图分类号：

TS2 [食品工业];

学科分类号：

0832 ;

摘要：

The study evaluated substrate cleavage product(s) generated by three botulinum neurotoxin serotype A (BoNT/A) medicinal drug products utilizing a novel and highly specific, light-chain activity, high-performance liquid chromatography (LCA-HPLC) method. Samples were reacted with a commercially available BoNT/A fluorescent substrate derived from the SNAP-25 sequence. Reaction products were separated by reversed-phase HPLC. The method detected an atypical cleavage pattern by one of the formulated drug products. IncobotulinumtoxinA produced two cleavage fragments rather than the single fragment typically generated by BoNT/A. Identification confirmed the secondary cleavage at a position corresponding to SNAP-25 Arg198-Ala199 (normal BoNT/A cleavage is Gln197-Arg198). Arg198-Ala199 is also the cleavage site for trypsin and serotype C toxin. Normal cleavage was observed for all other BoNT/A drug product samples, as well as 900-kD and 150-kD bulk toxin BoNT/A. The reason for this unexpected secondary cleavage pattern by one formulated BoNT/A drug product is unknown. Possible explanations include a contaminating protease and/or damage to the 150-kD type-A toxin causing nonspecific substrate recognition and subsequent cleavage uncharacteristic of type-A toxin. The BoNT/A drug products were also analyzed via the LCA-HPLC assay using a commercial BoNT/C fluorescent substrate derived from the syntaxin sequence. Cleavage of the serotype C substrate by incobotulinumtoxinA was also confirmed whilst neither of the other drug products cleaved the syntaxin substrate.

引用

页码：2198 / 2212

页数：15

共 10 条

[1] How botulinum and tetanus neurotoxins block neurotransmitter release [J].

Humeau, Y ;

Doussau, F ;

Grant, NJ ;

Poulain, B .

BIOCHIMIE, 2000, 82 (05) :427-446

[2] Potency of the botulinum toxin product CNBTX-A significantly exceeds labeled units in standard potency test [J].

Hunt, Terrence ;

Clarke, Kenneth .

JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY, 2008, 58 (03) :517-518

[3]

Hunt T, 2009, CLIN NEUROPHARMACOL, V32, P28, DOI [10.1097/WNF.0B013E3181692735, 10.1097/WNF.0b013e3181692735]

[4] New highly specific botulinum type C1 endopeptidase immunoassays utilising SNAP25 or Syntaxin substrates [J].

Jones, Russell G. A. ;

Liu, Yvonne ;

Sesardic, Dorothea .

JOURNAL OF IMMUNOLOGICAL METHODS, 2009, 343 (01) :21-27

[5] STRUCTURE AND FUNCTION OF CLOSTRIDIUM-BOTULINUM TOXINS [J].

OGUMA, K ;

FUJINAGA, Y ;

INOUE, K .

MICROBIOLOGY AND IMMUNOLOGY, 1995, 39 (03) :161-168

[6] Development of an in vitro activity assay as an alternative to the mouse bioassay for Clostridium botulinum neurotoxin type A [J].

Rasooly, Reuven ;

Do, Paula M. .

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2008, 74 (14) :4309-4313

[7] High-throughput assays for botulinum neurotoxin proteolytic activity: Serotypes A, B, D, and F [J].

Schmidt, JJ ;

Stafford, RG ;

Millard, CB .

ANALYTICAL BIOCHEMISTRY, 2001, 296 (01) :130-137

[8] Characterization of time-dependent enzymatic profiles under various liquid conditions for 150 and 900 kDa botulinum Type-A neurotoxins [J].

Shimizu, Gary ;

Rupp, David ;

Tam, Karen ;

Aoki, K. Roger ;

Hunt, Terrence .

TOXICON, 2008, 51 :21-21

[9]

Shine N.R., 2003, Substrate Peptides and Assays for Detecting and Measuring Proteolytic Activity of Serotype A Neurotoxin From Clostridium botulinum, Patent No. [6,504,006, 65040067]

[10] Proteolysis of SNAP-25 isoforms by botulinum neurotoxin types A, C, and E:: Domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage [J].

Vaidyanathan, VV ;

Yoshino, K ;

Jahnz, M ;

Dörries, C ;

Bade, S ;

Nauenburg, S ;

Niemann, H ;

Binz, T .

JOURNAL OF NEUROCHEMISTRY, 1999, 72 (01) :327-337

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