Shuowei Cai

Identification of β-turn and random coil amide III infrared bands for secondary structure estimation of proteins

Fourier transform infrared spectroscopy is increasingly becoming an important method to determine secondary structure of peptides and proteins. Among the spectral regions arising out of coupled and uncoupled stretching and bending modes of amide bonds, amide I and amide III spectral bands have been found to be the most sensitive to the variations in secondary structure folding. Amide I spectral region (1700-1600 cm-1), although most commonly used primarily because of its strong signal, suffers from several limitations, including a strong interference from water vibrational band, relatively unstructured spectral contour, and overlap of revolved bands correspondingly to various secondary structures. In contrast, amide III spectral region (1350-1200 cm-1), albeit relatively weak in signals, does not have the above limitations. Easily resolved and better defined amide III bands are quite suitable for quantitative analysis of protein secondary structure. While amide III region has been successfully used for determination of alpha-helix and beta-sheets (Fu, F.-N., et al. (1994) Appl. Spectrosc. 48, 1432-1441), bands corresponding to beta-turns and random coils have not been identified, so far. In this paper, we describe, for the first time, identification of amide III bands corresponding to beta-turns and random coils by selectively enhancing random coils by treatment with a denaturing reagent, and secondary structure estimation of several proteins by using the band assignments. The assignments of spectral bands were as follows: 1330-1295 cm-1, alpha-helix; 1295-1270 cm-1, beta-turns; 1270-1250 cm-1, random coils; and 1250-1220 cm-1, beta-sheets. The estimations of secondary structural elements by the above assignments correlated quite well with secondary structure estimations from X-ray crystallography data.

Botulism Diagnostics: From Clinical Symptoms to in vitro Assays

Botulinum neurotoxin (BoNT), which cause the deadly neuroparalytic disease, botulism, is the most toxic substance known to man. BoNT can be used as potential bioterrorism agents, and therefore, pose great threat to national security and public health. Rapid and sensitive detection of BoNTs using molecular and biochemical techniques is an essential component in the diagnosis of botulism, and is yet to be achieved. The most sensitive and widely accepted assay method for BoNTs is mouse bioassay, which takes 4 days to complete. This clearly can not meet the need for clinical diagnosis of botulism, botulinum detection in field conditions, and screening of large scale samples. Consequently, the clinical diagnosis of botulism relies on the clinical symptom development, thus limiting the effectiveness of antitoxin treatment. In response to this critical need, many in vitro methods for BoNT detection are under development. This review is focused on recently developed in vitro detection methods for BoNTs, and emerging new technologies with potential for sensitive and rapid in vitro diagnostics for botulism.

Immunological characterization of the subunits of type A botulinum neurotoxin and different components of its associated proteins

Botulinum neurotoxins (BoNTs) constitute a family of seven structurally similar but antigenically distinct proteins produced by different strains of Clostridium botulinum. Type A botulinum neurotoxin (BoNT/A) is produced along with 6 neurotoxin associated proteins (NAPs) including hemagglutinin (Hn-33) through polycistronic expression of a clustered group of genes to form a complex (BoNT/AC). The presence of NAPs enhances the oral toxicity of the neurotoxin significantly. Hn-33 makes up the largest fraction of NAPs in BoNT/AC and strongly protects BoNT/A against proteases of the GI tract. BoNT in its complex form is also used in therapeutic and cosmetic applications to treat several neuromuscular disorders. In this study immunological reactivity of BoNT/A in its purified and complex forms, neurotoxin associated proteins, and Hn-33 have been examined using enzyme-linked immunosorbent assay (ELISA). Antibodies raised against the whole complex reacted 60 times better with the complex and 35 times better with Hn-33 and NAPs compared to the purified neurotoxin suggesting stronger immunogenicity of NAPs over that of purified neurotoxin and a higher potential of BoNT/AC and its associated proteins to induce host immune response. This observation also suggests that Hn-33 and other NAPs could potentially be employed as adjuvants for development of vaccines against botulism and could be a good surrogate for botulinum diagnostics. ELISA binding curves of BoNT/AC and BoNT/A with antibodies raised against BoNT/A indicate that BoNT/A in its purified and complex forms induces equal immunogenic response and a 2.5-fold higher immunogenic response compared to BoNT/A light and heavy chains. We have also discovered a new protein, an intimin analog, present within the complex preparation of BoNT/A which shows dramatically high immunoreactivity.

In Vitro selection of RNA aptamers that inhibit the activity of type A botulinum neurotoxin

The category A agent, botulinum neurotoxin (BoNT), is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, we have identified three RNA aptamers through SELEX-process, which bind strongly to the light chain of type A BoNT (BoNT/A) and inhibit the endopeptidase activity, with IC(50) in low nM range. Inhibition kinetic studies reveal low nM K(I) and non-competitive nature of their inhibition. Aptamers are unique group of molecules as therapeutics, and this is first report of their development as an antidote against botulism. These data on K(I) and IC(50) strongly suggest that the aptamers have strong potential as antidotes that can reverse the symptom caused by BoNT/A.

Clostridial neurotoxins as a drug delivery vehicle targeting nervous system.

Several neuronal disorders require drug treatment using drug delivery systems for specific delivery of the drugs for the targeted tissues, both at the peripheral and central nervous system levels. We describe a review of information currently available on the potential use of appropriate domains of clostridial neurotoxins, tetanus and botulinum, for effective drug delivery to neuronal systems. While both tetanus and botulinum neurotoxins are capable of delivering drugs the neuronal cells, tetanus neurotoxin is limited in clinical use because of general immunization of population against tetanus. Botulinum neurotoxin which is also being used as a therapeutic reagent has strong potential for drug delivery to nervous tissues.

RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin.

A surface plasmon resonance based RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin is reported. Using detoxified recombinant type A botulinum neurotoxin as the surrogate, the aptasensor detects active toxin within 90 min. The detection limit of the aptasensor in phosphate buffered saline, carrot juice, and fat free milk is 5.8 ng/ml, 20.3 ng/ml and 23.4 ng/ml, respectively, while that in 5-fold diluted human serum is 22.5 ng/ml. Recovery of toxin from disparate sample matrices are within 91-116%. Most significant is the ability of this aptasensor to effectively differentiate the natively folded toxin from denatured, inactive toxin, which is important for homeland security surveillance and threat assessment. The aptasensor is stable for more than 30 days and over 400 injections/regeneration cycles. Such an aptasensor holds great promise for rapid detection of active botulinum neurotoxin for field surveillance due to its robustness, stability and reusability.

Analysis of genomic differences among Clostridium botulinum type A1 strains

BackgroundType A1 Clostridium botulinum strains are a group of Gram-positive, spore-forming anaerobic bacteria that produce a genetically, biochemically, and biophysically indistinguishable 150 kD protein that causes botulism. The genomes of three type A1 C. botulinum strains have been sequenced and show a high degree of synteny. The purpose of this study was to characterize differences among these genomes and compare these differentiating features with two additional unsequenced strains used in previous studies.ResultsSeveral strategies were deployed in this report. First, University of Massachusetts Dartmouth laboratory Hall strain (UMASS strain) neurotoxin gene was amplified by PCR and sequenced; its sequence was aligned with the published ATCC 3502 Sanger Institute Hall strain and Allergan Hall strain neurotoxin gene regions. Sequence alignment showed that there was a synonymous single nucleotide polymorphism (SNP) in the region encoding the heavy chain between Allergan strain and ATCC 3502 and UMASS strains. Second, comparative genomic hybridization (CGH) demonstrated that the UMASS strain and a strain expected to be derived from ATCC 3502 in the Centers for Disease Control and Prevention (CDC) laboratory (ATCC 3502*) differed in gene content compared to the ATCC 3502 genome sequence published by the Sanger Institute. Third, alignment of the three sequenced C. botulinum type A1 strain genomes revealed the presence of four comparable blocks. Strains ATCC 3502 and ATCC 19397 share the same genome organization, while the organization of the blocks in strain Hall were switched. Lastly, PCR was designed to identify UMASS and ATCC 3502* strain genome organizations. The PCR results indicated that UMASS strain belonged to Hall type and ATCC 3502* strain was identical to ATCC 3502 (Sanger Institute) type.ConclusionsTaken together, C. botulinum type A1 strains including Sanger Institute ATCC 3502, ATCC 3502*, ATCC 19397, Hall, Allergan, and UMASS strains demonstrate differences at the level of the neurotoxin gene sequence, in gene content, and in genome arrangement.

Effects of enzymatically inactive recombinant botulinum neurotoxin type A at the mouse neuromuscular junctions.

Botulinum neurotoxin A (BoNT/A) is used clinically to treat several neurological and metabolic diseases. However, the mechanisms that underlie the clinical use of the toxin remain still to be elusive. BoNT/A inhibits acetylcholine (ACh) release at the motor nerve terminals (MNT) and causes neuroparalysis. The toxic effects of BoNT/A at the MNT occur in sub-pico molar range, and it is invaluable to determine the half-life and the persistence of catalytic activity of the toxin to develop therapeutics against BoNT/A intoxication. However, the use of extremely low concentrations of BoNT/A in cellular, or animal models due to high toxicity makes it difficult to determine new cellular mechanisms and binding or interacting partners of BoNT/A. In order to address this, a catalytically deactivated, non-toxic version of BoNT/A, designated as DrBoNT/A, was characterized. DrBoNT/A lacks endoprotease activity (SNAP-25 cleavage) at concentrations as high as 46,875-fold, compared to wild-type BoNT/A. Unlike BoNT/A injection (3.2 pg), injection of the recombinant product (150 ng or 3.2 pg) into mouse hind limbs failed to cause neuroparalysis as exhibited by the lack of inhibition of toe spread reflex (ability of the mouse to spread its hindlimb toes), and inhibit ACh release at the MNT. The in vitro experiments also demonstrate that DrBoNT/A uptake (at concentrations equivalent to BoNT/A), internalization and localization at the MNT remained unaltered. In addition, modeling studies support that DrBoNT/A lacked the zinc binding ability, and the ability to directly participate in the hydrolysis of SNAP-25 substrate. Collectively, we demonstrate that DrBoNT/A is non-toxic to the MNT and can be used as a surrogate tool to understand the mechanism by which BoNT/A modulates signal transduction mechanisms.

Microarray analysis of differentially regulated genes in human neuronal and epithelial cell lines upon exposure to type A botulinum neurotoxin.

Among the seven serotypes (A-G), type A botulinum neurotoxin (BoNT/A) is the most prevalent etiologic agent and the most potent serotype to cause foodborne botulism, characterized by flaccid muscle paralysis. Upon ingestion, BoNT/A crosses epithelial cell barriers to reach lymphatic and circulatory systems and blocks acetylcholine release at the pre-synaptic cholinergic nerve terminals of neuromuscular junctions (NMJs) resulting in paralysis. One of the unique features of BoNT/A intoxication is its neuroparalytic longevity due to its persistent catalytic activity. The persistent presence of the toxin inside the cell can induce host cell responses. To understand the pathophysiology and host response at the cellular level, gene expression changes upon exposure of human HT-29 colon carcinoma (epithelial) and SH-SY5Y neuroblastoma cell lines to BoNT/A complex were investigated using microarray analysis. In HT-29 cells, 167 genes were up-regulated while 60 genes were down-regulated, whereas in SH-SY5Y cells about 223 genes were up-regulated and 18 genes were down-regulated. Modulation of genes and pathways involved in neuroinflammatory, ubiquitin-proteasome degradation, phosphatidylinositol, calcium signaling in SH-SY5Y cells, and genes relevant to focal adhesion, cell adhesion molecules, adherens and gap junction related pathways in HT-29 cells suggest a massive host response to BoNT/A. A clear differential response in epithelial and neuronal cells indicates that the genes affected may play a distinct role in BoNTs cellular mode of action, involving these two types of host cells.

The Identification and Biochemical Characterization of Drug-Like Compounds that Inhibit Botulinum Neurotoxin Serotype A Endopeptidase Activity

A robust, high-throughput, two-tiered assay for screening small molecule inhibitors against botulinum neurotoxin serotype A was developed and employed to screen 16,544 compounds. Thirty-four compounds were identified as potent hits employing the first-tier assay. Subsequently, nine were confirmed as actives by our second-tier confirmatory assay. Of these, one displayed potent inhibitory efficacy, possessing an IC(50)=16 microM (+/-1.6 microM) in our in vitro assay. This inhibitor (0831-1035) is highly water-soluble, and possesses an IC(50)=47 microM (+/-7.0 microM) in our primary cell culture assay (with virtually no cytotoxicity up to 500 microM), suggesting that this inhibitor is a good candidate for further development as a therapeutic countermeasure to treat botulism resulting from botulinum neurotoxin serotype A intoxication. An enzyme kinetics study indicated that this inhibitor exhibits mixed non-competitive inhibition, with a K(I)=9 microM.