Sandbox Reserved 403

TextToBeDisplayed</scene>' scene='THE HC FRAGMENT OF TETANUS TOXIN COMPLEXED WITH AN ANALOGUE OF ITS GANGLIOSIDE RECEPTOR GT1B' />

Clostidium tetani

The gram positive bacilli Clostridium tetani is the bacteria responsible for the disease state of tetanus. The presence of the bacteria does not cause the disease but instead the toxins it produces cause the disease state. C. tetani produces two toxins; tetanospasmin and tetanolysin. Tetanolysin is a cytolysin that increases the permeability of cellular membranes through cell lysis. Tetanospasmin is the cause of tetanus and is sometimes referred to as tetanus neurotoxin (TeNT), as it acts on the central nervous system. Tetanospasmin makes its way to the central nervous system via retrograde axonal flow beginning with α- motor neurons found in muscle and ending by binding to gangliosides found in the central nervous system.

Gangliosides

Gangliosides are in the category of glycosphingolipids and are found predominately in neuronal tissues. Gangliosides consist of sialic acid linked to a sugar (glucose, galactose, GalNAc, GlcNAc and/or fructose) backbone attached to a ceramide base. These gangliosides make up approximately 10% of a neuron’s total lipid content and like other lipids functions in cell signal transduction.

Tetanospasmin (TeNT)

Tetanospasmin is a 150-kDa toxin that is composed of one light chain (50-kDa) and one heavy chain (100-kDa). The light chain is responsible for the toxicity of the molecule, whereas the heavy chain is responsible for binding the toxin to the axonal membranes. The heavy chain can also be cleaved into 2 fragments Hn and Hc. The HN fragment is responsible for the translocation of the light chain across the axonal membrane, whereas the Hc fragment binds to the axonal membrane.

Hc and Ganglioside Interaction

Hc has two distinct domains:

1. Jelly-roll (amino end)

2. β-Trefoil (carboxyl end)

Studies have shown that the β-trefoil domain contains the ganglioside binding sites.

Binding studies have shown that a particular ganglioside, GT1-b, is necessary for the binding of the Hc fragment of tetanospasmin (TeNT).

An analogue of the GT1-b ganglioside was made in order to increase solubility because a crystal structure of the Hc and native GT1-b could not be obtained.

The Hc fragment has two binding sites in the β-trefoil domain:

At this site a narrow groove is formed where a number of hydrogen bonds can form.

Common hydrogen bonds are formed between the side chain of His1271 and OH-6, OH-4 and O-5 of Gal4 and between the main chain carbonyl oxygen of Thr1270 and OH-4 of Gal4. GalNAc3 interacts via a hydrogen bond between OH-4 andAsp1222 OD and between OH-4 and His1271. Ring stacking involving galactose also occurs in this site.

At this site a shallow pocket is formed where hydrogen bonding occurs.

Commonly hydrogen bonds form between OD-1 and OD-2 of Asp1147 and O-4 and the acetamido-N-5 of Sia6 and between ND-2 of Asn1216 and O-10 of Sia6. A salt bridge also forms between Arg1226 and the sialic acid of Sia7, also the carboxylate group and hydrogen bonds between O-1A and the amide NH of Asn1216; between O-4 and the carbonyl oxygen of Asp1214; and betweenOH-8 and Tyr1229 hydroxyl group on Sia7.