User:Marvin O'Neal/Antibody OspA and OspB

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Revision as of 18:08, 29 April 2012

Introduction

Borrelia burgdoferi

The causative agent of Lyme disease is Borellia burgdorferi, a spirochaete found in the gut of hard bodied ticks of genus Ixodes. A factor contributing to the severity of Lyme disease is its resistance to certain forms of complement-dependent immune response by the evasion of the alternative complement pathway, and the blocking of complement C3. ^[1] This resistance increases the importance of the complement independent immune response when combating B. burgdorferi in vivo. The fragment antigen binding regions (fab) of IgG and IgM monoclonal antibodies to OspA and OspB, outer surface proteins of B. burgdoferi leads to a complement-independent lysis of the bacteria.^[2]

Fragment Antigen Binding (fab) Reigon

Digestion of an antibody by Papain separates the fab reigons from the antibody

There are two fab reigons per antibody, each composed of a variable and constant reigon. The variable region contains the paratope, which recognizes the antigen epitope, located at the N terminus of both the heavy chain and light chain of the fab. The complement-independent fab regions that interact with these outer surface proteins are LA-2, which targets the C-terminal of OspA, and CB2 and H6831, which target the C-terminal of OspB. ^[3]

OspB and H6831

Complex Interaction between OspB and H6831

The consist of two main components, the outer surface protein , and the , subdivided into the and the . Most hydrogen bonds and electrostatic interactions that are responsible for the binding of H6831 to OspB are between the three adjacent surface-exposed at the C-terminal of OspB and some on the fab heavy chain that include tyrosine, tryptophan, glutamate, and histidine.

Fab-Osp complexes, such as the OspB-H6831 complex, consist of two main components; the (OspB in this case), and the , which is subdivided into the , and the . Most hydrogen bonds and electrostatic interactions that are responsible for the binding of H6831 to OspB are between the three adjacent surface-exposed at the C-terminal of OspB and some on the fab heavy chain that include tyrosine, tryptophan, glutamate, and histidine.

The majority of the electrostatic and hydrogen-bonded interactions are between loop 2 (residues 250-254) and the fab heavy chain. in loop 2 of OspB has a necessary and major role due to its central position in the exposed loops. A mutation at its position abrogates the binding interaction and causes the resistance of the bacteria to the bactericidal effect of the fab. Lys 253 binds to the two aromatic residues on the fab heavy chain, tyrosine (green link) and tryptophan (green link). It also makes hydrogen bonds with the glutamate (green link) in the heavy chain of the fab and forms an ionic bond. Carbonyl in loop 1 (green link) of the OspB interacts with histidine in the fab heavy chain. of OspB interacts with fab light chain. ^[4].

Conformational Changes to OspB

The binding leads to some conformational changes in OspB. Whereas Ding et al.^[5] found no changes in the C-terminal of OspA upon the binding to the fab. The most significant difference between the free and the complexed structure of OspB is the loss of the central strands 1-4. Both small positional shifts near the Fab binding site and a few larger structural changes away from the binding site were observed. The largest shifts (7– 8 Å) correspond to the repositioning of a loop opposite the Fab-binding site (residues 218 –220). In the free OspB structure, all regions that exhibit shifts are adjacent to the central sheet; in the OspB-H6831 complex they all shift toward, and slightly overlap, the position of the missing sheet. These observations suggest that the larger conformational changes are related to the loss of the central sheet, which could have happened through proteolytic cleavage or fortuitous crystal contacts ^[6]

Requirements for the Bactericidal Effect and Proposed Mechanism

The fab binding destabilizes the outer membrane (OM) of B. burdorferi, with subsequent formation of spheroplasts. It has been observed that the bactericidal action, but not the binding, requires the presence of bivalent cations (Mg2+ and Ca2+). Escudero et al. study demonstrated the inability of fab to kill bacteria in the absence of the bivalent cations. It was speculated that OspB- Cb2 (a fab similar to H6831) complexes could create physical openings in the OM allowing for rapid infusion of electrolytes, increasing the osmolarity of the periplasm and triggering bivalent cation dependent cascades ^[7]. Need to be rephrased and squeezed and may be a sentence about the cholestrol needs to be added and if there is a specific function for the charge triad it may be added

OspA and LA-2

spinqualitylabelsall models OspA-LA2 Complex Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

OspA

OspA is 53% similar to OspB. OspA is usually undetectable in the early stages of lyme disease, because it is down regulated when OspC is expressed (provide citation). Despite the similarity between OspA and OspB, both in vivo and vitro OspB is susceptible to cleavage by exogenous proteases, whereas OspA is relatively resistant (Becker, structural). This characteristic may have contributed to OspA being a better vaccine to lyme disease that is used to prevent transmission of B.burgodorferi from tick vector to mammalian host by complement-independent killing.

Interaction between OspA and LA-2

LA-2 is an IgM murine monoclonal antibody that interacts with three exposed loop on the C-terminal of OspA. These interactions include eight direct hydrogen bonds, four solvent-bridged hydrogen bonds, three ion pairs, and numerous van der Waals interactions (Ding 200).

Conformational changes

Conformational changes upon the binding of OspA and LA-2 show that LA-2 recognition of OspA involves an induced fit mechanism where loops 1-3 conformations shift to optimize complementarity to the antigen-combining site (Ding 2000). The overall structure of the C-terminal of OspA is unchanged upon the binding of LA-2 with comparison to the free OspA, except for the minor shift to accommodate for the binding. The maximum atomic shift is 4.7A at the site of Ser206