IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Generation of llama single domain antibodies that inhibit an intracellular bacterial toxin
Autor/es:
ALZOGARAY V, URRUTIA M, AGUIRRE A, BERGUER P, REYELT J, GARCÍA VÉSCOVI E, HAAG F, KOCH-NOLTE F, AND GOLDBAUM F.
Lugar:
Boston, USA
Reunión:
Congreso; PEGS: The Protein Engineering Summit; 2008
Resumen:
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Camelids (camels, dromedaries, llamas) produce
unusual antibodies composed only of heavy chains. The antigen combining site of
these antibodies is formed solely by the heavy-chain variable domain (VHH).
These VHH domains are easily produced as recombinant proteins and show similar
antigen binding affinity than their parent antibodies. Their CDR3s form long
finger-like extensions that can protrude into cavities on antigens, e.g. the
active site crevice of enzymes. Thus VHHs are a suitable fragment for the
development of enzyme inhibitors.
Salmonella enterica are intracellular bacteria that are pathogenic
for humans and animals, causing gastroenteritis and typhoid fever. These
bacteria express an enzyme called SpvB, which is essential for Salmonella
virulence. SpvB catalyzes the transference of the ADP-ribose moiety from NAD to
actin, causing the depolymerization of actin filaments and cell death.
To generate VHHs with inhibitory properties
against SpvB, we have isolated cDNA from lymphocytes of llamas immunized with
SpvB, obtaining a VHH library of 4x107 transformants. Phage display allowed an
enrichment of binders through consecutive rounds of panning. Clones recognizing
the antigen were sequenced and classified according to the length and
variability of their CDR3s.
Five independent clones were isolated, and the
VHH proteins codified by them were expressed and purified. All VHHs were able
to inhibit SpvB in enzymatic tests using radioactive probes. We quantitatively analyzed the inhibition of
SpvB activity by fluorescence using pyrene-labelled actin as substrate. For
each test, actin-pyrene, cold NAD, and both pure SpvB and the four VHHs were
used. The test showed strong inhibition for the clones named VHH5 and VHH6.
Furthermore, VHH5 was subcloned in a vector for
eukaryotic expression. As such, eukaryotic cells were transfected and cells
expressing the VHHs were infected with Salmonella enterica to test their
effect on virulence. Fluorescence microscopy analyses clearly show that VHH5 is
able to inhibit in vivo the ADP-ribosylation of actin by SpvB. This
result constitutes a proof of principle of the use of single domain antibodies
for the specific intracellular inhibition of normal or pathogenic enzymatic
functions.