Identification by Site

Identification by
Site-directed Mutagenesis of Amino Acid Residues Flanking RGD Motifs of Snake
Venom Disintegrins for Their Structure and Function

XU Cun-Shuan^*, RAHMAN Salman¹
( Life Science Institute, Henan Normal University, Xinxiang 453002,
China； ¹Hamophilia Research Center, University of
London, London SE1 7EH, United Kingdom )

Abstract    In order to
demonstrate that the amino acid residues flanking the RGD sequence were important
for inhibiting the ADP-induced platelet aggregation, we analyzed the role of
the amino acid residues in the domain preceding the RGD loop on the activity of
disintegrins. Our approach was to develop hybrids between the disintegrins
kistrin and elegantin targeting residues in this domain and within the RGD
loop. The basic sequence within elegantin KKKR⁴⁴T⁴⁵I⁴⁶/A⁵⁰RGDN⁵⁴P⁵⁵
was changed by mutagensis to SKAG⁴⁴T⁴⁵I⁴⁶/P⁵⁰RGDM⁵⁴P⁵⁵
and to SKAG⁴⁴I⁴⁶/P⁵⁰RGDM⁵⁴P⁵⁵,
thereby resembling the corresponding S³⁹RAGT⁴³/P⁵⁰RGDM⁵²P⁵³
sequence in kistrin. This changed KKKR⁴⁴T⁴⁵I⁴⁶/A⁵⁰RGDN⁵⁴P⁵⁵→SKAG⁴⁴T⁴⁵I⁴⁶/P⁵⁰RGDM⁵⁴P⁵⁵
dramatically reduced the activity of elegantin as an inhibitor of platelet
aggregation. In contrast, deletion of T⁴⁵(KKKR⁴⁴T⁴⁵I⁴⁶/A⁵⁰RGDN⁵⁴P⁵⁵→SKAG⁴⁴T⁴⁵I⁴⁶/P⁵⁰RGDM⁵⁴P⁵⁵)increased
activity of elegantin as an inhibitor platelet aggregation. It was further
shown that their electrophoresis properties were very different. These data
highlight the importance of the domain encompassing residues 39–5 and the amimo
acid residues flanking the RGD sequence on disintegrin structure-function.
Key words    site directed mutagenesis； integrin； disintegrin； RGD motif；viper venom

^*Corresponding author： Tel, 86-373-3326341； Fax, 86-373-3326524； e-mail, [email protected]