Cloning, expression, and characterization of a bi-functional disintegrin/alkaline phosphatase hybrid protein.
Integrins are transmembrane heterodimeric glycoproteins responsible for cellular communication; therefore, they play an essential role in many physiological events. Viper snake venoms contain integrin antagonists called disintegrins which bind and inhibit integrin function. They present a loop containing an RGD motif responsible for integrin binding. The engineering of disintegrins fused to a reporter enzyme will be an interesting approach to build integrin markers. Even more, the disintegrin scaffold could be used to present other protein binding motifs. In this work, we have obtained alkaline phosphatase (APv) tagged eristostatin (Er) by cloning and expressing eristostatin DNA into the pLIP6-GN vector. Eristostatin, a 49 residue disintegrin, binds selectively to alphaIIbbeta3 integrin, inhibiting its binding to fibrinogen. The resulting fusion protein Er/APv was identified by SDS-PAGE and by Western blotting using both anti-Er and anti-AP antibodies. This fusion protein showed enzymatic AP activity similar to that of wild APv and its potential use for an alphaIIbbeta3 integrin assay was tested in a one-step dot blot using immobilized cells incubated with the marker and developed by AP substrate. Er/APv showed selectivity towards platelets and alphaIIbbeta3 integrin transfected cells and reacted with the same region as unlabeled Er, as analyzed in competition assays. Our data present a novel tool, Er/APv, with potential use as molecular marker in processes where the alphaIIbbeta3 integrin is involved.
Butera
Diego
D
Laboratório de Imunopatologia, Instituto Butantan, Av Vital Brasil 1500, 05503-900 São Paulo, Brazil.
Skielka
Karen
K
McLane
Mary Ann
MA
Paquette-Straub
Carrie
C
Ducancel
Frédéric
F
da Silva
Ana Maria Moura
AM
D000469:Alkaline Phosphatase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D003001:Cloning, Molecular; D019483:Disintegrins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005822:Genetic Vectors; D006801:Humans; D010455:Peptides / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000032:analysis / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014757:Viper Venoms / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
D019483:Disintegrins; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D011993:Recombinant Fusion Proteins; D014757:Viper Venoms; C084060:eristostatin; D000469:Alkaline Phosphatase