Different regions of the class P-III snake venom metalloproteinase jararhagin are involved in binding to alpha2beta1 integrin and collagen.
SVMPs are multi-domain proteolytic enzymes in which disintegrin-like and cysteine-rich domains bind to cell receptors, plasma or ECM proteins. We have recently reported that jararhagin, a P-III class SVMP, binds to collagen with high affinity through an epitope located within the Da-disintegrin sub-domain. In this study, we evaluated the binding of jararhagin to alpha(2)beta(1) integrin (collagen receptor) using monoclonal antibodies and recombinant jararhagin fragments. In solid phase assays, binding of jararhagin to alpha(2)beta(1) integrin was detectable from concentrations of 20 nM. Using recombinant fragments of jararhagin, only fragment JC76 (residues 344-421), showed a significant binding to recombinant alpha(2)beta(1) integrin. The anti-jararhagin monoclonal antibody MAJar 3 efficiently neutralised binding of jararhagin to collagen, but not to recombinant alpha(2)beta(1) integrin nor to cell-surface-exposed alpha(2)beta(1) integrin (alpha(2)-K562 transfected cells and platelets). The same antibody neutralised collagen-induced platelet aggregation. Our data suggest that jararhagin binding to collagen and alpha(2)beta(1) integrin occurs by two independent motifs, which are located on disintegrin-like and cysteine-rich domains, respectively. Moreover, toxin binding to collagen appears to be sufficient to inhibit collagen-induced platelet aggregation.
Tanjoni
Isabelle
I
Laboratório de Imunopatologia, Instituto Butantan, Butantã, São Paulo, SP, Brazil.
Evangelista
Karla
K
Della-Casa
Maisa S
MS
Butera
Diego
D
Magalhães
Geraldo S
GS
Baldo
Cristiani
C
Clissa
Patrícia B
PB
Fernandes
Irene
I
Eble
Johannes
J
Moura-da-Silva
Ana M
AM
D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects; D003094:Collagen / Q000187:drug effects / Q000378:metabolism*; D003435:Crotalid Venoms / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D038982:Integrin alpha2beta1 / Q000187:drug effects / Q000378:metabolism*; D020014:K562 Cells / Q000187:drug effects / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection; D000097146:Bothrops jararaca Venom
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D003094:Collagen; D003435:Crotalid Venoms; D038982:Integrin alpha2beta1; D008666:Metalloendopeptidases; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; D000097146:Bothrops jararaca Venom