Disintegrin-like/cysteine-rich domains of the reprolysin HF3: Site-directed mutagenesis reveals essential role of specific residues.
Little is known about the biochemical properties of the non-catalytic domains of snake venom metalloproteinases (SVMPs). The ECD sequence of the disintegrin-like domain (D-domain) has been assigned as the disintegrin motif and, recently, the hyper-variable region (HVR) of the cysteine-rich domain (C-domain) was suggested to constitute a potential protein-protein adhesive interface. Here we show that the recombinant C-domain of HF3, a hemorrhagic SVMP from Bothrops jararaca, as well as three peptides resembling its HVR, inhibit collagen-induced platelet aggregation, which indicates a role for the C-domain and its HVR in targeting HF3 to platelets. Site-directed mutagenesis was used for the first time to identify charged residues essential for the functionality of the disintegrin-like/cysteine-rich domains (DC-domains). Residues of the disintegrin loop (E467 and D469), and of the HVR (K568, K569 and K575) of HF3 were individually mutated to Ala. Interestingly, only the mutant D469A was obtained in soluble form in Escherichia coli and this single mutation caused loss of two functional activities of the DC-domains: inhibition of platelet aggregation and increase of leukocyte rolling in the microcirculation. In summary we demonstrate that the C-domain and its HVR are critical for HF3 to affect platelets and leukocytes, however, the disintegrin loop may be important for the functionality of the D-domain in the context of the C-domain.
Menezes
Milene C
MC
Laboratório Especial de Toxinologia Aplicada-CAT/CEPID, Instituto Butantan, Av. Vital Brazil 1500, 05503-900 São Paulo, Brazil.
de Oliveira
Ana Karina
AK
Melo
Robson L
RL
Lopes-Ferreira
Mônica
M
Rioli
Vanessa
V
Balan
Andrea
A
Paes Leme
Adriana F
AF
Serrano
Solange M T
SM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology; D003094:Collagen / Q000494:pharmacology; D003435:Crotalid Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D003545:Cysteine / Q000032:analysis; D019483:Disintegrins / Q000737:chemistry*; D006801:Humans; D036904:Leukocyte Rolling / Q000187:drug effects; D008297:Male; D045726:Metalloproteases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D051379:Mice; D008833:Microcirculation / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010974:Platelet Aggregation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D003435:Crotalid Venoms; D019483:Disintegrins; D003094:Collagen; D045726:Metalloproteases; C543527:HF3 protein, Bothrops jararaca; D003545:Cysteine