The Disintegrin Domain, First Thrombospondin Type 1 Repeat, Cys-Rich and Spacer Domains of Adamts13 Are Essential for Cleavage of Von Willebrand Factor and for Determination of Substrate Specificity.

Abstract

ADAMTS13 consists of a reprolysin-type metalloprotease domain followed by a disintegrin domain, first thrombospondin type 1 repeat (TSP1), a Cys-rich domain and a spacer domain. The carboxyl terminus of ADAMTS13 has seven more TSP1 repeats and two CUB domains. ADAMTS13 cleaves the Tyr1605-Met1606 bond at the central A2 domain of von Willebrand factor (VWF). To determine the domains of ADAMTS13 required for substrate recognition and specificity, we have prepared a recombinant VWF73 peptidyl substrate from E. coli BL21Ai cells. This substrate consists of 73 amino acid residues (D1596~R1668) derived from the central A2 domain of VWF, flanked at its N-terminus by a glutathione s-transferase (GST) protein and at its C-terminus by a 6xHis epitope. It is highly specific and more sensitive than plasma VWF for determination of the proteolytic activity of ADAMTS13. Purified VWF73 substrate (200 nM) was incubated with the conditioned media from COS7 cells containing 14 nM of full-length ADAMTS13 and its various variants in 50 mM Tris-HCl and 150 mM NaCl at 37 °C for 3 hours. The cleavage of VWF73 at the specific Tyr-Met bond was determined by Western blotting with anti-GST. We showed that full-length ADAMTS13 and ADAMTS13 variants that were deleted after the TSP1 2 to 8 repeats (del1), spacer domain (del2), Cys-rich domain (del3) and first TSP1 repeat (del4) were all proteolytically active toward VWF73. ADAMTS13 variants deleted after disintegrin domain (del5) and after metalloprotease domain (del6) did not cleave VWF73 within 3 hours, but did cleave VWF73 upon a prolonged incubation (14~24 hours). Surprisingly, the del4 and del6 variants preferentially cleaved VWF73 at a site other than the Tyr-Met bond, resulting in a generation of an N-terminal fragment that was approximately 5 kDa shorter in length than expected. The cleavage of this alternative peptidyl bond by del4 and del6 was metal ion dependent and could be inhibited by an addition of 5 mM EDTA into the reaction. The conditioned medium from vector-transfected COS7 cells did not cleave VWF73 after 24 hours of incubation in the same condition. Time-course studies showed that full-length ADAMTS13, del1, del2 and del3 variants cleaved VWF73 with a similar efficiency, with an approximately 50% of VWF73 being cleaved in five minutes at 37 °C. The cleavage of VWF73 by ADAMTS13 variants del4, del5 and del6, however, was relatively slow with a less than 50% of VWF73 substrate being cleaved after 24 hours of incubation, suggesting that the Cys-rich/spacer domains participate in the substrate recognition to enhance the catalytic efficiency of ADAMTS13 metalloprotease. The other domains adjacent the Cys-rich/spacer domains upstream including the disintegrin domain and/or the first TSP1 repeat may also be critical for collaborative recoginition of the substrates, as the ADAMTS13 variants del5 and MT (consisting of only the metalloprotease domain and the first TSP1 repeat) cleaved VWF73 very slowly. In addition, the ADAMTS13 variant MCS that was deleted after the spacer domain, but lacking the disintegrin and the first TSP1 repeat was not proteolytically active toward VWF73 at all. We therefore conclude that the disintegrin domain, the first TSP1 repeat, the Cys-rich domain and the spacer domain are all important for substrate recognition to enhance the catalytic efficiency of ADAMTS13 metalloprotease domain and to improve substrate specificity.