Figure 2.
Figure 2. Expression and activity of the ADAMTS13 mutant. (A) COS-7 cells were transfected with plasmids encoding wild-type or mutant ADAMTS13. The ADAMTS13 in the conditioned media (top panel) and cell lysates (bottom panel) were analyzed by Western blotting. Controls were cells transfected with empty vector (lane 1), wild-type cDNA (lane 3), or mutant cDNA (lane 5) inserted in the reverse orientation. The band density of the secreted mutant (lane 4) was about 14% of the wild-type protein (lane 2). (B) ADAMTS13 activity in the conditioned medium of the transfected COS-7 cells was assayed using purified VWF. Reactions were resolved on 1% agarose and visualized by autoradiography (top panel) and 8% SDS-PAGE under reducing conditions and visualized by Western blotting (bottom panel). Purified rADAMTS13 (lanes 12-15 top panel; 7-9 bottom panel) was used to calibrate the assay. Wild-type conditioned medium (lanes 1-5 top panel; 3 and 4 bottom panel) had about 0.36 μg/mL VWF-cleaving proteinase activity, whereas mutant conditioned medium (lanes 7-11 top panel; 5 and 6 bottom panel) had about 0.04 μg/mL activity. Conditioned medium of cells transfected with wild-type ADAMTS13 cDNA in the reverse orientation had no activity (lane 2 bottom panel). Based on a secretion efficiency of 14% (panel A), the VWF-cleaving proteinase activity of the mutant was about 85% of the wild-type protein. (C) A 3-dimensional representation of the structure of acidic seminal fluid protein illustrating the CUB domain architecture. The β strands are shown as ribbons (blue) and the connecting loops as Cα traces (gray). The expected position of the free cysteine in the first CUB domain of ADAMTS13 is indicated with an arrow. The side chains of disulfides (SS1 and SS2) are shown in yellow. Note that SS2 can exist in the reduced dithiol form in the acidic seminal fluid protein and is absent from the second CUB domain in ADAMTS13. The 2 central β strands that are predicted to be deleted in the first or second CUB domain of ADAMTS13 as a result of the 3769-3770insT or 4143-4144insA mutation, respectively, are shown in red. C indicates C-terminus.

Expression and activity of the ADAMTS13 mutant. (A) COS-7 cells were transfected with plasmids encoding wild-type or mutant ADAMTS13. The ADAMTS13 in the conditioned media (top panel) and cell lysates (bottom panel) were analyzed by Western blotting. Controls were cells transfected with empty vector (lane 1), wild-type cDNA (lane 3), or mutant cDNA (lane 5) inserted in the reverse orientation. The band density of the secreted mutant (lane 4) was about 14% of the wild-type protein (lane 2). (B) ADAMTS13 activity in the conditioned medium of the transfected COS-7 cells was assayed using purified VWF. Reactions were resolved on 1% agarose and visualized by autoradiography (top panel) and 8% SDS-PAGE under reducing conditions and visualized by Western blotting (bottom panel). Purified rADAMTS13 (lanes 12-15 top panel; 7-9 bottom panel) was used to calibrate the assay. Wild-type conditioned medium (lanes 1-5 top panel; 3 and 4 bottom panel) had about 0.36 μg/mL VWF-cleaving proteinase activity, whereas mutant conditioned medium (lanes 7-11 top panel; 5 and 6 bottom panel) had about 0.04 μg/mL activity. Conditioned medium of cells transfected with wild-type ADAMTS13 cDNA in the reverse orientation had no activity (lane 2 bottom panel). Based on a secretion efficiency of 14% (panel A), the VWF-cleaving proteinase activity of the mutant was about 85% of the wild-type protein. (C) A 3-dimensional representation of the structure of acidic seminal fluid protein illustrating the CUB domain architecture. The β strands are shown as ribbons (blue) and the connecting loops as Cα traces (gray). The expected position of the free cysteine in the first CUB domain of ADAMTS13 is indicated with an arrow. The side chains of disulfides (SS1 and SS2) are shown in yellow. Note that SS2 can exist in the reduced dithiol form in the acidic seminal fluid protein and is absent from the second CUB domain in ADAMTS13. The 2 central β strands that are predicted to be deleted in the first or second CUB domain of ADAMTS13 as a result of the 3769-3770insT or 4143-4144insA mutation, respectively, are shown in red. C indicates C-terminus.

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