Figure 3
Figure 3. S1494C and S1534C form a disulfide bond that renders the A2 domain of VWF resistant to proteolysis by plasma proteases and trypsin. (A) Cysteine 1494 and 1534 are projected to form a disulfide bond linking the amino-terminal ends of the β1 and β2 strands, thereby making the unfolding of the β1-α1-β2 subdomain more difficult. The recombinant A2 domain used in our studies is flanked by the human vitronectin signal peptide (VnSP) and a carboxy-terminal His6 tag. Cysteine 1494 and 1534 are circled, as well as cysteine 1669 and 1670 (engaged in a vicinal disulfide bridge). (B) In the absence of reducing agent, neither A2/WT nor A2/p.S1494C-S1534C displays any Oregon green 488-maleimide labeling. However, on β-mercaptoethanol (β-ME) treatment and resulting sulfhydryl moieties exposure, both A2/WT and A2/p.S1494C-S1534C show Oregon-green 488-maleimide incorporation (n = 3). To quantify the difference in Oregon green 488-maleimide incorporation from one sample to the other, the mean immunofluorescence intensity of each bands was quantified with NIH ImageJ 1.41 software. The measured means for all 3 gels were then averaged and expressed as a fraction of the nonreduced A2/WT mean. Error bars indicate SE. The difference in signal (∼2) accounts for the number of disulfide bonds having been reduced in the 2 proteins (1 such bond in A2/WT and 2 for A2/p.S1494C-S1534C). The black and white image of the fluorescent gel was obtained through Photoshop grayscale mode adjustment and tone inversion of the original scan. The gel was subsequently stained with Coomassie blue as a loading control. (C) A2/WT and A2/p.S1494C-p.S1534C were incubated with platelet-poor plasma overnight at 37°C with or without prior β-ME and analyzed by SDS-polyacrylamide gel electrophoresis on a 17% acrylamide gel. Although A2/WT is cleaved under both conditions, A2/p.S1494C-p.S1534C is only cleaved after β-ME treatment (n = 3). (D) Similarly, A2/p.S1494C-p.S1534C shows stronger resistance to trypsin digestion than A2/WT. Statistical analysis shows that 62.06 ± 1.67% (mean ± SE) of A2/WT is proteolyzed by trypsin, whereas only 4.66 ± 0.84% (mean ±SE) of A2/p.S1494C-p.S1534C is degraded under the same condition. The probability associated with a Student t test is 3.82 × 10−5 (n = 7).

S1494C and S1534C form a disulfide bond that renders the A2 domain of VWF resistant to proteolysis by plasma proteases and trypsin. (A) Cysteine 1494 and 1534 are projected to form a disulfide bond linking the amino-terminal ends of the β1 and β2 strands, thereby making the unfolding of the β1-α1-β2 subdomain more difficult. The recombinant A2 domain used in our studies is flanked by the human vitronectin signal peptide (VnSP) and a carboxy-terminal His6 tag. Cysteine 1494 and 1534 are circled, as well as cysteine 1669 and 1670 (engaged in a vicinal disulfide bridge). (B) In the absence of reducing agent, neither A2/WT nor A2/p.S1494C-S1534C displays any Oregon green 488-maleimide labeling. However, on β-mercaptoethanol (β-ME) treatment and resulting sulfhydryl moieties exposure, both A2/WT and A2/p.S1494C-S1534C show Oregon-green 488-maleimide incorporation (n = 3). To quantify the difference in Oregon green 488-maleimide incorporation from one sample to the other, the mean immunofluorescence intensity of each bands was quantified with NIH ImageJ 1.41 software. The measured means for all 3 gels were then averaged and expressed as a fraction of the nonreduced A2/WT mean. Error bars indicate SE. The difference in signal (∼2) accounts for the number of disulfide bonds having been reduced in the 2 proteins (1 such bond in A2/WT and 2 for A2/p.S1494C-S1534C). The black and white image of the fluorescent gel was obtained through Photoshop grayscale mode adjustment and tone inversion of the original scan. The gel was subsequently stained with Coomassie blue as a loading control. (C) A2/WT and A2/p.S1494C-p.S1534C were incubated with platelet-poor plasma overnight at 37°C with or without prior β-ME and analyzed by SDS-polyacrylamide gel electrophoresis on a 17% acrylamide gel. Although A2/WT is cleaved under both conditions, A2/p.S1494C-p.S1534C is only cleaved after β-ME treatment (n = 3). (D) Similarly, A2/p.S1494C-p.S1534C shows stronger resistance to trypsin digestion than A2/WT. Statistical analysis shows that 62.06 ± 1.67% (mean ± SE) of A2/WT is proteolyzed by trypsin, whereas only 4.66 ± 0.84% (mean ±SE) of A2/p.S1494C-p.S1534C is degraded under the same condition. The probability associated with a Student t test is 3.82 × 10−5 (n = 7).

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