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. 2022 Mar 11;21(4):100223. doi: 10.1016/j.mcpro.2022.100223

Fig. 5.

Fig. 5

Validation of TAILS substrate EFEMP1 and cleavage site preference.A, EFEMP1/Fibulin-3 protein domains, amino acid sequence of the atypical EGF repeat linker, and location of the ADAMTS7 cleavage sites. Abbreviated EFEMP1 domains: signal peptide (SP), N-terminal region (N), EGF repeats (E). B, concentrated medium from HUVEC expressing Ad-Luc, Ad-mWT, or Ad-mEQ assessed by Western blot under nonreducing conditions. Anti-EFEMP1 antibody recognizes an epitope C-terminal to the ADAMTS7 cleavage sites. C, quantitation of semi-tryptic or semi-chymotryptic peptides from HUVEC medium matching novel cleavage sites from the endogenous EFEMP1 protein. The total area was greater for the 123.124 cleavage site compared to the adjacent 124.125 cleavage site. Additional cleavage events observed were also found in the Luc and EQ controls. D, in vitro cleavage of HA-EFEMP1 by purified full-length mouse ADAMTS7 S3A assessed by Western blot. The antibodies to the N-terminal HA epitope and C-terminal EFEMP1 epitope recognized the EFEMP1 more strongly under nonreducing conditions. A band at 100 kDa under nonreducing conditions is consistent with a purified HA-EFEMP1 dimer, which was also sensitive to ADAMTS7 cleavage. E, overnight digest of HA-EFEMP1 by mouse ADAMTS7 S3A assessed by Coomassie staining. F, quantitation of semi-tryptic or semi-chymotryptic peptides from the atypical EGF1 repeat region from HA-EFEMP1, showing a consistent preference for the 123.124 cleavage site. ADAMTS7, A disintegrin and metalloproteinase with thrombospondin motifs 7; E, EGF repeats; HUVEC, Human umbilical vein endothelial cells; N, N-terminal region; SP, signal peptides; TAILS, terminal amine isotopic labeling of substrates.