Extended Data Fig. 7: Carboxylation-sensitive antibodies identify functional motifs.
From: Multiplex and multimodal mapping of variant effects in secreted proteins via MultiSTEP
a, Multiple sequence alignment of Gla-domain containing proteins (UniProt) that bind the carboxylation-sensitive Gla-motif (ExxxExC) antibody using MUSCLE96,110. Antibody epitopes for both the carboxylation-sensitive FIX-specific antibody (ω-loop) and the carboxylation-sensitive Gla-motif antibody are shown. hF9, human coagulation factor IX (P00740); hF2, human prothrombin (coagulation factor II, P00734); hF7, human coagulation factor VII (P08709); hF10, human coagulation factor X (P00742); hPC, human protein C (P04070); hPS, human protein S (P07225); hBGP, human osteocalcin (P02818); bBGP, bovine osteocalcin (P02820); hGAS6, human growth arrest-specific protein 6 (P14393); ppVPA, Pseudechis prophyriacus venom prothrombin activator porpharin-D (P58L93); nsVPA, Notechis scutatis venom prothrombin activator notecarin-D1 (P82807); osVPA, Oxyuranus scutellatus venom prothrombin activator oscutarin-C (P58L96). b-c, Fluorescence of unrecombined negative control and WT FIX-expressing cells with and without warfarin pretreatment generated by staining cells with a carboxylation-sensitive FIX-specific (b) or carboxylation-sensitive Gla-motif antibody (c). d-f, Heatmaps showing carboxylation-sensitive FIX-specific carboxylation scores (d), carboxylation-sensitive Gla-motif carboxylation scores (e), or light chain secretion scores (f) for FIX propeptide variants. Furin cleavage site (Furin CS), ω-loop, ExxxExC motif, and aromatic stack (AS) are annotated above (d). Heatmap color indicates antibody score from 0 (blue, lowest 5% of scores) to white (1, WT) to red (increased). Black dots indicate the WT amino acid. Missing data are gray. g-i, Heatmaps showing carboxylation-sensitive FIX-specific carboxylation scores (g), carboxylation-sensitive Gla-motif carboxylation scores (h), or light chain secretion scores (i) for FIX Gla domain variants. Furin cleavage site (Furin CS) is annotated above (d). ω-loop, ExxxExC motif, and aromatic stack (AS) are annotated above (g). Heatmap color indicates antibody score from 0 (blue, lowest 5% of scores) to white (1, WT) to red (increased). Black dots indicate the WT amino acid. Missing data are gray.
