Tailored placement of a turn-forming PA tag into the structured domain of a protein to probe its conformational state

Abstract

Placement of a tag sequence is usually limited to either terminal end of the target protein, reducing the potential of epitope tags for various labeling applications. The PA tag is a dodecapeptide (GVAMPGAEDDVV) that is recognized by a high-affinity antibody NZ-1. We determined the crystal structure of the PA-tag-NZ-1 complex and found that NZ-1 recognizes a central segment of the PA tag peptide in a tight β-turn configuration, suggesting that it is compatible with the insertion into a loop. This possibility was tested and confirmed using multiple integrin subunits and semaphorin. More specifically, the PA tag can be inserted at multiple locations within the integrin α IIb subunit (encoded by ITGA2B) of the fibrinogen receptor α IIb β 3 integrin (of which the β 3 subunit is encoded by ITGB3) without affecting the structural and functional integrity, while maintaining its high affinity for NZ-1. The large choice of the sites for 'epitope grafting' enabled the placement of the PA tag at a location whose accessibility is modulated during the biological action of the receptor. Thus, we succeeded in converting a general anti-tag antibody into a special anti-integrin antibody that can be classified as a ligand-induced binding site antibody.

Lasso-grafting of macrocyclic peptide pharmacophores yields multi-functional proteins

“…We thus chose Cap loops for lasso-grafting since its replacement would disrupt the normal infectious ability of AAV, and thus the AAV-peptide fusion capsid will gain a new function originating from the nature of the grafted peptide. To explore the effect of exogeneous peptide insertion on the virus packaging and gene transduction ability, we rst inserted a 12-residue peptide tag (PA tag, 23 ) into the Cap of AAV serotype 2 (AAV2) at two locations (s1 and s2, Table S2) that had already been reported to tolerate a peptide insertion (Fig. 2j) 24 .…”

Development and characterization of anti‐glycopeptide monoclonal antibodies against human podoplanin, using glycan‐deficient cell lines generated by CRISPR/Cas9 and TALEN

“…The anti‐hPDPN mAb (NZ‐1) detects hPDPN with high specificity and sensitivity . Moreover NZ‐1, which is also useful for detecting the podoplanin/aggrus (PA) epitope tag , is efficiently internalized by glioma cell lines, accumulates in tumors in vivo , and is therefore a suitable candidate for therapy for malignant gliomas . Further, NZ‐1 inhibits tumor cell‐induced platelet aggregation and tumor metastasis .…”

Intermolecular autocatalytic activation of serine protease zymogen factor C through an active transition state responding to lipopolysaccharide

“…activation of factor C (22). In contrast, with the PA tag system it is possible to insert the PA tag into internal portions of a recombinant protein or boundary portions between the domains in a mosaic protein to maintain not only the antigenicity of the PA tag but also the physiologic activities of the tag-containing proteins (26). Accordingly, the PA tag was inserted into the peptide bond between Trp 758 -Leu 759 in the B chain of WT factor C (PA factor C), corresponding to the linker position between the two ␤-barrel structures composing the catalytic domain of serine proteases (1)(2)(3).…”