To begin to identify a smaller fragment in the carboxyl region of EBNA-1 that contains the epitope recognized by 3D4, we examined the binding of this MAb to three truncated carboxyl fragments; EBNA452 641, EBNA459 619, and EBNA459 607 (Figure 7A).
We observed two species, a major component with mass 128.83 ± 0.07 kDa corresponding to the unproteolysed tetramer of PanD-T57V binding to four equivalents of PanZ-CoA and a smaller component with mass 112.43 ± 0.07 kDa corresponding to the truncated tetramer of PanD-T57V bound to three equivalents of PanZ-CoA.
MAb 0211 was observed to bind all three truncated proteins indicating that it recognizes epitopes in both the amino and carboxyl regions of EBNA-1, however the binding to the amino proteins, LS7 and LS8 is better than the binding to the carboxyl protein, LS9 (Figure 6C).
The chimeras also showed specific binding to truncated Mcl-1 (ΔNΔC) and to truncated Bcl-2 (ΔC).
The full residue span within Cit573 may not be necessary for optimal ACPA binding, therefore two truncated linear peptides of 15 and 11 residues (Cit573Lin1 and Cit573Lin2), originating from the full-length Cit573, were tested and found to provide 75%% (IC50 51 μM ± 6, n = 3) and 69%% (IC50 123 μM ± 18, n = 2) ACPA inhibition, respectively (Fig. 3a).
Purified monoclonal antibodies were tested for binding to truncated amino (LS7 and LS8) and carboxyl regions (LS9, EBNA452 641, EBNA459 619, and EBNA459 607) of EBNA-1.
MAb, 0211 displays a similar binding pattern to the truncated carboxyl fragments of EBNA-1 with maximal binding to the two smallest fragments EBNA459 619 and EBNA459 607 (data not shown).
Hence, the distribution of tags should be fitted to the zero truncated Poisson distribution [ 59, 68].
In a companion study, we have determined the binding affinities of Tf 4-6) and Tf 4-7) for full-length and three truncated 5 S RNA molecules, the latter selected on the basis of the regions of the 5 S RNA molecule known to be important in the binding of TFIIIA.
However, it is noteworthy that all the three truncated αA-crystallins have shown decreased target protein binding.
