The difference between subcellular localization of HB-19 and endostatin could be accounted by the nature of interaction with surface nucleolin, since endostatin but not HB-19 binding to surface nucleolin requires heparan sulfate proteoglycans [21], [28].
Taken together, these results indicate that the existence of surface nucleolin in a high molecular weight complex is independent of ligand binding to surface nucleolin.
Indeed, after specifically binding to surface nucleolin, HB-19 and related Nucant pseudopeptides enter cells and accumulate in the cytoplasm without crossing the nuclear membrane.
Moreover, the binding affinity of Nucant to nucleophosmin is much lower compared to its binding affinity to surface nucleolin.
As Nucleolin is shown to bind Ca2+ [59], [59], and is involved in epidermal Ca2+ homeostasis [60], it remains to be shown whether binding of calcium to surface nucleolin is a necessary step for ligand internalization via surface nucleolin.
This and the capacity of HB-19 to bind surface nucleolin without dissociating the 500-kDa complex suggest that the binding of HB-19 to surface nucleolin could change the organization of the 500-kDa complex, and thus interfere with the proper functioning of nucleolin and the nucleolin associated proteins.
Consequently, the binding of HB-19 to surface nucleolin could also affect the organization of nucleolin-associated proteins in this 500-kDa complex and thus generate more inhibitory mechanisms.
The 500-kDa complex containing surface nucleolin was purified from the cell surface by the capacity of HB-19/Btn to bind specifically surface nucleolin [ 9, 16, 52].
The HB-19 pseudopeptide 5[Kψ(CH2N PR-TASP, for [Lysψ(CH2N Pro-Arg]-template-assembled synthetiCH2N Pro-Arg]-template-assembledrfaCH2N Pro-Arg]-template-assembledtion [ 9, 16, 37].
In different types of cells, HB-19 binds surface nucleolin in a dose-dependent manner with maximum binding occurring at 1 5 µM concentration [12], [13], [15], [45].
