"Zero-length" dimers of
ribonuclease A, a novel type of dimers formed by two
RNase A molecules bound to each other through a zero-length
amide bond [Simons, B. L., et al. (2007)
Proteins 66, 183-195], were further characterized and tested for their possible in vitro cytotoxic activity. Results obtained are the following. Besides dimers, also trimers and higher oligomers could be identified among the products of the covalently linking reaction, and the "zero-length" dimers prepared by us appear not to be a unique species. The product was indeed heterogeneous, and results obtained with two
RNase A mutants, E9A and K66A, indicated that amino and carboxyl groups others than those belonging to Lys66 and Glu9 are involved in the
amide bond. As for their functional properties, the "zero-length" dimers degrade
poly(A).
poly(U) (dsRNA) with an activity that increases with the increase of the oligomer's basicity and yeast
RNA (ssRNA) with an activity that instead decreases with the increase of oligomer's basicity, which is in agreement with previous data. No cytotoxicity of the
RNase A "zero-length" dimers could be evidenced in assays performed with various
tumor cells lines; the dimers, instead, become cytotoxic if cationized by conjugation with
polyethylenimine (PEI) [Futami et al. (2005) J. Biosci. Bioengin. 99, 95-103]. However, PEI derivatives of
RNase A "zero-length" dimers and PEI derivatives of native
RNase A resulted to be equally cytotoxic. In other words,
protein "dimericity" does not play any role in this case. Moreover, the acquired cytotoxicity does not seem to be specific for
tumor cells: PEI-cationized native
RNase A was also cytotoxic toward human monocytes.