Artificial fatty acylation of proteins has attracted significant attention during the last decade as a method for modification of protein specificity and efficacy of action on mammalian cells (A. V. Kabanov and V. Yu. Alakhov (1994) J. Contr. Release 28, 15-35). Horse radish peroxidase (HRP) is used in this work to study the interaction of a fatty acylated protein with various mammalian cells. The HRP is modified with the chloranhydride of the stearic acid in the reversed micelles of sodium bis-(2-ethylhexyl)sulfosuccinate (Aerosol OT) in octane, a convenient protocol allowing production of protein molecules with a controlled, low modification degree (A.V. Kabanov et al. (1987) Ann. N. Y. Acad. Sci. 501, 63-66). The influence of the hydrophobic group on the binding and internalization of HRP in MDCK, P3-X63-Ag8, CHO, and HepG2 cells is examined. The major results are as follows: (i) the fatty acylation of a protein significantly enhances its binding to all tested mammalian cell lines, with a line-specific efficiency; (ii) the binding efficiency can be modified by changing growth conditions in a defined medium; (iii) along with the enhancement of protein adsorption on the plasma membrane, fatty acylation increases internalization of the protein during incubations at 37 degrees C; (iv) internalized protein was observed in endocytic vesicles; no evidence was obtained for a cytoplasmic distribution. These results are discussed in connection with previously observed effects of the fatty acylated proteins on cell activity.

Acylation↗

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Efficient gene delivery by a peptide derived from a monoclonal anti-DNA antibody.

We recently reported that translocating murine polyreactive anti-DNA antibodies can be used as vectors for the transfer of macromolecules into cells growing in culture. We show here that two such monoclonal antibodies (J20.8 and F4.1) conjugated to polylysine with a high (93) but not a low (19) number of lysine residues can transfer genes in the presence of serum. A 30 amino acid long peptide, VAYISRGGVSTYYSDTVKGRFTRQKYNKRA (peptide P3), corresponding to joined heavy-chain complementary-determining regions 2 and 3 of F4.1 antibody and carrying 19 lysine residues at its N-terminal, was found to be an efficient vector for the transfection of the luciferase gene into 3T3 and CCL39 cells in the presence of serum. Addition of 0.23 M glycerol during transfection considerably enhanced gene delivery. These results show that conjugation of a short polylysine tail converted a spontaneously internalizing peptide into a potent nontoxic plasmid vector.

3T3 Cells↗

PubMedDate not supplied

Expression and induction of MOPC-460 idiotopes in different strains of mice.

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Animals↗