Abstract
BackgroundA variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL) (DvLPBaPPP)2LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma.ResultsAn analog of the SHAL (DvLPBaPPP)2LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more 111In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the 111In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus.ConclusionThe incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP)2LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP)2LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a short polypeptide.
Highlights
A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells
A biotin was attached to the ε-amino group of the terminal amine (A) on both (DvLPBaPPP)2LLA and (DvLPBaPPP)2LArg6AcLLA to produce biotinylated forms for use in cell and protein binding experiments. 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was attached to both (DvLPBaPPP)2LLA and (DvLPBaPPP)2LArg6AcLLA at the same site to enable the selective high affinity ligand (SHAL) to be labeled with 111In
The DOTA SHAL (DvLPBaPPP)2LLDo and the hexa-arginine SHAL analog (DvLPBaPPP)2LArg6AcLLDo (Figure 1) were labeled with 111In at high efficiency (>90%) with specific activities ranging from 70–85 μCi/μg SHAL
Summary
A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. Some of the earliest approaches used to enhance the cellular uptake of therapeutics and other molecules (fluorescent dyes, enzymes, antibodies and other proteins) involved introducing the molecules into liposomes or micelles [7,8] Such constructs have been shown to fuse with the cell's membrane, introducing the contents inside the cell or transferring the lipid-bound components into the cell's membrane. One of the more successful shuttles is a nuclear localization signal peptide derived from the SV40 T antigen [12] This sequence, other peptide sequences derived from the transduction domain of the HIV-1 protein Tat [13,14], penetratin [15], and intact proteins such as the herpes virus protein VP22 [16] and anti-DNA antibodies [17] are currently being used to facilitate the transport of liposomes, viruses, enzymes, antibodies and a variety of other proteins into cells. Considerable success has been achieved using synthetic cationic peptide transporters such as oligoarginine [18,19,20,21], lactosylated poly-L-lysine [22] and short peptide sequences selected from phage display libraries [23] that exhibit sequence similarities to know peptide shuttles
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