Hexa-arginine enhanced uptake and residualization of selective high affinity ligands by Raji lymphoma cells.
BACKGROUND
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. 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.


RESULTS
An 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.


CONCLUSIONS
The 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.
Balhorn
Rod
R
University of California, Department of Applied Science, Hertz Hall, Livermore, CA 94551, USA. rodbalhorn@hughes.net
Hok
Saphon
S
DeNardo
Sally
S
Natarajan
Arutselvan
A
Mirick
Gary
G
Corzett
Michele
M
Denardo
Gerald
G
D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D006684:HLA-DR Antigens / Q000378:metabolism; D059810:HLA-DR Serological Subtypes; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D016393:Lymphoma, B-Cell / Q000378:metabolism*; D018613:Microscopy, Confocal; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*
D006684:HLA-DR Antigens; D059810:HLA-DR Serological Subtypes; C421823:HLA-DR10 antigen; D006573:Heterocyclic Compounds, 1-Ring; D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D001120:Arginine