Identification of a natural killer enhancing factor (NKEF) from human erythroid cells.
We have previously shown that RBC significantly augment NK cell-mediated cytotoxicity when added at the initiation of cytotoxicity assays. We now report that RBC cytosol contains a soluble factor that mediates a similar enhancement of NK activity when the factor is precoated on a plastic surface. We have purified this NKEF by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration HPLC. While gel filtration HPLC shows that NKEF has an apparent molecular mass of between 300 and 400 kDa, SDS-PAGE analysis indicates an apparent molecular weight of 48 and 24 kDa under nonreducing and reducing conditions, respectively. Immune serum obtained from a rabbit immunized with purified NKEF blocks NKEF augmentation of NK activity. In contrast, preimmune serum fails to inhibit NKEF activity. Anti-NKEF antibody reacts with the cytosol of NK-sensitive erythroleukemic target cell K562. Western blot analysis indicates that this antibody recognizes a 24-kDa protein both in RBC and in K562. We have generated tryptic peptides from NKEF and obtained partial amino acid sequences from three of the peptides. Two of the sequences show no significant homology with any known sequences. The third sequence shows partial identity with a deduced sequence from a murine erythroleukemia-related gene. Thus, NKEF is a novel protein with unique amino acid sequence. Our results of NKEF in RBC suggest that RBC could play an important role in regulating NK function.
Shau
H
H
Division of Surgical Oncology, School of Medicine, University of California, Los Angeles 90024-1782.
Gupta
R K
RK
Golub
S H
SH
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002460:Cell Line; D003600:Cytosol / Q000737:chemistry; D003602:Cytotoxicity, Immunologic; D004912:Erythrocytes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D014357:Trypsin
D001798:Blood Proteins; D014357:Trypsin