EXTRA-NUCLEAR LOCATION OF HISTONES IN ACTIVATED HUMAN PERIPHERAL-BLOOD LYMPHOCYTES AND CULTURED T-CELLS

Dextrin-2-sulphate (D2S) is a sulphated polysaccharide which inhibits human immuno-deficiency virus type 1 infection of T-cells by binding t o the cell surface. During our investigations of the nature of this in teraction, a cell membrane fraction was prepared by ultracentrifugatio n from the T-cell line, HPB-ALL. Separation of membrane proteins by so dium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and ana lysis for binding proteins using ligand blotting showed that H-3-D2S b ound, in a saturable and displaceable manner, to two regions correspon ding to molecular weights of 14,000-18,000 and 28,000-32,000. The N-te rminal sequences of two of the major protein components in the 14,000- 18,000 region were consistent with those of histones H2B and H3. The p resence of histone H2B in the cell membrane preparation was confirmed by immunoblotting and enzyme-linked immunosorbent assay using a specif ic antibody. Histone standards were used to determine the level of eac h histone in the cell membrane fraction. In addition, the binding of H -3-D2S to purified histone standards was quantified. These results sho w that all of the binding of H-3-D2S to proteins in the 14,000-18,000 region of the cell membrane preparation can be attributed to the histo nes present. In contrast to HPB-ALL cells, a cell membrane fraction fr om freshly isolated human peripheral blood lymphocytes contained very low levels of histones. However, after culture with phytohaemagglutini n for 3 days the cell membrane fraction contained greatly increased le vels of histones. To exclude the possibility of contamination of the c ell membrane preparation with histones derived from the nucleus, cell membranes were also prepared using an affinity-based method using poly ethyleneimine-cellulose. Immunoblotting of adsorbed plasma membranes s howed the presence of histone H2B. SDS-polyacrylamide gels stained for protein also indicated that the preparation contained histones H1, H2 A, H3 and H4. In further experiments whole cells were used to avoid co ntamination from nuclear proteins. Lactoperoxidase mediated I-125 labe lling, a method specific for radiolabelling cell surface proteins, con firmed the presence of histones H2B, H3 and H4 on the surface of HPB-A LL cells. Also, incubation of HPB-ALL cells or phytohaemagglutinin-act ivated peripheral blood lymphocytes with D2S caused displacement of hi stones from the cell surface into the supernatant without altering cel l viability. In addition, immunocytochemistry of freshly isolated peri pheral blood lymphocytes showed that histone H2B was located predomina ntly in the nucleus. However, in phytohaemagglutinin activated periphe ral blood lymphocytes immunoreactive material was also prominent in th e endoplasmic reticulum and on the plasma membrane. These results demo nstrate that histones are not confined to a nuclear location.