Biochemical SocietyTransactions (1 994) 22
Histones located on the plasma membrane of T-cells.
KEITH WATSON, ROBERT J. EDWARDS, DAVID C.
PARMELEE', SUNK SHAUNAK', NIGEL J. GOODERHAM
and DONALD S. DAVIES
Departments of Clinical Pharmacology and Infectious Diseases',
Royal Postgraduate Medical School, London W12 ONN, U.K.
'National Cancer Institute, Bethesda, MA 20892, U.S.A.
Several sulphated polysaccharides have been shown to block
the infection of T-cells by the human immunodeficiency virus
(HIV)-l [1,2]. Dextrin-2-sulphate (D2S) is a synthetically
sulphated polysaccharide which inhibits infection of human T-cell
lines by a variety of HIV-1 cell-free isolates [3]. Although its
mechanism of action has not been established, this compound
appears to prevent HIV-1 infection by binding to the cell surface.
Most sulphated polysaccharides interfere with the binding of
m i 2 0 to CD4 [2], but D2S appears to act by a different
mechanism [4]. The aim of this study was to identify cell surface
proteins which bind D2S and may be involved in the entry of
HIV- 1 into cells.
A plasma membrane fraction was prepared from a human Tcell line, HPB-ALL, by ultracentrifugation [5]. This was
subjected to ligand blotting in which the membrane fraction was
separated by SDS-polyacrylamide gel electrophoresis (SDSPAGE) using 15% (w/v) gels, electrotransferred onto
nitrocellulose filters and incubated with 'H-D2S. It was found
that 'H-D2S bound in a specific, displaceable manner to proteins
of molecular weights 17 kDa and 29 kDa. Microsequencing was
attempted on proteins present in these regions. In the 17 kDa
region the N-termini of two proteins was successfully determined.
These were PEPAKSAPAPKKGXKKXVTKA and
ARTKQTARKSTGGKAPRKQLAT, which correspond to the Ntermini of histones H2B and H3, respectively. In addition, an
antibody was raised against the N-terminal sequence of histone
H2B (PEPAKSAPAPKKGSKKAVTKAQK).
The antibody
bound to purified histone H2B, but not to the cell surface or to
chromatin. This may be explained by occlusion of the antibody
binding site in complexed histones. Immunoblotting with this
antibody showed that histone H2B was present in the membrane
preparation.
To exclude the possibility that the histones in the membrane
preparation was due to contamination by nuclear proteins, HPBALL membranes were prepared using an affinity based method
of purification using polyethyleneimine-resin [6]. This method is
based on interaction of the negatively charged cell membrane
with the positively charged resin. HPB-ALL cells were
suspended in 15 mM sodium acetate, pH 5.0 containing 220 mM
sucrose and incubated with polyethyleneimine-resin for 1 h at
room temperature. Free binding sites were blocked by the
addition of 1 mg/ml polyglutamic acid. The cells were then lysed
in a hypotonic buffer (10 mM Tris-HCI, pH 7.4). The
polyethyleneimine-resin with plasma membrane material bound
to it was then washed extensively with the same buffer. SDSPAGE of the bound material revealed the presence of histones
H2A, H2B, H3, and H4 by comparison with the electrophoretic
mobility of purified calf thymus histones. Also, immunoblotting
using the anti-histone-H2B antibody showed the presence of
histone H2B.
Further, the surface of HPB-ALL cells with >95% viability
(determined using Trypan blue exclusion) was labelled with '*'I
using the lactoperoxidase. method [7]. The cells were then
washed 3-times with phosphate-buffered saline and the membrane
fraction prepared by ultracentrifugation as described above.
Proteins in the membrane fraction were separated by SDS-PAGE,
stained using Coomassie blue, dried and then subjected to autoradiography. Proteins corresponding to the migration positions of
histones H2B, H3, and H4 were radiolabelled and this coincides
with the bands which bind 'H-D2S. Interestingly, histone H2A
was not iodinated. This observation is consistent with a previous
study in which chromatin was radiolabelled under similar
conditions to those used here [8]. It was concluded that tyrosine
residues in histone H2A are inaccessible due to complexing of
histone H2A with H2B. Thus, histones on the cell surface may
also be complexed with one another or other proteins.
There was a linear relationship between the binding of 'HD2S and the concentration of each of the purified histones H2A,
H2B, H3, and H4 following SDS-PAGE and analysis by ligand
blotting. Further, measurement of the amount of each of these
histones in Coomassie blue-stained gels by laser densitomeny
showed that the binding of 'H-D2S to the 17 kDa region of the
membrane fraction was entirely accounted for by binding to
histone proteins.
Surface expression of nuclear material including histones has
been reported on a variety of cell types, including cytotoxic T
cells [9], endothelial cells [lo], human monocytes 11 11, sperm
[12], and mouse B cells [13]. In addition, heparin has been
shown to bind proteins present in cell membrane preparations
which resemble histones [14].
The possibility that D2S may prevent the infection of T-cells
by HIV-1 by binding to histones present on the cell surface is
currently being investigated.
This work was supported by ML Laboratories.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Abbreviations used: HIV, human immunodeficiency virus; D2S,
dexmn-2-sulphate; SDS-PAGE, sodium dodecyl sulphatepolyacrylamide gel electrophoresis.
199s
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