Download Heat shock protein (Hsp)65-70: dominant self

Biochemical Society Transactions (1995) 23 215s
Heat shock protein (Hsp)65-70 dominant self-antigen candidate in the
selection of 76 lymphocytes?
CLARA G.H. BELL
UIC Coll. Med. at Chicago, Chicago, IL., 60612 USA
The function of the immune systeiii is to protect the host from exogenous
invaders and endogenous aberrations and it is related to the capacity of the
system to recognize foreign antigens (Ags) and discriminate them from selfAgs. Central to this function are the T cells. The major set with the most
diverse clonal receptor weapons against foreign Ag invaders, are the cells
that differentiate intrathymically from fetal liver or bone marrow-derived
stem cells, via a series of steps that lead to the rearrangement and expression
of the T cell receptor (TcR) a(3 (that convey the clonal Ag-specificity), and
of the differentiation CD4 and CD8 Ags, which are skewed to recognition
of the Ag in the context of the self-major histocompatibility complex (MHC)
class I1 and class I gene products. The thymus is central' for acquisition of
the TcRap, and of the CD4 and CD8 Ags -which permit division of the
thymocytes into the four subsets recognized through the distinct cellular
phenotypes during maturation: CD4 CD8 ; CD4+CD8+; C D 4 T D 8 ; CD4CD8'. The thymus is central also for the dual (positive and negative)
selection of the ap' at the CD4+CD8+ level, which is dependent on
intrathymic events skewed toward governing self-reactivity and which results
in the CD4+ and CD8+ mature thymocytes that are functional against nonself. In contrast. the ligand recognized by, and the function of, the minor
thymocyte population -which rearranges a TcRy8, which fails to exhibit the
TcRaB classical MHC-restricted Ag recognition- is still a matter of debate.
It is unclear whether the TcRy8, that are found primarily in the CD4-CD8subset--that appears unrestricted to the polymorphic MHC determinants that
restrict the TcRa&would be selected according to the same criteria as the
TcRap.
Here, I report a study showing that the flow cytometry and Western
immunoblotting distribution of the TcRy8 in murine tissues (intestinal
intraepithelial[IELl> =skin> >liver> =lung>thymus> > >spleen>kidney=heart), which is distinct from that of the TcRaB (spleen1 > >IEL>
=skin) directly parallels the expression (by Western immunoblotting ) of
heat shock proteins (Hsp)68-702members (Fig. 1) (that are ubiquitous stress
proteins present in multiple copies within the mouse genome). I speculate
that the ubiquity and predominance within the murine yB locales, of the
highly conserved murine Hsp68-70 (which share sequence homology with the
Drosophila and yeast Hsps), suggest an important function for the murine
endogenous Hsp68-70, such as reactivity with the TcRy8, and function as a
ligand in the TcRy8 selection.
Over recent years, Hsps, that were initially recognized in Drosophila by
the increased expression after exposure to elevated temperatures (assumed
to provide the cells with protection during recovery from stress), but that
now are identified as structurally conserved elements that are constitutively
expressed in prokaryotic and eukaryotic organisms even in the absence of
any kind of stress, have emerged as important biological members. Hsps,
whose functions, under normal cellular, or under stress conditions, are
suggested to have important general roles, are now the focus of biological
and biochemical research.
Different Hsp families are distinguished on the basis of their size: a
heterogeneous family group of small Hsp (18-30 kDa); Hsp90 (that may play
a role in the physiology of the steroid family of receptors for glucocorticoid,
progesterone, estrogen, dioxin); Hsp60; and the disperse Hsp70 multigene
family (that is the most highly conserved family). Hsp7O comprises at least
four major eukaryotic stress proteins: Hsp72 (referred to as heat-shock
cognate Hsp70. a constitutively produced, heat-shock enhanced, major
responsive protein); Hsp73 (referred to as cognate or constitutive, which is
present at high levels in normal unstressed animal tissues, and which
functions in regulation pathways essential to the unstressed cells to facilitate
protein folding/assembly , ATP-dependent protein translocation, recognition.
binding, and turnover of denatured proteins); grp75 (the glucose regulated
protein functioning in the assembly or disassembly of target proteins in
mitochondria); grp80 (that exhibit > 45 % sequence homology to the bacterial
hsp7O members, Dnak); grp78 (also referred to as grp80) which is identical
or similar to the heavy (H) chain-binding protein (BiP). Under normal
conditions BiP is abundant in the lumen of the endoplasmic reticulum
[ER].BiP is increased under malfolding signaling. In general, the Hsp7O
family functions within similar cytoplasmic and nucleus locales and in similar
biochemical fashion binding tightly ATP and associating in an ATPdependent manner with cellular proteins in the ER, functioning as molecular
chaperons to facilitate assembly of native monomeric proteins into oligomeric
structures. The significant conservation of the Hsp7O family genes, and the
M.Western immunoblotting of homogenates prepared h
m tissues
derived from C57BW6 euthymic (+I+), C57BU6 nude (4-1.and C57BY6
nude/+ (J+) heterozygous littermate mouse strain, dissolved in 100 mM
Tris pH 6.8, 4% sodium dodecyl sulfatte (SDS), 20% glycerol, 200 mM
dithiothreitol (D'TT). fractionated by SDS polyacrylamide gel (13%)
electrophoresis as described by Laemmli, transferred to nitrocellulose
sheets, and reacted with polyclonal antibody against bovine brain heat
shock protein (Hsp70).
Depicted are the molecular weights against the migration of markers.
ubiquity of the Hsp68-70 in the examined murine tissues (intestine, skin,
liver, lung, spleen, heart, kidney [Fig. I]) derived from both thymus+
bearing (euthymic) (+/+) mice (whose functionally mature a@' CD4' or
CD8+ T are selected on the thymic epithelium geared towards governing
self-MHC), as well as from thymus lacking (nude) (-/-) mice (whose thymic
ap' T counterpart are essentially lacking, and in which mice, the role in
keeping the tolerant state is a function of extrathymic MHC-unrestricted y8
maturation), suggest that Hsp70 plays an important function. Clearly, the
expression of the Hsp, coded by genes that are highly conserved during
evolution (both in their protein-coding sequences and in their regulatory
sequences), though universal, may be regulated in different ways in the
different tissues. Fig. 1 depicts somewhat different Hsps fractions detected
with the anti-Hsp70 antibody in the various murine tissues; whether these
reflect specific biological characteristics is unknown. But because of the
present awareness and evidence that aspects of the heat-shock response may
be of potential relatedness both to the normal physiology and to disease,
besides of the already known Hsp70 protecting function of cells from the
toxic effects of stress, I plan to further analyze the precise relation of the
bands delineated in the tissue sonicates (Fig. I ) to the T subsets (detected by
flow cytometry), by using this time purified T sonicates. rather than the
whole tissue sonicates that comprise among other elements B lineage cell
too. I plan to clarify whether the smaller kDa molecular sizes proteins
reactive with the antibody against bovine brain Hsp70 (that reacts with the
Hsp68-70 band by Western) represent Hsp7O break down fragments
indicative of a primary function, or of a secondary function because of
shared epitopes with those of bacterial origin? I plan to asses whether the
antibody used depicts also BiP proteins -thought to function as chaperons in
normal immunoglobulin (Ig) H and L chain assembly or oligomerization.
assisting protein folding and assembly processes that protect the cell from
damaging effects. BiP exhibits some 60% sequence homology to the
cytoplasmic Hsp70, as also to the non heat-induced grp, which is
ubiquitously expressed in different tissues.
1. Smodgrass, H.R., Dembic, Z., Steinmetz. M. & von Boehmer, H.
(1985) Nature (Lond). 315, 232
2. Lindquist, S. & Craig, E.A. (1988) AM. Rev. Genet. 22, 631-677;
Lewis, M.J.& Pelham, H.R.B. (1985) EMBO 1. 4, 3137-3143