The studies undertaken in this research project characterised as fully as possible the
antigen identified by the monoclonal antibody BOB78. Earlier studies (Hart, Ross et al.
2000) have linked BOB78 to the identification of apoptotic cells. The present project
extended this finding by further studies utilising various antibody -based techniques on
cancer -derived cell lines in different stages of apoptosis.
The BOB78 antigen was confirmed to be present in normal cells and to be multi -lineage.
BOB78 antigen is normally expressed within the cytosol of nucleated cells, but absent in
anucleated red blood cells which do not undergo apoptosis. Following depolarisation of
mitochondrial transmembrane potential, a key event in the initiation of apoptosis,
BOB78 antigen is detected on the surface of the outer cell membrane. This surfacing of
the BOB78 antigen parallels that of phosphatidylserine residues, presently the most well
characterised marker for apoptosis in analysis with flow cytometry.
Immunocytochemistry corroborated this finding by showing the translocation of the
BOB78 antigen to membrane blebs of apoptotic cells. Observations using agents which
disrupt the endomembrane synthetic pathways suggest that BOB78 is probably not
trafficked through the Golgi apparatus or processed for secretion. Although BOB78 is
an IgM antibody, it does not identify a carbohydrate epitope and is therefore likely to be
specific for a multi -lineage protein.
Membrane blebbing in apoptotic cells appears to share certain characteristics with the
budding of platelets from megakaryocytes. For example, the production of platelets
during the terminal differentiation of megakaryocytes involves activation of caspases.
Interestingly, BOB78 was found by flow cytometric analysis to be present in platelets as
they develop from the membranes of the MEG -01 megakaryocytes. BOB78 was also
expressed on the surface of senescent platelets. These MEG -01 platelets were therefore
used for purification of BOB78 through immunoprecipitation. The captured BOB78
antigen was sequenced using MALDI -TOF, which identified it as chaperonin, also
known as heat shock protein 60 (hsp60).
Heat shock protein 60 is a highly conserved stress protein which has chaperone
functions in prokaryotes as well mammalian cells. Expression of hsp60 on the surface of
apoptotic cells may serve novel functions or purposes akin to molecular chaperoning.
Mechanisms underlying the translocation of hsp60 to the cell membrane have been
characterised in various prokaryotes. Homologous pathways of movement for this
highly conserved entity are likely to exist in mammalian cells. The observation that
hsp60 is expressed on the surface of senescent platelets suggests a possible role as a
recognition signal in phagocytosis. Platelets are equipped with caspases and when
senescent display membrane changes typical of apoptotic cells. Like apoptotic cells,
platelets are normally cleared from tissues by macrophages. Surface expression of hsp60
is probably an essential change marking cellular entities for uptake by phagocytes. It
remains to be studied if engulfment of bodies displaying an endogenous moiety like
hsp60 might play a key role in mediating a non -inflammatory response observed in
apoptosis. It may also be speculated that hsp60 serves the function of molecular
mimicry as many intracellular bacteria display surface hsp60, the expression of which
has been linked to pathogenic invasiveness. The ability of these microbes to evade
immune clearance may be linked to the non -inflammatory apoptotic disguise which
surface hsp60 confers on them.