The purification of early‐pregnancy factor to homogeneity from human platelets and identification as chaperonin 10

Abstract

Early‐pregnancy factor (EPF), first discovered in the early stages of gestation, is associated with and necessary for cell proliferation in a wide variety of biological situations. Like many other growth factors, EPF is present in platelets, and, by titration studies with a neutralising anti‐EPF monoclonal antibody, platelets were identified as an extremely rich source of this growth factor. EPF has been purified from clinically outdated human platelets by heat extraction, ion‐exchange and affinity chromatographies on SP‐Sephadex and heparin‐Sepharose respectively, high‐performance hydrophobic interaction chromatography and three reverse‐phase HPLC steps, with an average yield of 15 μg/100 platelet units (equivalent to ∼ 50 1 blood). Using SDS/PAGE, EPF migrated as a single band with approximate Mr 8500, coincident with biological activity. Mass spectrometry provided an accurate and precise determination of the molecular mass as Mr 10843.5±2, along with definitive evidence of the homogeneity of the preparation. Attempts at Edman degradation indicated that the molecule was blocked at the N‐terminus and sequencing of proteolytic fragments was undertaken. The amino acid sequence of approximately 70% of the molecule was determined which, with a single exception, is identical with rat chaperonin 10. This structural relationship was shown to extend to functional identity by studies using chaperonin 10 and its functional associate chaperonin 60. Investigations with the latter confirmed that chaperonin 10 is the moiety in pregnancy serum which initiates response in the EPF bioassay. Our studies identify EPF as a member of the highly conserved heat‐shock family of molecules and demonstrate a molecular chaperone performing an extracellular role.

Expression in Escherichia coli, purification and functional activity of recombinant human chaperonin 10

“…In light of recent findings suggesting that cpnl0 are multifunctional molecules one may also speculate that N-terminal acetylation could be relevant in functions other than assisting cpn60 in protein folding. Comparative studies of natural and recombinant cpnl0 in those biological systems that have been studied so far only with the natural form [13] may help clarify this issue. The availability of recombinant human cpnl0 will facilitate to address these studies.…”

“…A recent report has shown that human cpnl0 is identical to the early pregnancy factor (EPF) [13]. EPF has been known for long time to be involved in a number of physiological processes which are apparently unrelated to the role of cpnl0 in protein folding, but its characterisation has been elusive due to the difficulties encountered in its purification [13]. Here, we report the high-level expression of human cpnl0 in Escherichia coli.…”

“…It showed a 100% homology with the bovine cpnl0 [11] and possessed a single amino acid substitution, serine in place of glycine at position 52, compared to rat cpnl0 [12]. A recent report has shown that human cpnl0 is identical to the early pregnancy factor (EPF) [13]. EPF has been known for long time to be involved in a number of physiological processes which are apparently unrelated to the role of cpnl0 in protein folding, but its characterisation has been elusive due to the difficulties encountered in its purification [13].…”

Unfolding the relationship between secreted molecular chaperones and macrophage activation states

“…Indeed, some 30 years ago, 1 year before the discovery of the first molecular chaperone, a circulating immunosuppressive activity, termed early pregnancy factor (EPF), was discovered (Morton et al 1977). This turned out to be the molecular chaperone, chaperonin (Cpn)/heat shock protein (Hsp)10, a mitochondrial protein (Cavanagh and Morton 1994). Co-incidentally, both Hsp10 and the co-chaperone Hsp60 are now implicated as cell surface proteins essential for the capacitation of sperm, revealing a 'prior role' for these proteins in pregnancy (Walsh et al 2008).…”

Recombinant EPF/chaperonin 10 promotes the survival of O4-positive pro-oligodendrocytes prepared from neonatal rat brain

“…Early pregnancy factor (EPF) is a molecule that is present, from soon after conception, in the sera of all pregnant mammals that have been tested (Morton et al 1992;Cavanagh 1996;Cavanagh 1997); EPF has immunomodulatory properties and growth factor properties (Morton et al 1982;Morton 1998). EPF was found to be homologous to cpn 10 (Cavanagh and Morton 1994).…”