The beta-subunit of human chorionic gonadotrophin exists as a homodimer

Abstract

The free -subunit of human chorionic gonadotrophin (hCG ) is well recognised as a product of many epithelial tumours. Recently, it has been shown that this ectopic production may have a functional relationship to tumour growth. The growth-promoting activity of hCG may be explained by its structural similarity to a family of growth factors which all contain the same distinct topological fold known as the cystine-knot motif. Since the other members of this family all exhibit their activities as homo-and heterodimers, it is possible that the same may be true for hCG .Using size-exclusion chromatography, low stringency SDS-PAGE and matrix assisted laser desorption/ionisation (MALDI) time-of-flight (TOF) mass spectrometry (MS) we have shown that pure preparations of hCG contain hCG homodimers. Size-exclusion chromatography revealed asymmetric elution profiles with a forward peak corresponding to the size-exclusion characteristic of a globular protein with an approximate mass of 44-54 kDa and a late shoulder centered around an elution position expected for a globular protein of approximately 29 kDa. Two immunoreactive hCG species, of approximately 32 and 64 kDa, were clearly resolved by SDS-PAGE and Western blotting. When analysed by MALDI-TOF MS a 23 kDa monomer and a 46 kDa dimer were identified.Formation of hCG homodimers is consistent with the behaviour of other cystine-knot growth factors and strengthens the inclusion of the glycoprotein hormones within this superfamily. It has yet to be determined whether it is this dimeric molecular species that is responsible for growth-promoting activity of hCG preparations in tumours.

The Free Monomeric Beta Subunit of Human Chorionic Gonadotrophin (hCGβ) and the Recently Identified Homodimeric Beta-Beta Subunit (hCGββ) both Have Autocrine Growth Effects

“…Batch purity details supplied by Sigma UK indicated 99% purity with only minimal potential contamination of ·-subunit. Our own previous examination of this preparation revealed the presence of monomeric and dimeric hCGß, with a similar profile to that of urinary derived pregnancy hCG, and no other contaminating proteins [5].…”

“…Lyophilised aliquots, separated by size exclusion, were reconstituted in distilled deionised water allowing a 10! concentration of protein, and separated on 12% acrylamide gels under low stringency conditions as described previously [5] using the Mighty Small II gel system (Hoefer, Amersham Pharmacia Biotech, St. Albans, UK). 5-Ìl samples of the recombinant hCGß preparation (100 Ìg/ml) were separated as a positive control, along with 14 C rainbow molecular weight markers (Amersham Pharmacia Biotech).…”

“…The dimeric species were observed following three different methods of molecular mass analysis: size exclusion chromatography, low stringency sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry [5]. Homodimers of bovine luteinizing hormone subunits · and ß (LH· and LHß) have also been noted following size determination by sedimentation equilibrium ultracentrifugation [6] and dimeric aggregates of hCGßcf have also been reported and blamed for interassay discrepancies in Down's syndrome screening [7].…”

“…Three distinct observations have led to the suggestion that hCGß may have a similar mode of action to the cystine knot growth factors transforming growth factor-ß (TGFß), nerve growth factor (NGF) and platelet-derived growth factor (PDGF-B): The structure of hCGß is topologically similar to these known growth factors [4], hCGß has been shown to increase epithelial cell populations by inhibition of apoptosis [2,3] and hCGßß homodimerisation has also been described [5].…”

Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

“…However, Butler et al (34) showed that homodimeric hCG␤ secreted by certain epithelial tumors has receptor binding and uncontrolled growth-promoting activities. It appears that the proximity of two ␤-subunits in hCG␤␤ has resulted in a conformation equivalent to that seen in the heterodimeric hormone necessary for receptor binding.…”