Invited critical reviewhCG, five independent molecules
Highlights
► This paper reviews every aspect of 5 variants of hCG. ► The biological function and detection of hCG are examined. ► The paper examines the structure, biological function and detection of hyperglycosylated hCG. ► The biological functions of sulfated hCG are examined and functions during the menstrual cycle. ► The function of hCGß and hyperglycosylated hCGß, two cancer promoters, is investigated.
Introduction
The name hCG, today refers to 5 independent molecules, each having identical amino acid sequence, thus all are named hCG, yet each varies greatly in structure and biological function. This review examines each of these 5 hCG variants carefully. hCG is the only example in biochemistry of one amino acid sequence describing 5 independent molecules. There are clear reasons that cause this anomaly. The first is that hCG is the most glycosylated glycoprotein. Research shows that 28% to 39% of the molecular weight can be due to sugar side chains [1], [2], [3], [4], [5], [6] (Table 1). hCG and its sister molecules with separate functions, hyperglycosylated hCG and sulfated hCG, just vary in carbohydrate structure [1], [2], [3], [4], [5], [6]. Other independent hCG molecules, hCGß and hyperglycosylated hCGß vary in subunit combination and carbohydrate structure [7].
Much of the reason that hCG has multiple molecules is hCG's evolutionary origin. hCG and sulfated hCG normally bind the hCG/luteinizing hormone (LH) receptor as a hormone [2], [8], [9]. hCG has molecular evolutionary origins with TGFß, and has sequence in common with this growth factor [10], [11]. Furthermore, as shown when hCG's crystal structure was determined [12], hCG shares a unique 4 peptide cystine knot structure with TGFß and three other cytokines [12]. As such, it is no surprise that hyperglycosylated hCG, hCGß and hyperglycosylated hCGß function as autocrines by antagonizing a TGFß receptor [7], [13], [14]. Basically, there are two groups of hCG molecules. hCG is produced by syncytiotrophoblast cells of the placenta. Sulfated hCG is made by gonadotrope cells of the pituitary. Both are independent molecules and both are hormones that bind the hCG/LH receptor. Hyperglycosylated hCG is produced by placental cytotrophoblast cells, and hCGß and hyperglycosylated hCGß which are produced by all advanced cancer cells (other than choriocarcinoma and germ cell malignancies). All three are autocrines and antagonize a TGFß receptor [7], [13], [15].
To introduce the hormone hCG, the first of the five hCG molecules, it is a dimeric glycoprotein of molecular weight 37,180 made by placental syncytiotrophoblast cell (Table 1). It is an unusually acidic glycoprotein with an isoelectric point (pI) of 3.5. Acids are negatively charged molecules at physiological pH that are repelled by the glomerular basement membrane, which is also negatively charged. The acidity also gives hCG a long circulating half-life of 36 h (Table 1). hCG has multiple hormonal functions during pregnancy, ranging from maintenance of corpus luteal progesterone production, to promotion of growth and differentiation of the uterus, placenta and fetus, growth of uterine and umbilical blood vessels and blockage of macrophage destruction by maternal tissues of feto-placental components as foreign tissues [9].
By contrast, sulfated hCG, the second hCG-related molecule is produced by pituitary gonadotrope cells. Sulfated hCG production seemingly follows LH production in men and women [16], [17], [18]. Sulfated hCG functions to supplement LH actions. Interestingly, sulfated hCG is produced at approximately 1/50th the level of LH [16], [17], [18]. Yet hCG has 50-fold greater biopotentcy (circulating half-life) than LH [2]. It is concluded that sulfated hCG may be the co-promoter of ovulation, androstenedione production in the follicular phase of the menstrual cycle and progesterone production in the luteal phase of the menstrual cycle.
Hyperglycosylated hCG is the third hCG-related molecule. It is produced by root cytotrophoblast cell, and is an autocrine that promotes implantation during pregnancy [15], [19]. Hyperglycosylated hCG promotes invasion of the uterus at pregnancy implantation, it also promotes growth of placental tissue during pregnancy. Hyperglycosylated hCG also drives growth and malignancy of choriocarcinoma and germ cell malignancies. These are cancers of cytotrophoblast tissue, or cancers that take on cytotrophoblast histology [15], [20], [21]. Research indicates that hyperglycosylated hCG antagonizes a TGFß receptor on cytotrophoblast cells. [7], [13]. In summary, hyperglycosylated hCG is an autocrine produced by cytotrophoblast tissue and circulates before acting back on this same tissue by promoting growth and invasion by antagonizing a TGFß receptor.
All advanced cancers (except choriocarcinoma and germ cell malignancies) produce hCGß or hyperglycosylated hCGß in variable proportions [22], [23], the fourth and fifth hCG molecules. These molecules are major cancer promoters, promoting advanced cancer growth and malignancy by antagonizing a TGFß receptor, like hyperglycosylated hCG [7], [13].
We ask why all these 5 very different molecules are called hCG. Under World Health Organization (WHO) regulations all molecules with a set amino acid sequence are given the same name. Initially, with the discovery of hyperglycosylated hCG in 1997 [1], we called the molecule ITA or invasive trophoblast antigen, as the molecule that promotes invasive trophoblast. Four years later, WHO told us that the name had to be changed to a name that included the word hCG. We then renamed it hyperglycosylated hCG as the over-sugarized form of hCG.
In this review, we now examine these 5 hCG related molecules in detail. We examine and compare their structures, examine and compare their different site of production and their different functions. Finally we examine different hCG assays and their specificities.
Section snippets
Five different hCG structures
This section presents the structures of the five different bioactive forms of hCG in (Table 1, Fig. 1, Fig. 2, Fig. 3, Fig. 4). The structures all share one common α-subunit and ß-subunit amino acid sequence [4]. As shown, hCGα has one gene on chromosome 6 (6q14–q21), while hCGß is represented by 8 genes on chromosome 19, alongside the single LHß gene (19q13.32). Large discrepancies are noted in the 8 duplicate genes in intron and exon sequences. It has been demonstrated that hCGß genes 3, 5
hCG the pregnancy hormone
hCG is a hormone made by placental syncytiotrophoblast cells [25]. It comprises 2 oligosaccharides 92 amino acid α-subunit and a 6 oligosaccharides 145 amino acid ß-subunit (Table 1, Fig. 1). The ß-subunit of hCG, while structurally similar to the ß-subunit of LH, differentiates hCG from other glycoprotein hormones. hCG, like LH, is a hormone, and binds a common hCG/LH hormone receptor.
For the first 3 weeks following implantation of pregnancy, hCG promotes production of progesterone by ovarian
Hyperglycosylated hCG, the invasion promoter
Hyperglycosylation leads to incomplete folding of hCG. This causes exposure of sequences otherwise hidden. These are the common evolutionary TGFß structures on hCG. Hyperglycosylated hCG is an autocrine, and not a hormone like hCG. Hyperglycosylated hCG binds and antagonizes TGFß receptors on the cytotrophoblast cells, the cells that make hyperglycosylated hCG [7], [9], [19], [26], [27], [29], [21], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64]. This is part
hCGß and hyperglycosylated hCGß the cancer promoters
At the same time as research has been progressing showing the relationship between hyperglycosylated hCG and choriocarcinoma and germ cell malignancies, other researchers have been investigating the role of hCG ß-subunit (hCGß) and large forms of hCGß and other malignancies. As found, all other malignancies produce hCGß or large hCGß in advanced stages of malignancy [3], [7], [13], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], [83],
Sulfated hCG a pituitary hormone
A fifth variant of hCG is made by pituitary gonadotrope cells during the normal menstrual cycle [2], [16], [17], [18]. This is the sulfated variant of hCG with sulfated oligosaccharides as shown in Fig. 2[2]. Research using an exceptionally sensitive total hCG assay shows that sulfated hCG is secreted during the length of the menstrual cycle [16], [17], analogous to the secretion pattern of LH. hCG and LH both bind a joint receptor, the hCG/LH receptor. Research in my laboratory shows that in
hCG, hyperglycosylated hCG and abnormal pregnancies
The hCG doubling test remains today the recommended marker for identifying a failing pregnancy [103], [104], [105], [106]. This test, however, has very limited sensitivity in detecting biochemical pregnancies, spontaneous abortion and ectopic pregnancy. The hCG doubling test is commonly used (in normal pregnancy hCG levels should double, 4–8 weeks gestation, within 2 days). Depending on the report, 62–78% sensitivity is claimed, and by an unacceptably high false-positive rate, 26–40% [103], [104]
hCG assays: detection of pregnancy and cancer hCG
As published, today's laboratory automated laboratory total hCG tests invariable detect the multiple forms of hCG and their variants [118], [119], [120]. As demonstrated, no real improvement has been made in the laboratory hCG test since the 1970s radioimmunoassay [118]. Yes, the assays have been very much sped up and automated, but no regard has been placed by most tests to maintaining the wide specificity of the radioimmunoassay, limiting modern automated pregnancy tests to in some cases, to
Sulfated hCG and menopause
Sulfated hCG, produced by the pituitary, is barely detectable during the menstrual cycle. At the time of the LH peak, hCG level in 277 menstrual cycles averaged 1.54 ± 0.90 IU/L [18]. In menopause, with the absence of estrogen and progesterone feedback to the hypothalamus, gonadotropin releasing hormone (GnRH) pulses become maximal. The result is promotion of excess LH, hCG and FSH by gonadotrope cell due to GnRH pulses. Serum LH increases from, 1–90 IU/L to > 100 IU/L in menopause, serum FSH
Early pregnancy tests
Fig. 6 shows serum hCG and hyperglycosylated hCG levels during the course of pregnancy [113], [114]. As shown, in the first 3 weeks of pregnancy, hyperglycosylated hCG production predominates. Hyperglycosylated hCG rises to a 10 week peak at approximately 1/10th hCG levels (10 week serum, hyperglycosylated hCG 10% of total hCG). Levels continue to diminish, hyperglycosylated hCG concentration diminishing to approximately ½% of serum hCG levels in the third trimester of pregnancy.
In the weeks
Men and women positive for hCG outside of pregnancy
There are multiple reasons why men and women may be positive in a serum pregnancy or urine pregnancy test, after clinical pregnancy and ectopic pregnancy (in women) have been eliminated. In women explanations include —
- 1.
Menopausal or pre-menopausal hCG (see Section 9. Sulfated hCG and menopause).
- 2.
Quiescent gestational trophoblastic disease 3–9 months following an ectopic pregnancy or spontaneous abortion [63], [124].
- 3.
A false positive hCG test (see following segment, Section 12).
- 4.
Choriocarcinoma or
False positive serum hCG
The biggest issue faced by the USA hCG Reference Service 1998–2003 was the abundance of false positive serum hCG cases [124], [128], [129]. It all came down to a problem in the design of the Abbott Axsym total hCG test, at that time the leading test used by the USA (College of American Pathologists report). Undiluted samples, because of the test design lacked any added serum or non-specific antibody to prevent interaction with heterophilic antibodies [129]. The problem was fixed by Abbott in
hCG in Down syndrome screening
In 1995, I presented the discovery of hyperglycosylated hCG and the availability of a new hyperglycosylated hCG assay at a Yale University Obstetrics and Gynecology grand rounds. I was approached by a member of the Maternal Fetal Medicine division to test out a library of 2nd trimester Down syndrome pregnancy samples and controls. As a result, the first application found for the hyperglycosylated hCG test was in screening pregnancies for Down syndrome [130]. I ask, why should hyperglycosylated
The degradation of hCG and related molecules
Trophoblast cells make large concentrations of hCG and hyperglycosylated hCG. These concentration of hCG promote progesterone production by corpus luteal cell, hyperglycosylated hCG promotes invasion as part of implantation. hCG promotes uterine spiral artery angiogenesis, uterine growth, trophoblast differentiation, myometrial muscle relaxation, and suppression any macrophage or immune reaction to the foreign feto-placental unit (145, see 3 hCG the pregnancy hormone, 4 Hyperglycosylated hCG,
Evolution of hCG and hyperglycosylated hCG and their role in human evolution
This section is a summary of the published role of hCG and hyperglycosylated hCG in human evolution [35], [36], [155]. The earliest primates, prosimian primates like lemurs, had small brains like most earlier mammals, 0.07% of body weight (Table 8). This happens because prosimian primates and earlier mammals used an ineffective placentation system, non-invasive epitheliochorial placentation. In this placentation system, maternal nutrients had to travel through multiple layers of decidua,
Conclusion
This review tells the complete story of hCG and associate molecules. The only sauce known of a more complete review is the recent book I published on hCG [174]. Multiple chapters from this book have been cited. hCG is an extremely unusual molecule, it is the most glycosylated glycoprotein (Table 1) and the most acidic protein. It is the only known glycoprotein that has 5 independent variants, each sharing the same amino acid sequence (Table 1). hCG is unusual in that it plays multiple roles, as
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