The Isoforms Variants And Nicking Of Human Chorionic Gonadotrophin

The Isoforms Variants And Nicking Of Human Chorionic Gonadotrophin

Human Chorionic Gonadotrophin consists of two units, alpha hCG and beta hCG which combine
to form the biologically active hormone hCG.
Several factors have to be considered in the relationship of hCG to pregnancy nausea and vomiting. 

hCG exhibit’s a considerable heterogeneity in maternal blood during pregnancy (1). Typical isoelectric focusing (IEF) pattern of immuno-reactive (IR) hCG is sera of normal pregnant women at 7 weeks of pregnancy (these components were designated for convenience). A PI (isoelectric point) 3.9, B PI 4.1, C PI 4.4, D PI 4.7, E PI 5.0, F PI 5.8, G approx PI 6.7. In general the reactive amounts of B and C were large while A, D, E, F and G were small (2).

The isoforms of hCG produced in early and late pregnancy are different (1). The isoforms of hCG
in sera from early pregnancy were more acidic (have a lower isoelectric point) than those obtained
from later pregnancy (1).  The hCG molecules with the highest in vivo biological activity were
produced during the first trimester (1).  hCG from early pregnancy has a longer half-life (when
tested in mice) than hCG from late pregnancy which explains the higher activity in the in vivo
bioassay of hCG in early pregnancy (1).

In the alpha unit of hCG, carbohydrate constitutes 30% of the total weight of which 27.4% is sialic
acid.  Similarly, the beta subunit contains 36% carbohydrate of which 28% is sialic acid.  The sialic
acid residues as well as the entire carbohydrate moiety of hCG have been shown to be essential for
the full expression of hCG’s in vitro and in vivo gonadotrophic activities (3).

The electrophoretic technique measures the overall charge of the isoforms of hCG and the change in the median charge of the isoforms of hCG reflects a change in the carbohydrate of the polypeptide structure (1). This change of iso-electric charge during pregnancy is not a continuous process but occurred at a limited period of time, namely 11-15 weeks of gestation. All the values of median mobility were higher at weeks 6-10 than at weeks 16-43 of gestation. The mean values of the degree of charge of heterogeneity of hCG was significantly (P<0.05 and P<0.01 respectively) higher at 11-15 weeks and at 16-43 weeks than at 6-10 weeks (1). The concentration of serum hCG decreased at the same period of gestation as the median charge of hCG changed. There was a significant (r-o 394; P<0.01; n=3g) positive correlation between the median mobility and the concentration of hCG in serum during the weeks 6-10 or the weeks 16- 43 of gestation (1).

In patients with hyperemesis gravidarum (HG), ten European and ten Samoan women the hCG profiles differed significantly from those without HG (p<0.001). The HG subjects had higher concentrations in the acidic third when compared with control subjects. Peak 6 (PH 3.3) was observed only in hCG profiles of women suffering from HG. Peak 5 (PH 3.6) occurred significantly more frequently in hCG profiles of HG women, than with control subjects (p<0.05). Therefore, the acidic forms might be responsible for pregnancy related nausea either by direct effect on the brain stem centres or in intestinal mobility or by indirect effects through secretion of hormones normally related to other glycoproteins, such as thyroid hormones (4).

 Binding of hCG variants to Liver Receptors

The biological activities of desialyted forms of hCG are greatly reduced in vivo due to the high
affinity of asiolo-glycoprotions for hepatic receptors and their consequent rapid clearance from the
circulation.  Intact purified hCG on the other hand has little affinity for hepatic receptors and has a
relatively long life in vivo (5).

The ß-subunit sialic acid seems to be more critical than the alpha-sialic acid in preventing hepatic
binding and prolonging plasma half-life (7).  Intact hCG exhibited the slowest metabolism as, very
little, if any, of it was removed from the circulation during the thirty minute observation period (in
mice). Asiolo (desialyted) hCG by contrast was rapidly downgraded and only 2% of the initial
concentration was detectable after the first two minutes and less than 0.4% remaining after 30
minutes.  Intact alpha asiolo-beta was also rapidly removed as about 1% of its residual
concentration was detectable even after 30 minutes. Asiolo-alpha intact beta was cleared much
more slowly as more than 30% of the initial concentration was present at two minutes, whereas, at
30 minutes nearly 10% was still detectable (5).
 

Yoshimura et al state other mutations of hCG cause it to be unable to dimerise i.e. for alpha and beta subunits to join which they must for normal activity, such mutations may manifest as elevated free β-hCG. This is seen in molar gestations. They found that compared to 23 gestational age-matched controls, 39 patients with hyperemesis had elevated free β-hCG 101  70ng/ml, v31  31ng/ml p<0.001. There was also a significant difference between groups for total hCG 9,327  3,613 ng/ml, 5,543  2,290 ng/ml p<0.01 but not for free alpha hCG 399  231, 377  ng/ml (6).

What was commonly referred to as immuno-reactive hCG is a mixture of free-beta hCG, free-alpha
hCG, deglycosylated hCG, desialyted (asiolo) hCG and hCG completely lacking the four sites of
glycosylation (BCTP).  Molar tissue in which only 3% of the measured hCG is fully glycosylated
and a majority of hCG is not intact.  Large amounts of the basic fractions of hCG were much more
potent than intact hCG in stimulating the release of cyclic ‘5’ adenosine monophosphate (c’AMP)
when combined with human TSH or LH Receptors.  These basic fractions correspond to the
deglycosylated hCG =4()'&8$-.*$/-CTP.  Thus, part of the puzzle of the relationship between hCG
and NVP may lie in the fact that measurements of intact hCG do not reflect various fractions of
different potency (6).

Alpha and Beta hCG gene profiles

Whereas the human alpha-gene is present as a simple copy gene on chromosome 6 there are six
genes which enclose hCG beta-like products on chromosome 19.  Each of these hCG-beta genes
are transcribed in vivo but with highly variable levels of expression.  The genes hCG beta 5 and
hCG beta 3 are expressed at about a 7:1 ratio.  Furthermore, analysis of first trimester RNA
expression using gene-specific oligonucleotide indicated a 20:1 ratio for the two hCG-beta genes.  
Could it be that the increased beta hCG unit found in molar gestations, or the change in charge of
hCG isoforms around 13 weeks of gestation are due to a change in the relative expression between
the genes encoding different hCG proteins?  (1)

“Nicking of hCG”

A proportion of hCG molecules in pregnancy serum and urine samples have nicks or a missing
peptide linkage between either beta subunit residues 44 and 45 or beta subunit residues 47 and 48.  
The nick causes a rapid dissociation of hCG into free alpha and free beta subunits, with consequent
ablation of the steroidogenic activity of hCG (7).
 
Once nicked, hCG rapidly dissociates into free alpha and beta subunits.  Standard hCG (batch CR
127, 20% nicked) and hCG preparation C5 (100% nicked) were incubated for varying times in
whole blood.  C5 hCG dissociated rapidly into free alpha and beta subunits (dissociation half-life
22 + 5.2 hours) over 30 times faster than standard hCG (dissociation half-life 700 hours).  It was
inferred that nicked hCG rapidly dissociates and that the relative amount of nicked molecules
produced by trophoblast may considering circulating (37-41h) and dissociation half-lives be 3.4-
3.6 times higher than measured in serum samples (7).


Levels of total hCG (nicked and non-nicked) and intact hCG (non-nicked) were determined in 233
serum and 168 urine samples from 4-40 weeks of pregnancy.  A linear relationship was indicated
between advancing weeks of gestation and increasing extent of nicking.  Minimum ‘nicking’ was
observed in serum from the first two months of pregnancy (mean = 9% of hCG molecules) and
increased ‘nicking’ in the months thereafter, with maximum ‘nicking’ in samples from the last
months of pregnancy (mean = 21% of hCG molecules P<0.00005).  It was concluded that nicking
is more prevalent after hCG peak (after two months of pregnancy) (7).
 
The increased degree of ‘nicking’ of the hCG molecules was reported to be a gradual process
throughout gestation, whereas, the median charge changed at a restricted period and then remained
constant throughout the second and third trimesters.  It, therefore, seems unlikely that the two
processes are related (1).


The Source of “Nicking”

Human leukocyte elastase secreted by neutrophils can ‘nick’ hCG.  Type IV collagenases secreted
by macrophages are also elastases with the same specificity as the leukocyte enzyme.  This enzyme
may also ‘nick’ hCG.  We postulate that an elastase or type IV collagenase like enzyme, associated
with or present in trophoblast tissue, specifically ‘nicks’ and thus deactivates hCG.  The
progressive increase in proportions of ‘nicked’ hCG may simply reflect the increase of placental
mass that occurs throughout pregnancy.  We infer that ‘nicking’ occurs before or immediately upon
secretion of hCG by trophoblast tissue (7).

Summary 

hCG exhibits considerable heterogeneity in maternal blood during pregnancy.  Isoforms of hCG in
sera from early pregnancy were more acidic than those obtained from later pregnancy.  This acidic
hCG is also more glycosylated hCG and has a longer half-life than basic hCG.  A significant
change in the charge of hCG and its glycosylation takes place between 11-15 weeks gestation, the
hCG becoming more basic with altered glycosylation. 

Desialyted (asiolo) hCG has a high affinity for specific liver receptors and is, consequently, rapidly
removed from the circulation, while intact hCG has little affinity for these liver receptors and,
therefore, has a relatively long circulation half-life.  hCG can also be ‘nicked’ when a peptide
linkage becomes missing at the beta subunit residues 44 and 45 or 47 and 48.  The nick causes a
rapid dissociation of hCG into free alpha and free /-subunits with consequent ablation of hCG’s
steroidogenic activity.  This ‘nicking’ is minimal until 8 weeks of gestation and gradually increases
throughout pregnancy.  
 
If either of these processes (a) desialyation of hCG with increased binding to liver receptors or (b)
‘nicking’ of hCG does not act efficiently, the resultant fully glycosylated hCG may continue later
into pregnancy, possibly causing nausea and vomiting of pregnancy to persist longer than usual
until these changes occur.   

REFERENCES


1. WIDE L, LEE J-Y, RASMUSSEN C.

 A change in the isoforms of Human Chorionic Gonadotrophin.

 Occurs Around the 13th Week of Gestation.

 J. Clin. Endocrinol Metab. 1994;78:1419-1423.

2. YAZAKI K, YAZAKI C, WAKABAYASHI K, IGARASHI M.

 Isoelectric Heterogeneity of Human Chorionic Gonadotrophin:

 Presence of Choriocarcinoma Specific Components.

 AM J Obstet Gynecol. 1980;138:189-194.

3. HOERMANN R, KEUTMANN H T, AMIR S M.

Carbohydrate Modifications Transform Human Chorionic Gonadotrophin into a Potent Stimulator of Adenosine 3,’5’ - Monophosphate and Growth Repsonses in

 FRTL-5 Thyroid Cells.

 Endocrinol. 1991;128:1129-1135.

4. JORDAN V, GREBE SKG, COOKE R R, FORD H C, LARSEN P D,

 STONE P R, SALMOND C E.

Acidic Isoforms of Chorionic Gonadotrophin in European and Samoan Women are Associated with Hyperemesis Gravidarum and may be Thyrotrophic.

 J. Clin. Endocrinol. 1999;50:619-627.

5. HOERMANN R, KUBOTA K, AMIR S M.

Role of Subunit Sialic Acid in Hepatic Binding, Plasma Survival Rate and In Vivo Thyrotropic Activity of Human Chorionic Gonadotrophin.

 Thyroid. 1993;3(i):41-47.

6. YOSHIMURA M, PEKARY E, PANG X-P, BERG L, GOODWIN M J, HERSHMAN J M.

 Thyrotropic Activity of Basic Iso-Electric Forms of Human Chorionic Gonadotrophin Extracted From Hydatidiform Mole Tissues.

 J. Clin. Endocrinol Metab. 1994;78:862-866.

7. COLE A, KARDANA A, PARK S-Y, BRAUSTEIN G-D

The Deactivation of hCG by Nicking and Dissociation.

J. Clin. Endocrinol Metab. 1993; 76:704-710.

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