Summer Research Fellowship Programme of India's Science Academies 2017
Analyzing functionality of Ig super family domain in CEA
antigen evaluated through PROVEAN 1.1 and SIFT
Narisetty Madhansai
Vignan’s University, Guntur, Andhra Pradesh, India
Guided by
Dr. Arnab Bhattacharya
Department of Computer Science, Indian Institute of Technology, Kanpur, India
Abstract
Intent: To predict promiscuous peptide sites determinants of T-helper cell from carcinoembryonic
antigen to find its influence on tumor marking function.
CEA (carcinoembryonic antigen) is a forerunner of the inflammatory response and its effect on
colorectal cancer hepatic metastasis. There is significant raise in percentage of colorectal cancer and
most of deaths are embodying with liver metastasis. CEA also has a role in protecting tumor cells from
the effects of anoikic (programed cell death) and this affords a selective advantage for tumor cell
survival in the circulation.
CEA acts in the liver through its interaction with its receptor (CEAR) called carcinoembryonic antigen
receptor, a protein that is related to the hnRNP M family of RNA binding proteins. In the liver CEA
binds with hnRNP M on Kupffer cells and causes activation and production of pro- and anti-
inflammatory cytokines like inter leukin-10) (IL-10), IL-6 and TNF-α.
These cytokines affect the up-regulation of adhesion molecules like other CAM (Cellular adhesion
molecule) on the hepatic sinusoidal endothelium and protect the tumor cells against cytotoxicity by
nitric oxide (NO) and other reactive oxygen radicals.
To analyze the functionality of this important biological molecule by altering aminoacid at genomic
level to predict its function particularly in Ig super family domain site with the aid of provean
computational tool. The amino acid length of 14 from 625 to 639 which is said to be T-helper
determinants base pair in entire 702 length of protein sequence and analyze that there is strong influence
of unstable amino acids serine(s), Arginine(r), cysteine(c) which do not tolerated towards any function
and allows us to know the forerunner in T-helper determinants.
The possibility of Interaction of CEA antigen with its receptor is deduced with
following scores obtained from SIFT (Scale of variant feature transform) based on its
impact.
Key words: Carcinoembryonic antigen, T-helper determinants, Provean, SIFT.
1. Introduction
Cancer of the large intestine (colon, rectum and anus) is a major public health problem world wide,
with approximately 147,000 new cases and 50, 000 deaths. The incidence and mortality from colorectal
cancer has remained constant over the past decade.
The role of carcinoembryonic antigen (CEA) which is a forerunner of the inflammatory response and
its effect on colorectal cancer hepatic metastasis. There is significant raise in percentage of colorectal
cancer and the majority of deaths are embody with liver metastasis.
If left untreated, median survival is only six to 12 months. Resection of liver metastases offers the only
chance for cure. If even treated there is high possibility of further tumor recurrence. The molecular
mechanisms associated with colorectal cancer metastasis to the liver are largely unknown and still in a
constant debate. However, CEA is proved as a factor in production of an increased metastatic potential
of colorectal cancers to the liver.
CEA also has a role in protecting tumor cells from the effects of anoikic (programed cell death) and
this affords a selective advantage for tumor cell survival in the circulation (and other factors are also
indulged with example like protein machinery produced during cellular metabolism).
A majority of deaths from colorectal cancer are associated with liver metastasis and approximately
25% of patients have liver metastasis at the time of presentation. If metastatic disease is left untreated,
median survival is six to 12 months with a 5 year survival of only 9%.
Resection of liver metastases offers the only chance for cure, although ~70% of patients who undergo
a hepatic lobectomy will have further tumor recurrence (5 year survival ranges between 25 and 39%).
This raises the possibility of early tumor-cell spread at the time of, or before, surgical resection of the
primary tumor. Understanding the mechanisms involved in liver metastasis would provide the
opportunity to intervene before resection of the primary tumor.
Fig 1. Interactions of CEA in the hepatic sinusoid. CEA released by the tumor cell binds with hnRNP
M (CEAR) on the Kupffer cell surface resulting in release of the cytokines IL-1, IL-6, IL-10 and TNF-
α. B Effect of CEA induced cytokines on tumor cell interactions in the hepatic sinusoid. Cytokines IL-
1, IL-6, IL-10 and TNF-α produced by Kupffer cells have a number of effects on the tumor cell
microenvironment. These include up-regulation of adhesion molecules on hepatic sinusoidal
endothelial cells. The most important of these seem to be E-selectin and ICAM-1.
T-cell immunotherapy is one of most promising tools in cancer therapy. Different preclinical and
clinical studies have demonstrated the ability of T lymphocytes to control tumor growth. Because
tumor-specific CTLs are responsible for the lysis of tumor cells, many research groups have channeled
their efforts to the induction of CTL responses and to the identification of T-cytotoxic cell determinant
peptides from human tumor antigens.
These TCds are presented at the surface of tumor cells in the form of a complex TCd-MHC-I, which
is recognized by the T-cell receptor of CTL. However, despite the well-established role of CD4 T cells
to induce immune responses, and in particular antitumor responses (1620), much less effort has been
put to identify T-helper determinants (THds) from tumor antigens.
2. Carcinoembryonic antigen
Carcinoembryonic antigen was first discovered in 1965 by Gold and Freeman. It is a glycoprotein that
is a member of a large gene family that consists of 29 genes divided into three subgroups that include
the CEA-like glycoproteins and the pregnancy-specific glycoproteins (PSGs).
All these proteins are members of the much larger immunoglobulin supergene family. CEA is also
known as CD66 or CEACAM5 and the nomenclature for the entire CEA and PSG families can be
found in Beauchemin et al. CEA has a molecular mass of 180200 kD depending on the extent of its
glycosylation. The protein consists of a series of immunoglobulin-like domains, at the N-terminus is
108 amino acid v-domain followed by three pairs of c2-like domains each of 178 amino acids. Each c2
loop domain is held in conformation by a single disulphide bond.
There is a small hydrophobic C-terminal domain (26 amino acids) that is modified to give a GPI-linked
membrane anchor. This anchor can be cleaved by phospho-inositol specific phospholipases C and D to
release CEA in a soluble form. The structural features of the protein also allow up to 28 tetra-antennary
complex carbohydrate chains of the N-linked type.
The complete gene for CEA has been cloned and includes a promoter region that appears to confer
organ and cell type-specific expression. Since its discovery in 1965 a very large number of studies have
been carried out to determine the effectiveness of CEA as a clinically useful tumor marker. Serum
elevations of CEA are seen in about 60% of presenting colorectal cancer patients.
While not considered useful as a cancer screen for the general population due to high false positive and
false negative rates, CEA is used to monitor tumor recurrence following surgery and as a marker for
prognosis.
A small rise in CEA can be predictive of recurrence following curative surgery for colorectal cancer
and can result in detection of recurrence up to a year before the onset of clinical symptoms. A recent
study has even suggested that elevated serum CEA levels in people over 50 are predictive of increased
mortality.
CEA mRNA expression has recently been shown to be useful as an early marker for recurrence in
pancreatic cancer. Other members of the CEA gene family (e.g. CEACAM6 or NCA-90) can also be
used for prognosis and as predictors of tumor recurrence.
CEA is widely used as a target antigen for radio-immunodetection of occult metastasis and for radio-
immunotherapy in patients with CEA producing cancers. More recently it has been used as a preferred
antigen for the development of anti-colorectal cancer vaccines.
Elevated serum CEA levels are associated with liver metastasis and are used as a prognostic indicator
[19]. In the normal colon CEA is produced by mature colonocytes and is localized to the apical
membrane. In colon cancers and in fetal gut polarity is lost and CEA is found on all surfaces of the
cell. The normal function of CEA has been a mater of some debate.
There is a large body of evidence that shows that it can act as a receptor for bacteria including Neisseria
gonorrhoea and Escherichia coli CEA family members expressed in rodents also act as receptors for
viruses, including mouse hepatitis virus.
It seems unlikely that such a large complex glycoprotein (180 kD with 28 complex tri-antennary
carbohydrate chains) would be secreted into the gut without a purpose.
Thus a role for CEA in protecting the gut mucosa by binding potentially harmful bacteria seems to be
a possible function in the normal individual.
Carcinoembryonic antigen (CEA) is a protein found in many types of cells but associated with tumors
and the developing fetus.
CEA is tested in blood. The normal range is <2.5 ng/ml in an adult non-smoker and <5.0 ng/ml in a
smoker.
Benign conditions that can increase CEA include smoking, infection, inflammatory bowel disease,
pancreatitis, cirrhosis of cancer, and some benign tumors (in the same organs which have cancers with
increased CEA). Benign disease does not usually cause a CEA increase over 10 ng/ml.
The main use of CEA is as a tumor marker, especially with intestinal cancer. The most common cancers
that elevate CEA are in the colon and rectum. Others: cancer of pancreas, stomach, breast,