CLAUDIN-1 REQUIRED FOR HCV VIRUS
ENTRY HAS HIGH POTENTIAL FOR
PHOSPHORYLATION AND O-
GLYCOSYLATION
Abstract
HCV is a leading cause of hepatocellular carcinoma and cirrhosis all over the world. Claudins belong to family of
tight junction’s proteins that are responsible for establishing barriers for controlling the flow of molecules around
cells. For therapeutic strategies, regulation of viral entry into the host cells holds a lot of promise. During HCV
infection claudin-1 is highly expressed in liver and believed to be associated with HCV virus entry after HCV binding
with or without co-receptor CD81. The claudin-1 assembly with tight junctions is regulated by post translational
modifications. During claudins assembly and disassembly with tight junctions, phosphorylation is required at C-
terminal tail. In cellular proteins, interplay between phosphorylation and O-b-GlcNAc modification is believed to be
functional switch, but it is very difficult to monitor these functional and vibrant changes in vivo. Netphos 2.0 and
Disphos 1.3 programs were used for potential phosphorylation; NetPhosK 1.0 and KinasePhos for kinase prediction;
and YinOYang 1.2 and OGPET to predict possible O-glycosylation sites. We also identified Yin Yang sites that may
have potential for O-b-GlcNAc and phosphorylation interplay at same Ser/Thr residues. We for the first time
proposed that alternate phosphorylation and O-b-GlcNAc modification on Ser 192, Ser 205, Ser 206 ; and Thr 191
may provide an on/off switch to regulate assembly of claudin-1 at tight junctions. In addition these
phosphorylation sites may be targeted by novel chemotherapeutic agents to prevent phosphorylation lead by HCV
viral entry complex.
Introduction with host receptor CD81 and scavenger receptor class B
HCV, the deadly virus has infected almost 3% of the world member I (SR-BI). It is reported that these two receptors
population. Most of the infected patients develop chronic are not sufficient for its entry and later another receptor
infection leading to end stage hepatocellular carcinoma. A claudin-1 was discovered which play an important role in
better understanding of mechanism of infection and the viral entry lately after viral binding to the CD81 [6,7].
potential host co-factors facilitating its replication is an Claudins are transmembrane proteins which play important
urgent need of the hour for the release of disease burden
role in tight junction formation and act as barrier in cellular
and vaccine development [1-3].
permeability. Tight junctions are the combination of
In multicellular organisms, movement of ions, proteins
transmembrane and peripheral proteins tied with
and water is controlled by barriers known as tight junctions
cytoskeleton. Several classes of claudin interact with other
(TJs) formed by epithelial and endothelial cell monolayers
proteins to form tight junction and regulate permeability of
[4]. While tight junctions require the coordinated activity
TJs. It is also observed that the expression of claudin
of several different proteins, the specificity of tight
proteins found to be differentially regulated in several
junction’s permeability is regulated by transmembrane
cancers. Claudin-1 expression was observed up-regulated
proteins known as claudins [5]. Entry of HCV in to the in liver, stomach, thyroid, pancreas and cervix tumor
hepatocytes is a complex process and involves interaction formations [8-12]. Claudin-1 removal in mouse epidermis
of HCV glycoproteins E1 and E2 results in dramatic transepidermal water loss, inferring its
indispensable role in
* Correspondence: creating and maintaining the epidermal barrier [13].
1
Applied and Functional Genomics Lab, Centre of Excellence in Molecular In biological systems, protein localization, activity, their
Biology, University of the Punjab, Lahore-53700, Pakistan interaction with other proteins and overall
, Full list of author information is available at the end of the article
© 2011 Ahmad et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
turnover is determined by post translational modifications The FASTA sequence of human claudin-1 was retrieved
(PTMs) [14]. Several PTMs like phosphorylation, from the SWISS-PROT sequence database [23] with entry
glycosylation, acetylation and methylation are some well name CLD1_human. The primary accession number for
known examples. Phosphorylation in claudin protein this sequence was O95832. Homology search was made
family is well observed and believed to be modulating TJs using the BLAST at NCBI database with default
permeability on both charged and uncharged ligands and parameters [24]. The search was made for all organisms’
molecules [9,15]. Several enzymes like protein kinase A sequences. A total of 250 hits were retrieved for claudin-1
(PKA), protein kinase C (PKC), protein phosphatase 2A with highest bits score and zero expected values. Out of 70
(PP2A), MAPK etc are involved during claudin retrieved sequences, seven were selected representing
phosphorylation [16-18]. Phosphorylation has dual effect major mammalian or vertebrate groups. The accession
on TJs functionality i.e. phosphorylation on some claudins numbers for eight selected sequences (Table 1) were
increased paracellular permeability or enhanced barrier O95832 (Human), Q6L708 (Bovine), D6RU0 (Sheep),
function [19]. It is reported that claudin-1 phosphorylation C3VMK8 (Pig), O88551 (Mouse), P56745 (Rat),
enhances its barrier functions while dephosphorylation Q5ZMG2 (Chick) and Q5FW44 (Xentr). ClustalW [25]
leads to detergent solubility and enhanced paracellular was used for multiple alignments of all the sequences of
permeability [20]. claudin-1 to get the conservation status.
O-glycosylation is also very important PTM of nuclear The claudin-1 sequence used in this study was
and cytoplasmic proteins. During O-glycosylation one “MANAGLQLLGFILAFLGWIGAIVSTALPQWRIYSY
molecule of N-acetylglucosamine (O-GlcNAc) is AGDNI
introduced on Ser or Thr residue by enzyme OGT (O- VTAQAMYEGLWMSCVSQSTGQIQCKVFDSLLNLS
GlcNAc transferase). Addition of O-b-GlcNAc can inhibit STLQATRALMVVGILLGVIAIFVATVGMKCMKCLE
phosphorylation on Ser or Thr residue. Interplay between D
O-b-GlcNAc modification and phosphorylation on the DEVQKMRMAVIGGAIFLLAGLAILVATAWYGNRIV
QEFYDPMTPVNARYEFGQALFTGWAAASLCLLGG
same amino acid residues has been observed in several
nuclear and cytoplasmic proteins [21]. These PTMs are ALLCCSCPRKTTSYPTPRPYPKPAPSSGKDYV”.
dynamic and result in temporary conformational changes
and regulate many functions of the proteins. The Post-translational modifications prediction methods We used
interchange of these two modifications on the same or more than one bioinformatics tools to access the post-
neighboring residue may modulate the specific function of translational modification on claudin-1 to get best results.
the proteins either by enhancing or inhibiting the functional Prediction of phosphorylation residues and related kinases
capacity. Residues where O-b-GlcNAc and Phosphorylation potential for human claudin-1 was
phosphorylation compete for each other are known as Yin predicted by using NetPhos 2.0 [26] and Disphos 1.3
Yang sites [22]. These Yin Yang sites can be predicted and server [27]. These are neural network-based programs that
analyzed using various computer-assisted neural network- predict the potential phosphorylation sites for each Thr,
based programs, which can help us to determine proteins Ser and Tyr residues. The minimum threshold value used
regulatory functions by accessing their modification to predict phosphorylation is 0.5 for NetPhos 2.0.
potentials. The present work describe potential Kinase specific phosphorylation sites in human claudin-
phosphorylation, O-glycosylation and their possible 1 were predicted by NetPhosK 1.0 [28] and KinasePhos 2.0
interplay sites which may influence claudin-1 interaction server [29]. These servers predict the kinase specific
with TJs and their possible effects on HCV entry and future acceptor substrates including Ser, Thr and Tyr.
therapeutics. For the evaluation of experimentally verified
phosphorylation sites on human claudin-1, Phospho.ELM
Materials and methods database was used [30]. This database contains a collection
ENTRY HAS HIGH POTENTIAL FOR
PHOSPHORYLATION AND O-
GLYCOSYLATION
Abstract
HCV is a leading cause of hepatocellular carcinoma and cirrhosis all over the world. Claudins belong to family of
tight junction’s proteins that are responsible for establishing barriers for controlling the flow of molecules around
cells. For therapeutic strategies, regulation of viral entry into the host cells holds a lot of promise. During HCV
infection claudin-1 is highly expressed in liver and believed to be associated with HCV virus entry after HCV binding
with or without co-receptor CD81. The claudin-1 assembly with tight junctions is regulated by post translational
modifications. During claudins assembly and disassembly with tight junctions, phosphorylation is required at C-
terminal tail. In cellular proteins, interplay between phosphorylation and O-b-GlcNAc modification is believed to be
functional switch, but it is very difficult to monitor these functional and vibrant changes in vivo. Netphos 2.0 and
Disphos 1.3 programs were used for potential phosphorylation; NetPhosK 1.0 and KinasePhos for kinase prediction;
and YinOYang 1.2 and OGPET to predict possible O-glycosylation sites. We also identified Yin Yang sites that may
have potential for O-b-GlcNAc and phosphorylation interplay at same Ser/Thr residues. We for the first time
proposed that alternate phosphorylation and O-b-GlcNAc modification on Ser 192, Ser 205, Ser 206 ; and Thr 191
may provide an on/off switch to regulate assembly of claudin-1 at tight junctions. In addition these
phosphorylation sites may be targeted by novel chemotherapeutic agents to prevent phosphorylation lead by HCV
viral entry complex.
Introduction with host receptor CD81 and scavenger receptor class B
HCV, the deadly virus has infected almost 3% of the world member I (SR-BI). It is reported that these two receptors
population. Most of the infected patients develop chronic are not sufficient for its entry and later another receptor
infection leading to end stage hepatocellular carcinoma. A claudin-1 was discovered which play an important role in
better understanding of mechanism of infection and the viral entry lately after viral binding to the CD81 [6,7].
potential host co-factors facilitating its replication is an Claudins are transmembrane proteins which play important
urgent need of the hour for the release of disease burden
role in tight junction formation and act as barrier in cellular
and vaccine development [1-3].
permeability. Tight junctions are the combination of
In multicellular organisms, movement of ions, proteins
transmembrane and peripheral proteins tied with
and water is controlled by barriers known as tight junctions
cytoskeleton. Several classes of claudin interact with other
(TJs) formed by epithelial and endothelial cell monolayers
proteins to form tight junction and regulate permeability of
[4]. While tight junctions require the coordinated activity
TJs. It is also observed that the expression of claudin
of several different proteins, the specificity of tight
proteins found to be differentially regulated in several
junction’s permeability is regulated by transmembrane
cancers. Claudin-1 expression was observed up-regulated
proteins known as claudins [5]. Entry of HCV in to the in liver, stomach, thyroid, pancreas and cervix tumor
hepatocytes is a complex process and involves interaction formations [8-12]. Claudin-1 removal in mouse epidermis
of HCV glycoproteins E1 and E2 results in dramatic transepidermal water loss, inferring its
indispensable role in
* Correspondence: creating and maintaining the epidermal barrier [13].
1
Applied and Functional Genomics Lab, Centre of Excellence in Molecular In biological systems, protein localization, activity, their
Biology, University of the Punjab, Lahore-53700, Pakistan interaction with other proteins and overall
, Full list of author information is available at the end of the article
© 2011 Ahmad et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
turnover is determined by post translational modifications The FASTA sequence of human claudin-1 was retrieved
(PTMs) [14]. Several PTMs like phosphorylation, from the SWISS-PROT sequence database [23] with entry
glycosylation, acetylation and methylation are some well name CLD1_human. The primary accession number for
known examples. Phosphorylation in claudin protein this sequence was O95832. Homology search was made
family is well observed and believed to be modulating TJs using the BLAST at NCBI database with default
permeability on both charged and uncharged ligands and parameters [24]. The search was made for all organisms’
molecules [9,15]. Several enzymes like protein kinase A sequences. A total of 250 hits were retrieved for claudin-1
(PKA), protein kinase C (PKC), protein phosphatase 2A with highest bits score and zero expected values. Out of 70
(PP2A), MAPK etc are involved during claudin retrieved sequences, seven were selected representing
phosphorylation [16-18]. Phosphorylation has dual effect major mammalian or vertebrate groups. The accession
on TJs functionality i.e. phosphorylation on some claudins numbers for eight selected sequences (Table 1) were
increased paracellular permeability or enhanced barrier O95832 (Human), Q6L708 (Bovine), D6RU0 (Sheep),
function [19]. It is reported that claudin-1 phosphorylation C3VMK8 (Pig), O88551 (Mouse), P56745 (Rat),
enhances its barrier functions while dephosphorylation Q5ZMG2 (Chick) and Q5FW44 (Xentr). ClustalW [25]
leads to detergent solubility and enhanced paracellular was used for multiple alignments of all the sequences of
permeability [20]. claudin-1 to get the conservation status.
O-glycosylation is also very important PTM of nuclear The claudin-1 sequence used in this study was
and cytoplasmic proteins. During O-glycosylation one “MANAGLQLLGFILAFLGWIGAIVSTALPQWRIYSY
molecule of N-acetylglucosamine (O-GlcNAc) is AGDNI
introduced on Ser or Thr residue by enzyme OGT (O- VTAQAMYEGLWMSCVSQSTGQIQCKVFDSLLNLS
GlcNAc transferase). Addition of O-b-GlcNAc can inhibit STLQATRALMVVGILLGVIAIFVATVGMKCMKCLE
phosphorylation on Ser or Thr residue. Interplay between D
O-b-GlcNAc modification and phosphorylation on the DEVQKMRMAVIGGAIFLLAGLAILVATAWYGNRIV
QEFYDPMTPVNARYEFGQALFTGWAAASLCLLGG
same amino acid residues has been observed in several
nuclear and cytoplasmic proteins [21]. These PTMs are ALLCCSCPRKTTSYPTPRPYPKPAPSSGKDYV”.
dynamic and result in temporary conformational changes
and regulate many functions of the proteins. The Post-translational modifications prediction methods We used
interchange of these two modifications on the same or more than one bioinformatics tools to access the post-
neighboring residue may modulate the specific function of translational modification on claudin-1 to get best results.
the proteins either by enhancing or inhibiting the functional Prediction of phosphorylation residues and related kinases
capacity. Residues where O-b-GlcNAc and Phosphorylation potential for human claudin-1 was
phosphorylation compete for each other are known as Yin predicted by using NetPhos 2.0 [26] and Disphos 1.3
Yang sites [22]. These Yin Yang sites can be predicted and server [27]. These are neural network-based programs that
analyzed using various computer-assisted neural network- predict the potential phosphorylation sites for each Thr,
based programs, which can help us to determine proteins Ser and Tyr residues. The minimum threshold value used
regulatory functions by accessing their modification to predict phosphorylation is 0.5 for NetPhos 2.0.
potentials. The present work describe potential Kinase specific phosphorylation sites in human claudin-
phosphorylation, O-glycosylation and their possible 1 were predicted by NetPhosK 1.0 [28] and KinasePhos 2.0
interplay sites which may influence claudin-1 interaction server [29]. These servers predict the kinase specific
with TJs and their possible effects on HCV entry and future acceptor substrates including Ser, Thr and Tyr.
therapeutics. For the evaluation of experimentally verified
phosphorylation sites on human claudin-1, Phospho.ELM
Materials and methods database was used [30]. This database contains a collection
