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-β-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-β-GlcNAc and phosphorylation interplay at same Ser/Thr residues. We for the first time proposed that alternate phosphorylation and O-β-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.

Figure 1

Multiple alignments of seven vertebrates sequences (Human, Bovine, Sheep, Pig, Mouse, Rat, Chick and Xentr). These sequences were ordered as in aligned results from ClustalW. The consensus sequence is marked by an asterisk, conserved substitution by a double dot, and semiconserved substitution by a single dot.

Figure 2

Graphic representation of the potential Ser, Thr, and Tyr residues for phosphorylation and O-glycosylation modification for human claudin-1. A) Predicted potential sites for phosphate modification on Ser and Thr residues. The light gray horizontal line indicates the threshold for modification potential. The blue, green and red vertical lines showed the potential phosphorylated Ser, Thr and Tyr residues, respectively. B) Predicted potential sites for O-glycosylation modification of Ser and Thr. O-β-GlcNAc modification potential of Ser/Thr residues is shown by green vertical line, while the light blue wavy line indicates the threshold for modification potential. C) The Yin Yang sites that were positively predicted are shown with red asterisk at the top, while the NP-Yin Yang site are shown with purple asterisk on the top of vertical lines. The green vertical lines show the O-β-GlcNAc potential of Ser/Thr residue and the light blue horizontal wavy line indicates the threshold for modification potential.

Figure 3

A homology model of human claudin-1 utilizing automated protein modeling option was retrieved through I-TASSER server. Through this option five models were received from the server utilizing five different templates namely: model 1-5. Among the five, one that covered all amino acids with alpha helix structure and beta pleated sheet, and high C-value was selected. This model showed that predicted Yin Yang sites have high surface accessibility for the phosphorylation and O-glycosylation interplay. The Ser and Thr residues are denoted by red and green colors.