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Amino acid residue Val362 plays a critical role in maintaining the structure of C terminus of connexin 50 and in lens epithelial-fiber differentiation.

ABSTRACT: We have previously shown that connexin (Cx) 50, unlike the other two lens connexins, Cx43 and Cx46, promotes chicken lens epithelial-fiber differentiation in a channel-independent manner. Here, we show that deletion of the PEST motif at the C terminus (CT) domain of Cx50 attenuates the stimulatory effect of Cx50 on lens fiber differentiation. Valine 362, a residue located within the PEST domain, is functionally involved. The structure of the Cx50 CT predicted by molecular modeling revealed four alpha-helices and Val(362) was found to be located in the middle of the 3rd helix. Replacement of Val(362) with amino acid residues that disrupt the alpha-helical structure predicted by molecular modeling, such as arginine, glutamate, or phenylalanine, attenuated the stimulatory effects of Cx50 on lens differentiation, whereas replacement with threonine, isoleucine, leucine, or proline, which maintain the structure preserved the function of Cx50. Circular dichroism (CD) studies supported the structural predictions and showed that the substitution with Glu, but not Thr or Pro, disrupted the alpha-helix, which appears to be the structural feature important for lens epithelial-fiber differentiation. Together, our results suggest that Val(362) is important for maintaining the helical structure and is crucial for the role of Cx50 in promoting lens epithelial-fiber differentiation.

SUBMITTER: Shi Q

PROVIDER: S-EPMC2881767 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Amino acid residue Val362 plays a critical role in maintaining the structure of C terminus of connexin 50 and in lens epithelial-fiber differentiation.

Shi Qian Q Banks Eric A EA Yu X Sean XS Gu Sumin S Lauer Janelle J Fields Gregg B GB Jiang Jean X JX

The Journal of biological chemistry 20100415 24

We have previously shown that connexin (Cx) 50, unlike the other two lens connexins, Cx43 and Cx46, promotes chicken lens epithelial-fiber differentiation in a channel-independent manner. Here, we show that deletion of the PEST motif at the C terminus (CT) domain of Cx50 attenuates the stimulatory effect of Cx50 on lens fiber differentiation. Valine 362, a residue located within the PEST domain, is functionally involved. The structure of the Cx50 CT predicted by molecular modeling revealed four ...[more]

PMID: 20395299

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Project description:Tembusu virus (TMUV) is a flavivirus responsible for panzootic outbreaks of severe egg-drop and fatal encephalitis of domestic waterfowl in China. Although TMUV can be attenuated by in vitro passaging, experimental evidence supporting the role of specific genetic changes in virulence attenuation is currently lacking. Here, we performed site-directed mutagenesis on five envelope (E) protein amino acid residues in accordance with the attenuated TMUV generated in our recent study. Our results showed that the Thr-to-Lys mutation of residue 367 in E protein (E367) plays a predominant role in viral cell adaptation and virulence attenuation in ducks compared with mutations in other residues. We further demonstrated that the positively charged basic amino acid substitution at E367 enhanced the viral binding affinity for glycosaminoglycans (GAGs) and reduced viremia levels and the efficiency of replication in major target organs in subcutaneously inoculated ducks. Interestingly, the T367K mutation increased viral neutralization sensitivity to the early immune sera. Together, our findings provide the first evidence that a basic amino acid substitution at E367 strongly impacts the in vitro and in vivo infection of TMUV.IMPORTANCE Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses in the production of domestic waterfowl since the virus was first recognized in China in 2010. To control TMUV infection, a live-attenuated vaccine candidate of TMUV was developed in our previous study, but the mechanisms of virulence attenuation are not fully understood. Here, we found that the Thr-to-Lys substitution at E367 is a crucial determinant of TMUV virulence attenuation in ducks. We demonstrated that the T367K mutation attenuates TMUV through reducing viral replication in the blood, brain, heart (ducklings), and ovaries. These data provide new insights into understanding the pathogenesis of TMUV and the rational development of novel TMUV vaccines.

Dataset Information

Amino acid residue Val362 plays a critical role in maintaining the structure of C terminus of connexin 50 and in lens epithelial-fiber differentiation.

Publications

Amino acid residue Val362 plays a critical role in maintaining the structure of C terminus of connexin 50 and in lens epithelial-fiber differentiation.

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