Merck

Claudin-2 pore function requires an intramolecular disulfide bond between two conserved extracellular cysteines.

American journal of physiology. Cell physiology (2013-05-17)
Jiahua Li, Susanne Angelow, Anna Linge, Min Zhuo, Alan S L Yu
RESUMEN

Claudins constitute a family of tight junction transmembrane proteins whose first extracellular loop (ECL1) determines the paracellular permeability and ion selectivity in epithelia. There are two cysteines in the ECL1 that are conserved among all claudins. We hypothesized that these extracellular cysteines are linked by an intramolecular disulfide bond that is necessary for correct pore folding and function. To test this, we mutated C54 and C64 in claudin-2, either individually or together to alanine or serine, and generated stable Madin-Darby canine kidney (MDCK) I Tet-off cell lines. Immunoblotting showed a higher molecular mass band in the mutants with a single cysteine mutation, consistent with a claudin-2 dimer, suggesting that the two conserved cysteines normally form an intramolecular disulfide bond in wild-type claudin-2. By immunofluorescent staining, the alanine mutants were mislocalized intracellularly, while the serine mutants were expressed at the tight junction. Thus dimerization of both C54A and C64A did not require tight junction expression, suggesting that C54 and C64 are located near an intermolecular interface involved in cis-interaction. The conductance and Na(+) permeability of the serine mutants were markedly lower than the wild type, but there was no difference between the single mutants and the double mutant. We conclude that the disulfide bond between the conserved extracellular cysteines in claudin-2 is necessary for pore formation, probably by stabilizing the ECL1 fold, but is not required for correct protein trafficking. We further speculate that this role is generalizable to other claudin family members.

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
L-Cysteine, ≥97%, FG
Sigma-Aldrich
L-Cysteine, 97%
SAFC
L-Cysteine
Sigma-Aldrich
L-Cysteine, BioUltra, ≥98.5% (RT)
Sigma-Aldrich
L-Cysteine, from non-animal source, BioReagent, suitable for cell culture, ≥98%
Supelco
L-Cysteine, certified reference material, TraceCERT®
Sigma-Aldrich
L-Cysteine, produced by Wacker Chemie AG, Burghausen, Germany, ≥98.0%
Sigma-Aldrich
L-Cysteine hydrochloride monohydrate, reagent grade, ≥98% (TLC)
Sigma-Aldrich
L-Cysteine hydrochloride monohydrate, BioUltra, ≥99.0% (RT)
Sigma-Aldrich
L-Cysteine hydrochloride monohydrate, from non-animal source, suitable for cell culture, meets EP, USP testing specifications
Sigma-Aldrich
L-Cysteine hydrochloride monohydrate, Produced by Wacker Chemie AG, Burghausen, Germany, Life Science, 98.5-101.0%
Supelco
L-Cysteine hydrochloride monohydrate, Pharmaceutical Secondary Standard; Certified Reference Material
Cysteine hydrochloride monohydrate, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
L-Cysteine hydrochloride monohydrate, FG, produced by Wacker Chemie AG, Burghausen, Germany, Food, 20 mesh