A Comparative Study of Water Permeability Between Aquaporin 5 Wild Type and Mutant Proteins Causing Palmoplantar Keratoderma

Abstract

Multiple missense mutations in the tetrameric transmembrane protein human aquaporin 5 (HsAQP5), which is a member of the water conducting aquaporin superfamily, is reported to cause nonepidermolytic palmoplantar keratoderma (NEPPK), which is a diffuse and autosomal-dominant skin disease. A white appearance and spongy texture of the skin arise after exposure to water which is thought to be caused by the gain-of-function of the different AQP5 mutants. However, this has not been shown for the isolated protein and there is no deduced molecular mechanism of the gain-of-function. In this report, the increased water permeability of a AQP5 mutation, Tyr178Cys, was either confirmed or confuted compared to the wild type variant of AQP5. This was also thought to be performed on another AQP5 mutation, Trp35Ser, but was not accomplished due to lack of time. Furthermore, several molecular mechanisms of the supposed gain-of-functions of these two mutants were proposed. This was done by stripping membranes from Pichia pastoris cells and isolating tetrameric AQP5 by immobilised metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Stopped-flow measurements was then performed on proteoliposomes of the wild type and the Tyr178Cys mutant, as well as control liposomes without protein. In parallel crystallisation trials were set up with the isolated wild type AQP5. The wild type variant obtained a rate constant difference between its proteoliposomes and control liposomes of 3.74 and the Tyr178Cys mutant obtained a difference of 4.28 and an unpaired t-test comparing these two differences gave a P-value of less than 0.0001. This means that the Tyr178Cys mutant has a statistically highly significant increased water permeability in comparison to the wild type, which is confirming a gain-of-function in the mutant. No AQP5 crystals were obtained, so molecular mechanisms were proposed based on an existing AQP5 wild type crystal structure, in which loss of existing and newly formed interactions caused by the two mutants are discussed under the assumption that the Trp35Ser mutant also has an increased water permeability compared to the wild type.

Popular Abstract

In the human body, there is a certain type of a protein family called the aquaporins (AQPs). The function of the aquaporins is that they transport predominantly water across the cell membrane. In a member of this protein family, aquaporin 5 (AQP5), it has been found that certain single amino acids have been replaced by other amino acids which has been reported to contribute to a skin disease. These altered proteins are called mutant proteins. Individuals that have this disease is developing a white appearance and spongy texture of the skin after exposure to water. Studies have suggested that these altered AQP5 proteins are channelling more water across the cell membrane in those affected individuals in comparison to healthy individuals that do not have these altered AQP5 proteins. Researchers are not quite sure of how the structure of the AQP5 protein is altered in individuals having this disease. This work is relevant because knowledge about how the mutant protein is altered in structure and function would provide a better understanding of how the permeability of AQP5 is regulated and could also be valuable information for production of a drug that would possibly cure this disease.
The aim of this study is to confirm or reject higher water transport ability of two AQP5 mutants in comparison to the non-mutated AQP5. One mutant is changed on position 178 in the amino acid sequence where a tyrosine residue has been replaced by a cysteine residue and the other mutant deviates on position 35 where a tryptophan residue has been replaced by a serine residue. Proposals are also going to be made for how the structure of the mutant proteins may have been altered. This is done by performing measurements of how high the water transport ability is of the AQP5 mutant on position 178 and the non-mutated AQP5. These three protein variants are going to be extracted from yeast cells, in which the protein has been produced, by isolating their cell membranes and then isolating the different AQP5 variants by chromatography methods based on binding properties and size. It turned out that the mutant altered at position 178 has a higher water transport ability in comparison to the non-mutated AQP5 which is confirming that this mutant would cause the skin disease. No measurements were performed on the mutant changed at position 35 due to lack of time. Proposals of how the structure of the protein may have been altered on a molecular level of the two mutants were also made in which old and new interactions between amino acids are discussed.

• author: Gripvall, Simon
• supervisor: Urban Johanson
• organization: Department of Chemistry
• course: KEML10 20211
• year: 2021
• type: M2 - Bachelor Degree
• subject: Chemistry
• keywords: aquaporin 5 mutants, biochemistry, molecular mechanisms, skin disease, stopped-flow, transmembrane protein
• language: English
• id: 9049078
• date added to LUP: 2021-06-16 10:23:42
• date last changed: 2021-06-16 10:23:42