Expression and functional studies of the wild type and C-terminus truncated rat aquaporin 5 (AQP5)‎ water channel proteins

مقدم أطروحة جامعية

Bouchoutrouch, Nadiyah

مشرف أطروحة جامعية

Delporte, Christine
Barradah, Fuad
Perret, Jason

أعضاء اللجنة

Sindidi, Khalid Sadaqa
al-Asli, Abd al-Ghani
Fechtali, Tawfiq

الجامعة

جامعة الأخوين

الكلية

كلية الهندسة و العلوم

القسم الأكاديمي

التكنولوجيا الحيوية

دولة الجامعة

المغرب

الدرجة العلمية

ماجستير

تاريخ الدرجة العلمية

2010

الملخص الإنجليزي

Aquaporins are integral membrane channel proteins responsible for water movement across biological membranes; where they play an important role in maintaining homeostasis.

AQP5 is an apical membrane water channel that is highly expressed in the cell membrane of numerous secretory glands, such as salivary and lachrymal glands.

Several pathologic situations have been associated with a dysfunction in AQP5 proteins like Sjögren's Syndrome.

In fact, patients suffering from this disease manifest a decrease and an abnormal distribution of these aquaporins.

A better understanding of the mechanisms behind the regulation of AQP5 channel is essential in order to elucidate Sjögren's syndrome and other potentially associated diseases.

The purpose of the present work is to set up an immunofluorescence technique in order to determine the localization of the aquaporin5 proteins expressed from different molecular constructs in MDCK transfected cells (Madin-Darby Canine Kidney) in different conditions; and set up a functional assay based on the flux measurement of water through membranes.

Another goal is to determine the function of the wild type AQP5 proteins and a naturally occurring mutated variant, as well as N-terminus tagged and C-terminus truncated forms of AQP5 proteins in a Xenopus oocytes swelling test.

Finally, generate stable transfected MDCK cells with an expression vector containing one of the following recombinant genes: the N-terminus fused tag (HA and VSV) to AQP5, and the C-terminus truncated forms of AQP5 proteins.

Confocal microscopy analysis revealed that the transfected MDCK-rnAQP5koz expressed the protein at both the plasma membrane and the cytoplasm.

Following a treatment with indomethacin (10-4M) which induces a decrease of cAMP levels in the cells, the ratio of AQP5 expression in the membrane fraction versus the one in the cytoplasmic fraction seemed to be increased.

In addition, a treatment with forskolin (10-5M), a cAMP activator, revealed that the expression of the AQP5 proteins in the cytoplasm was higher than in the membranes.

Also the localization of the AQP5 proteins in transfected MDCK-rnHA-AQP5 was not affected, while it was altered in transfected MDCK- rnHA-AQP5 T1 and T2 cells.

Using the Xenopus swelling assay, we were able to determine the water permeability of the following aquaporins: the AQP5 koz, AQP5 HA, AQP5 VSV, AQP5 M0, AQP5 T1, AQP5T2, AQP5T3, and AQP5T4.

We observed that, AQP5 koz, AQP5 HA, AQP5 VSV, AQP5 M0, AQP5 T1, AQP5 T3, AQP T4 proteins showed good water permeability, while AQP5T2 proteins seemed to exhibit a higher water permeability.

Different stably transfected MDCK cells, consisting of the HA-AQP5, VSV- AQP5, AQP5T1, AQP5T2, AQP5T3, AQPT4, HA-AQP5 T1, HA- AQP5 T2, HA-AQP5 T3, and HA-AQP5 T4, were generated and tested for the expression of the AQP5 proteins.

RT-PCR and Western blot analysis revealed that our MDCK transfected cells stably expressed AQP5 proteins.

The results of the first part of the present work allowed us to show the localization of AQP5koz wild type, HA-AQP5, HA-AQP5 T1, and HA-AQP5 T2 proteins in MDCK cells.

The preliminary results with indomethacin and forskolin suggest that the fisrt one induces a plama membrane localization of the AQP5 proteins, and that the second one leads to a cytoplasmic localization of AQP5 proteins.

.

We were also able to measure functional differences in terms of water permeability between our different aquaporins proteins.

التخصصات الرئيسية

الأحياء
تكنولوجيا المعلومات وعلم الحاسوب

الموضوعات

• التكنولوجيا الحيوية
• البروتينات
• الأغشية الحيوية

عدد الصفحات

50

قائمة المحتويات

• APA
• MLA
• AMA

لغة النص

الإنجليزية

نوع البيانات

رسائل جامعية

رقم السجل

BIM-644619