CORDIS Archive : Biotechnology / Contract Nr : BIO4CT965122

REGULATION OF POST-TRANSLATIONAL POLYMERIZATION AND SECRETION OF MUCINS IN CELL LINES

Host Laboratory	Scientific Supervisor
University of Goteborg Department of Medical Biochemistry - Faculty of Medecine MEDICINAREGATAN 9 413 90 Goteborg Sweden	Mr. Gunnar C. Hasson Tel : 46/31 773 34 88 / Fax : 46/31 41 61 08 Email : GUNNAR.HASSON@MEDKEM.GU.SE
	Grant Holder
	Ms. Daniella Steel (British) Tel : 46 31 773 34 61 / Fax : 46 31 426 108
Abstract The major mucins giving the mucus its gel-like properties are large and polymeric in nature. The protein core of mucins is assembled into dimers in the endoplasmic reticulum, before being O-glycosylated in the Golgi. In the secretory vesicles the glycosylated dimers are forming oligo- and polymers, probably by linking two N-termini to each other. Recently it has been shown in Goteborg that this process is an intrinsic property of both MUC2 and MUC5AC mucins and that mucin polymerisation could be modified by altering the pH or calcium ion concentration. The internal milieu of secretory vesicles can probably also affect the glycosylation and thus properties of mucins. Project. To study the cellular assembly and glycosylation of mucins in relation to the internal milieu of the secretory vesicles of cells. The group in Goteborg has an international strong position in the studies of the structure, assembly and glycosylation of mucins. The applicant project is going to study the regulation of pH and ion concentration (especially calcium) in secretory vesicles of mucin secreting cells, the role of CFTR chloride channels, effect of different drugs, and possible signal transduction pathways regulating this. Recently equipment for video monitoring and measuring cellular alterations of pH and calcium ion concentration using intracellular probes has been purchased to Goteborg, techniques that the applicant has trained. A primary goal is to study the effect presence/absence of normal and AF508CFTR on calcium concentrations (using the fura 2 probe) in the secretory vesicles, as a control the effect on pH will also be studied. Several types of cell lines naturally expressing different types of mucins are available together with cells with different types of defects in the cystic fibrosis chloride channel and with and without the controlled secretory pathway. The turnover, assembly and regulation of mucin secretion will be studied using different drugs affecting vesicle pH and secretion in the available cell lines, including the recent discovered potent secretagouge ATP. Endogenous signalling pathways that alter the vesicle milieu will also be studied as well as the role of inflammatory mediators These processes will also be studied in cell lines with transfected miniMUC2 mucins. The applicant will also construct expression vectors encoding miniMUC2 mucins fused with a fluorescent peptide (GFP) (Heim and Tsien, 1996). This peptide is very suitable for the study of proteins secreted via the regulated pathway as it need some time to mature and become fluorescent. This approach will greatly facilitate the above described studies. Contract number : BIO4CT965122 Duration : 24 Months from 01-12-96 -- Category 30 (B30)

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