Topological Structure of Lens Fibre Cell Membrane Protein MIP

Authors: Nihat DİLSİZ, Sait ÇELİK

Abstract: The topological structure of the MIP (major intrinsic protein) family was investigated. The MIP family is a group of transmenbrane proteins which are also known as aquaporins because of their role as water channels. To enable study of the topological structure of MIP, a heterologous-expression cell system using Escherichia coli was used as a tool for the production and assembly of MIP into the cytoplasmic membrane. A set of specific antibodies was used to establish the topological model of lens MIP. Antipeptide antisera were prepared for four hydrophilic regions of rat MIP according to its predicted residues (1). Monoclonal Anti-Flag M2 antibody was used against the PCR based FLAG peptide fused to either the N- or C-terminal end of MIP. Two peptides, HNPA l (Ser-Gly-Ala-His-Val-Asn-Pro-Ala-Val-Thr) and NPAR 2 (Thr-Gly-Ala-Gly-Met-Asn-Pro-Ala-Arg-Ser), were synthesised according to NPA boxes HNPA1 amino acids 61-70 represent a portion of the second cytoplasmic loop of rat MIP while NPAR 2 amino acids 178-187 represent a portion of the fifth extracellular loop of MIP containing a predominance of polar and charged amino acids. The data presented in this study are not consistent with all aspects of the previously proposed model. Based on the data in this work, and previously reported information regarding MIP, a new topological model is proposed for the assembly of MIP in the lens fibre cel membrane. This may provide a revised model for investigating the role of MIP in the formation of cararacts with age and diabetes.

Keywords: Lens MIP protein, topology, NPA boxes, spheroplast, immunofluorescence.