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Occludin TM4^-: an isoform of the tight junction protein present in primates lacking the fourth transmembrane domain

M. Reza Ghassemifar^1,*,, Bhavwanti Sheth^1,, Tom Papenbrock¹, Henry J. Leese², Franchesca D. Houghton² and Tom P. Fleming^1,

¹ Division of Cell Sciences, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK
² Department of Biology, University of York, PO Box 373, York YO10 5YW, UK
^* Present address: Division for Infection, Inflammation and Repair, School of Medicine, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK
These authors contributed equally to this work

Author for correspondence (e-mail: tpf{at}soton.ac.uk )

Accepted 6 May 2002

The tight junction protein occludin possesses four transmembrane domains, two extracellular loops, and cytoplasmic N- and C-termini. Reverse transcription-PCR analysis of human tissues, embryos and cells using primers spanning the fourth transmembrane domain (TM4) and adjacent C-terminal region revealed two products. The larger and predominant product corresponded in sequence to canonical occludin (TM4⁺), while the smaller product exhibited a 162 bp deletion encoding the entire TM4 and immediate C-terminal flanking region (TM4^-). Examination of the genomic occludin sequence identified that the 162 bp sequence deleted in TM4^- coincided precisely with occludin exon 4, strongly suggesting that TM4^- is an alternative splice isoform generated by skipping of exon 4. Indeed, the reading frame of downstream exons is not affected by exclusion of exon 4. The presence of both TM4⁺ and TM4^- occludin isoforms was also identified in monkey epithelial cells but TM4^- was undetected in murine and canine tissue and cells, indicating a late evolutionary origin for this alternative splicing event. Conceptual translation of TM4^- isoform predicts extracellular localisation of the C-terminus. Immunocytochemical processing of living human Caco-2 cells using a C-terminal occludin antibody revealed weak, discontinuous staining restricted to the periphery of subconfluent islands of cells, or islands generated by wounding confluent layers. In occludin immunoblots, a weak band at 58 kDa, smaller than the predominant band at 65 kDa and corresponding to the predicted mass of TM4^- isoform, is evident and upregulated in subconfluent cells. These data suggest that the TM4^- isoform may be translated at low levels in specific conditions and may contribute to regulation of occludin function.

Key words: Occludin, Tight junction, Epithelium, Isoform, Alternative splicing, Embryo

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