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

Abstract

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.