Occludin TM4(-): an isoform of the tight junction protein present in primates lacking the fourth transmembrane domain.
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 approximately 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.
Division of Cell Sciences, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK. Department of Biology, University of York, PO Box 373, York YO10 5YW, UK.
M Reza
MR
Ghassemifar
Bhavwanti
B
Sheth
Tom
T
Papenbrock
Henry J
HJ
Leese
Franchesca D
FD
Houghton
Tom P
TP
Fleming
D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D000906:Antibodies; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004285:Dogs; D004622:Embryo, Mammalian / Q000378:metabolism; D004727:Endothelium / Q000378:metabolism* / Q000648:ultrastructure; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D005091:Exons / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D062793:Occludin; D011323:Primates / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D019108:Tight Junctions / Q000378:metabolism*
D000906:Antibodies; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568986:Ocln protein, mouse; D020033:Protein Isoforms