Occludin TM4(-): an isoform of the tight junction protein
present in primates lacking the fourth transmembrane domain
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 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.
occludin, tight junction, epithelium, isoform
alternative splicing, embryo
3171-3180
Ghassemifar, M.R.
219dba14-d37d-4062-b687-bcdcc6889045
Sheth, Bhavwanti
2ca6ed58-a992-47b7-b3a5-3c5df82aada7
Papenbrock, T.
ff77ce9c-910b-4e64-ac29-7a2cdcd52d48
Leese, H.J.
1f369c23-4361-4534-a093-54699ec5eceb
Houghton, Franchesca D.
53946041-127e-45a8-9edb-bf4b3c23005f
Fleming, T.P.
1431b2dc-b145-4bc2-aea4-bc360fc370e9
1 August 2002
Ghassemifar, M.R.
219dba14-d37d-4062-b687-bcdcc6889045
Sheth, Bhavwanti
2ca6ed58-a992-47b7-b3a5-3c5df82aada7
Papenbrock, T.
ff77ce9c-910b-4e64-ac29-7a2cdcd52d48
Leese, H.J.
1f369c23-4361-4534-a093-54699ec5eceb
Houghton, Franchesca D.
53946041-127e-45a8-9edb-bf4b3c23005f
Fleming, T.P.
1431b2dc-b145-4bc2-aea4-bc360fc370e9
Ghassemifar, M.R., Sheth, Bhavwanti, Papenbrock, T., Leese, H.J., Houghton, Franchesca D. and Fleming, T.P.
(2002)
Occludin TM4(-): an isoform of the tight junction protein
present in primates lacking the fourth transmembrane domain.
Journal of Cell Science, 115 (15), .
(doi:10.1242/jcs.115.15.3171).
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.
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Published date: 1 August 2002
Additional Information:
All the work for this paper took place within my laboratory and funded by my grants (Tom Fleming)
Keywords:
occludin, tight junction, epithelium, isoform
alternative splicing, embryo
Identifiers
Local EPrints ID: 24590
URI: http://eprints.soton.ac.uk/id/eprint/24590
ISSN: 0021-9533
PURE UUID: 781f7213-b873-4300-beb2-13428f1e7775
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Date deposited: 31 Mar 2006
Last modified: 16 Mar 2024 03:49
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Author:
M.R. Ghassemifar
Author:
T. Papenbrock
Author:
H.J. Leese
Author:
T.P. Fleming
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