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

M. Reza Ghassemifar1,*,{ddagger}, Bhavwanti Sheth1,{ddagger}, Tom Papenbrock1, Henry J. Leese2, Franchesca D. Houghton2 and Tom P. Fleming1,§

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
{ddagger} These authors contributed equally to this work



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  		Fig. 1. Composite image of single-stage RT-PCR analysis of human tissue, embryo and cell line RNA using occludin primers shown in Table 1. Primers within the C-terminus (H5/H6) amplify a single product corresponding to the published occludin sequence, while primers flanking the TM4 and immediate proximal C-terminus region (H1/H2 and H3/H4) amplify two products, the upper corresponding to the published cDNA sequence while, in the lower band, the TM4 and immediate 3'-flanking sequence are deleted. B, embryo blastocyst stage; BE, bronchial epithelium; C, cervix; Ca, Caco-2 cells; HBE, 16HBE cells; HuV, HuVEC cells; K, kidney; M, marker lane; Mo, embryo morula stage; U, uterus.

 


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  		Fig. 2. (A) Schematic of human occludin mRNA coding sequence showing complete (TM4+) and TM4- isoforms based on RT-PCR sequence data. Black segments represent transmembrane domains and the shaded segment the proximal C-terminal domain deleted with TM4 in the TM4- isoform. (B) Nucleotide and deduced amino acid sequence of the region deleted in human and African Green Monkey (AGM) TM4- occludin isoform. AGM sequence identities with the human are represented by dots, while different residues are shown. Numbering of human nucleotide sequence refers to the published cDNA sequence (accession no. NM_002538.2). The entire TM4 domain is contained within the deleted region and is highlighted in bold.

 


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  		Fig. 3. Generation of occludin TM4- isoform by skipping of exon 4. (A) Aligment of human genomic occludin sequence with TM4+ and TM4- cDNAs obtained by RT-PCR. The region of interest from the genomic sequence of human occludin (AC010355.4) is shown in the top line. Central parts of introns are omitted as indicated by double slashes. Above the genomic sequence, the exon/intron structure is indicated in accordance with the annotation of pertinent genomic contig (accession no. NT_006497.6). Exons are labelled and their extent is marked by square brackets over their first and last nucleotide. Introns are labelled and their extent is marked by indicating conserved dinucleotides of splice donor (GT) and acceptor (AG) sites, respectively. Underneath the genomic sequence, the corresponding sequence of the longer (TM4+) RT-PCR product is shown. TM4+ product is 100% identical to published occludin cDNA sequence (NM_002538.2), and the numbering shown refers to that sequence. The third line shows the corresponding sequence of smaller RT-PCR product TM4-, which is 100% identical to TM4+ save for a 162 nucleotide deletion in TM4- compared with TM4+. Note that this deletion coincides precisely with the extent of exon 4. Below the alignment, translation of coding sequences is shown based on published cDNA sequence (NM_002538.2). Note that removal of exon 4 is not predicted to affect the reading frame of downstream coding region. (B) Schematic of proposed mechanism. 3' part of exon 3, exon 4, and 5' part of exon 5 of human occludin gene are represented by numbered boxes. Top: proposed alternative splicing event gives rise to TM4- mRNA isoform by skipping exon 4. Bottom: canonical splicing leads to inclusion of exon 4 in TM4+ mRNA isoform.

 


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  		Fig. 4. Composite image of RT-PCR analysis of occludin TM4- isoform expression in different species using primer sets shown in Table 1. Both TM4+ and TM4- isoforms are detectable in African Green Monkey (AGM) BSC1 epithelial cells but only the canonical TM4+ isoform is detectable in mouse tissues, cells and embryos, and in canine-derived MDCK cells. B, embryo blastocyst stage; BS, AGM BSC1 cells; CM, CMT64/61 cells; K, kidney; L, liver; M, marker lane; MK, MDCK cells; Mo, embryo morula stage.

 


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  		Fig. 5. Immunocytochemical analysis of occludin and ZO-1 in Caco-2 cells. (A,B) Brightfield and occludin labelling of subconfluent island after fixation showing typical TJ pattern of staining at cell borders. (C,D) Brightfield and ZO-1 staining of subconfluent island after fixation showing typical TJ pattern. (E-J) Brightfield and protein labelling of subconfluent islands immunolabelled prior to fixation. (E,F) Occludin labelling; note weak staining of the island periphery. (G,H) ZO-1 labelling showing absence of peripheral staining. (I,J) Negative control (secondary antibody only) showing absence of peripheral staining. (K,L) Confluent monolayer wounded to produce island and cultured for 30 minutes (K) or 1 hour (L) before labelling for occludin prior to fixation, showing weak staining around cells close to the wound edge (arrows). Bars, 20 µm.

 


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  		Fig. 6. (A) Occludin immunoblot of human Caco-2 cells, monkey BSC1 cells, canine MDCK cells, murine CMT64/61 cells and lung (mLung). In each case, a major band at 65-67 kDa is evident as well as other bands, as shown previously in occludin blots, including one at ~62 and ~58 kDa. The 58 kDa band (arrow) is where the TM4- isoform would be expected to migrate. (B) Equally loaded lanes of confluent (Con) and subconfluent (Sub) Caco-2 cells with the major (65-67 kDa) and lowest migrating (58 kDa) bands indicated by arrows. Densitometric analysis reveals equivalent expression of the major band in confluent and subconfluent cells but significant upregulation of the 58 kDa band in subconfluent cells (*P<0.001, n=12 replicates of each condition; experiment conducted three times).

 

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© The Company of Biologists Ltd 2002