In this study, we have shown that MarvelD3, a four-pass transmembrane protein, co-localises with the tight junction protein occludin, apical to the adherens junction protein E-cadherin, indicating that MarvelD3 is a third tight junction-associated Marvel domain protein. A functional analysis using RNA interference-mediated depletion indicates that MarvelD3 is not essential for junction assembly and the formation of a functional paracellular diffusion barrier, but the observed increase in TER in depleted cells indicates that MarvelD3 is a determinant of paracellular ion permeability.
Our data demonstrate that MarvelD3 associates with the junctional complex of intestinal and corneal epithelial cells, and its expression was detected in multiple mouse tissues and cultured epithelial and endothelial cell lines. MarvelD3 thus seems to be a widely expressed protein and hence likely to be a component of tight junctions with different functional properties.
MarvelD3 is expressed as two isoforms and both were recruited to the cell periphery in transfected cells in a manner similar to the endogenous protein, suggesting that both isoforms associate with junctions. Our attempts to generate isoform-specific antibodies, which are required to study endogenously expressed isoforms at the protein level, have so far failed. Based on reverse transcription PCR results, however, both isoforms seem to be widely expressed. An aim of future studies will be to differentiate between MarvelD3 isoforms at the protein level and to determine whether there are any isoform-specific differences in expression, localisation and/or function, as has been suggested for occludin [46, 47].
Occludin and tricellulin not only localise to tight junctions, but have been shown to associate with the junctional intramembrane strands observed in freeze fracture replicas [27, 48]. It will thus be important to determine whether MarvelD3 also associates with these structures and whether the Marvel domain, which is found in all three proteins, is important for strand association . The Marvel domain is found in proteins such as MAL that are thought to associate with cholesterol-rich microdomains in cell membranes . As occludin has previously also been shown to associate with cholesterol-rich microdomains , it is possible that Marvel domain proteins affect tight junctions via such membrane microdomains. At present, however, the precise role of cholesterol in tight junctions is not clear [50–52].
Given the membrane morphology of the tight junction in which the outer leaflet of the plasma membrane becomes closely apposed to that of an adjacent cell, proteins with potential properties to influence close membrane/membrane contacts, such as Marvel domain proteins, may prove of significant interest in understanding the structure and function of tight junctions. Tricellulin has been suggested to play a structural role in tricellular corners, which would be compatible with a role in forming and/or stabilising membrane/membrane contacts [27, 39]. Surprisingly, however, the analysis of patients carrying tricellulin mutations has so far only revealed defects in the cochlea and not other organs . In the case of occludin, depletion and knockout experiments as well as expression studies with dominant mutations have thus far not suggested a role in tight junction structure [28, 34, 37, 53]. However, it has recently been reported that depletion of occludin results in a more even distribution of tricellulin along the entire junctional length . Hence, different Marvel domain proteins may be able to compensate for each other. With this in mind, it will be important to design approaches to study Marvel domain proteins as a whole and to elucidate a function for the Marvel domain itself.
Depletion of MarvelD3 in Caco-2 cells resulted in an increase in TER but did not affect permeability to fluorescent dextran tracers. Since occludin overexpression was previously observed to increase TER , we wondered whether the increased TER observed in the present study could be attributable to elevated levels of occludin expression. Immunoblotting cell lysates from MarvelD3 knockdown cells however showed occludin levels, as well as levels of ZO-1, ZO-2 and ZO-3, to be unchanged. Neither did we observe any significant changes in the distribution of these proteins by immunofluorescence. However, at this point it cannot be excluded that more subtle changes in occludin distribution might have caused the increase in TER. Occludin knockdown has been shown to reduce expression levels of claudins 1 and 7 and increase levels of claudins 3 and 4 in MDCK cells . Given the importance of claudin family members for paracellular ion permeability [12–14], it is possible that MarvelD3 depletion affected claudin expression or function; though we have not been able to detect differences in claudin-1 expression. However, it is possible that other claudins are affected or that claudin activity is regulated by MarvelD3.
MarvelD3 shows distant structural similarity to occludin. Occludin and tricellulin are located in tandem on human chromosome 5 and it has been speculated that they may have arisen from gene duplication during phylogenetic evolution . In contrast, MarvelD3 is located on chromosome 16 but is also expressed by all vertebrates, including birds, fish, amphibians and mammals. In contrast to occludin and tricellulin, all known isoforms and variants of MarvelD3 have short C-terminal cytoplasmic domains that are also distinct from each other. It is therefore unlikely that they are able to interact with ZO-1 as those of occludin and tricellulin do [38, 43]. In contrast, the N-terminal domain of MarvelD3, which is shared by both isoforms, is long in comparison to that of occludin. Intriguingly, the N-terminal domain of MarvelD3 is not as well conserved as the rest of the protein; however, it contains regions that are better conserved than others, which might be of functional relevance. In the case of occludin, this domain is thought to have a regulatory function and interacts with a ubiquitin ligase [32, 54]. As the N-terminal domains of tight junction-associated Marvel domain proteins are distinct in length and structure, they might provide structural links to different types of junctional regulatory mechanisms.