In the present study, we found 19 tetraspanins to be expressed in megakaryocytes and raised the first antibodies to the previously uncharacterized tetraspanin Tspan9, which was originally named NET-5 [27
]. Tspan9 protein expression was relatively specific to platelets when compared to other blood cell types. Moreover, biochemical analyses demonstrated that Tspan9 was a novel component of tetraspanin microdomains on the platelet surface.
This is the first characterization of the tetraspanin Tspan9 at the protein level through the generation of antibodies. Of the 33 tetraspanins in the human genome, antibodies have been reported for less than half. As such, we do not know the full complement of tetraspanins that are expressed in a given cell type and which other proteins are recruited to tetraspanin microdomains. The probable reasons for the lack of antibody reagents are firstly the relatively small size of tetraspanins and their association with larger proteins in tetraspanin microdomains [39
]. This may render them virtually inaccessible to antibody recognition in the context of intact cells, which are the predominant immunogens used to date for the generation of tetraspanin monoclonal antibodies. Secondly, tetraspanin protein sequences are highly conserved during evolution [40
], so the number of tetraspanin non-self epitopes is very low. However, we managed to overcome these problems by raising antibodies in rabbit and chicken to the intracellular C-terminal tail of human Tspan9. Although this approach was successful for Tspan9, it is not applicable to all tetraspanins, since some have cytoplasmic tails that are predicted to contain as few as four amino acids and are therefore too short to be used as immunogens. Moreover, we have failed to raise good antibodies to Tspan3, Tspan15, Tspan17, Tspan32 and Tspan33 using this method, although an antibody that recognized both Tspan13 and Tspan31 was successfully generated (M.B.P. and M.G.T., unpublished work).
In order to identify platelet tetraspanins in the absence of antibodies, we measured mRNA levels in megakaryocytes, the platelet progenitor cells, since platelets have extremely low levels of mRNA [10
]. To do this, we took advantage of a mouse megakaryocyte SAGE library that we had generated previously [11
], and more recent DNA microarray analyses of human megakaryocytes and seven other haematopoietic cell types [12
]. SAGE, which detects essentially all expressed genes in a quantitative manner, identified the four platelet tetraspanins, CD63, CD9, CD151 and Tspan32, amongst the six most highly expressed mouse megakaryocyte tetraspanins. We identified an additional 15 tetraspanins, at generally lower expression levels, most of which have not been reported on megakaryocytes or platelets [9
], but which nevertheless may have important roles in, for example, the regulation of G protein-coupled receptors that are expressed at relatively low levels. A total of 19 tetraspanins in megakaryocytes may be typical for other cell types. For example, a figure of 20 tetraspanins has been estimated for leukocytes [41
], and we have found 22 for endothelial cells upon analyses of 11 publicly-available SAGE libraries (J.M.J.H., R.B. and M.G.T., unpublished data). Unlike SAGE, the microarray data is not a reliable indicator of relative tetraspanin expression in megakaryocytes, due to the potential for differential hybridization efficiencies for different genes. However, the microarrays do enable a comparison of the expression levels of individual tetraspanins between different cell types. Those tetraspanins that have been reported to be expressed in human platelets using antibodies (CD9, CD63, CD151 and Tspan32) [9
], or by mass spectrometry peptide sequencing (Tspan9, Tspan15 and Tspan33) [11
], were relatively highly represented in human megakaryocytes. In contrast, a number of tetraspanins were not detected by either SAGE or DNA microarray, for example the CD9-related Tspan2 and the CD151-related Tspan11, suggesting that they are not expressed by the megakaryocyte/platelet lineage and thus do not compensate for CD9 and CD151 when these genes are knocked out in platelets. Finally, CD37, CD81 and Tspan4 were identified at the mRNA level in megakaryocytes but are absent at the protein level from human platelets (results not shown). It is possible that these tetraspanins are also not expressed at the protein level in megakaryocytes. Alternatively they may be megakaryocyte-expressed but actively excluded from platelets during platelet formation.
Genomic approaches, such as SAGE and DNA microarrays, do not prove expression at the protein level. However, using our new antibodies to Tspan9, we confirmed its expression on megakaryocytes and platelets by immunofluorescence microscopy, Western blotting and immunoprecipitation from surface-biotinylated cells. Consistent with its relative mRNA specificity for megakaryocytes within the haematopoietic lineage, Tspan9 was not readily detected in human B- and T-cell lines, or mouse thymus, and only weakly in spleen. Tspan9 was only detected in primary megakaryocytes and proplatelets, and not human megakaryocyte-like cell lines, highlighting that these cells are not good models for primary cells. Tspan9 is not entirely restricted to megakaryocytes and platelets, though, since expression was also detected in mouse lung, brain and kidney. In human platelets, we found that Tspan9 expression was tightly regulated between donors at a level of 6% of CD9, 43% of CD151 and three times the level of CD63 (this relatively low protein expression level for the latter in platelets contrasts with its high mRNA expression in megakaryocytes, for reasons that are currently unknown). Since we also showed that CD9 is the major platelet tetraspanin at 49000 cell surface copies per cell, we can estimate a Tspan9 copy number of approximately 2800 on the platelet surface, assuming that the plasma membrane to intracellular ratio is the same for each protein. Therefore it seems unlikely that the major platelet glycoproteins, such as integrin αIIbβ3 (80000 copies) [35
] or GPIb-IX-V (25000 copies) [36
], would be direct binding partners for Tspan9. We did detect 43 and 60 kDa proteins in relatively stringent co-immunoprecipitations with Tspan9, and we are working to identify these proteins using proteomics.
Immunoprecipitation of tetraspanins CD9, CD151 and Tspan9, and the CD151-associated integrin, α6β1, from surface biotinylated platelets revealed a distinct pattern of proteins in the relatively weak detergent Brij 97, in which tetraspanin microdomains are thought to remain largely intact [5
]. These proteins are likely to represent the major components of tetraspanin microdomains on the platelet cell surface. Using specific antibodies, we found that these tetraspanin-associated proteins do not include three of the major platelet glycoproteins, namely GPIb-IX-V, α2β1 or αIIbβ3. The latter result was somewhat surprising, since αIIbβ3 was reported to co-immunoprecipitate with CD151 in HEL cells lysed in the mild detergent CHAPS [42
], and with CD151 and Tspan32 in mouse platelets lysed in Triton X-100 [7
]. However, we did identify GPVI as a novel component of tetraspanin microdomains using co-immunoprecipitation experiments with CD9 and CD151 in Brij 97. GPVI interaction with CD9 and CD151 was lost in more stringent Triton X-100 detergent, which does not preserve tetraspanin–tetraspanin interactions and hence microdomain integrity [5
]. This suggests that either the GPVI-tetraspanin interaction is relatively weak or that GPVI interacts more strongly with an unidentified tetraspanin. If the latter is true, Tspan9 is unlikely to be the direct binding partner for GPVI because we could not detect Tspan9 in GPVI immunoprecipitates (results not shown). The functional consequences of GPVI-tetraspanin association are currently unknown. Interestingly, the antigen receptors on B- and T-lymphocytes, which signal via an ITAM (immunoreceptor tyrosine-based activation motif)-based mechanism in common with the GPVI/FcRγ complex [43
], are dependent on tetraspanins for their normal function [44
]. Particularly well characterized is the role of the tetraspanin CD81 on B-cells, where it interacts with the CD19 glycoprotein. CD19 is a signalling molecule which forms a complex with the complement receptor CD21, and is recruited to the B-cell receptor complex upon its engagement by complement-opsonized antigen. In the absence of CD81, the activated B-cell receptor fails to localize correctly to lipid raft microdomains and fails to induce sustained signalling [44
]. Moreover, CD19 biosynthetic maturation and trafficking to the plasma membrane are impaired [49
]. Our future studies will aim to determine whether a specific platelet tetraspanin can similarly regulate GPVI maturation, trafficking and/or signal transduction.
In summary, we have shown that Tspan9 is relatively highly expressed in the megakaryocyte/platelet lineage and is a component of tetraspanin microdomains on the platelet surface. Tspan9 is remarkably highly conserved during evolution, even relative to most other tetraspanins. A feature of conserved amino acids is their involvement in intra-molecular or inter-molecular interactions [50
]. This raises the possibility that almost the entire surface of Tspan9 is engaged in protein–protein interactions. Indeed, we hypothesize that Tspan9 has multiple simultaneous interacting partners and functions as an interaction ‘hub’ within tetraspanin microdomains. Tspan9 may therefore be essential for microdomain organization and for the function of microdomain-associated proteins on the platelet surface. The generation of Tspan9-deficient mice is in progress to test this hypothesis.