Investigating the Structure and function of Tetraspanin CD63

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  1. Tetraspanins

Tetraspanins are the small cell surface proteins (Hemler, 2005) that can be found over a great extent in all metazoans (Huang et al. 2005). Until today, scientists have discovered 33 members of tetraspanin superfamily expressed in humans and mice (Charrin et al. 2009). Tetraspanins are expressed in all human cell types and can be found in the exocytotic pathway, cell surface, endosomal system, secretory lysosomes (Pols and Klumperman, 2009) and they are enriched in exosomes (van Niel et al. 2006). The tetraspanin protein structure comprises of four transmembrane domains attached to a short extracellular loop (EC1), a very short intracellular loop, and a longer extracellular loop (EC2) (Hemler, 2005) (Figure 1.1). This protein structure is flanked by comparatively short N- and C-terminal cytoplasmic tails (Hemler, 2005). Except the second transmembrane domain, the other transmembrane domains of the most tetraspanins undergo palmitoylation to their membrane-proximal cysteine residue during post-translational modification (Hemler, 2005). The addition of palmitate enables the tetraspanins to associate with each other and various integral proteins, organizing an interacting network at the cell surface called “tetraspanin-enriched microdomains” (TEMs) (Rubinstein et al. 1996; Hemler, 2005; Charrin et al. 2009). Within TEMs, also known as “tetraspanin web”, tetraspanins act as molecular facilitator, interacting with integrins and other proteins to enable them mediating the cells in extracellular matrix (ECM) for adhesion, cell motility, invasion, fusion and signalling (Boucheix and Rubinstein, 2001; Hemler, 2003).

Figure 1.1: Tetraspanin structure. Tetraspanin protein structure consists of 200-350 amino acids. It comprises of four transmembrane protein domains attach to two different sizes of extracellular loops and one intracellular loop. The small extracellular loop, EC1, made up of 32-53 amino acids while the large extracellular loop, EC2 made up of 105-209 amino acids. This structure is flanked by short N-terminal and C-terminal cytoplasmic tails.

  1. Structure and function of CD63

Tetraspanin CD63 (also known as LAMP-3, LIMP-1, ME491 or Pltgp40) was the first characterized tetraspanin derived from the early stages of human melanoma tumour cells (Hotta et al. 1988). The presence of 6 cysteine residues in the EC2 and conserved polar residues in the transmembrane domains differentiate CD63 from the other tetraspanins (Hemler, 2003). Until now, CD63 protein is found to be expressed ubiquitously on all types of mammalian cell that have been studied (Pols and Klumperman, 2009). It is localized at the cell surface and due to the C-terminal lysosomal targeting motif, it present abundantly in lysosomal membranes and intraluminal vesicles (ILVs) of multi vesicular bodies (MVBs) within the endosomal system (Pols and Klumperman, 2009). Upon cell activation and secretion, fusion of MVBs with the plasma membrane will release the ILVs to the extracellular space which then termed as exosomes (Pols and Klumperman, 2009). Trafficking of intracellular CD63 to the cell surface has made the CD63 as an activation marker of several hematopoietic cells (Lettau et al. 2007) while surface exposure of CD63 on basophils function as degranulation and diagnostic marker in allergic diseases (Ebo et al. 2002; Sturm et al. 2004; Eborlein-Konig et al. 2006).

Like any other tetraspanins, CD63 interacts with diverse type of proteins including other type of tetraspanins, cell surface receptors, kinases and adaptor proteins (Pols and Klumperman, 2009). Within TEMs, CD63 was found to be interacting directly with integrins, syntenin-1 and membrane metalloproteases (Latysheva et al. 2006; Jung et al. 2008) thus it is believed that CD63 might regulate or be involved in cell adhesion, motility and phagocytosis (Yoshida et al. 2008). In vitro study in the 1990 found that culturing the myeloid cell lines with the human CD63 monoclonal antibody (mAb), anti-hCD63 (previously known as MAb 710F) increase the spreading of monocytic cells and make them adhere strongly to the serum-coated plastic (Koyama et al. 1990). However, the same antibody was found to impair the adhesion of neutrophilic granulocytes to the pre-treated endothelium (Toothill et al. 1990). Then again, rapid internalization of hCD63 along with enhanced motility among dendritic cells was observed using different anti-hCD63 (Manteggazza et al. 2004). On the other hand, mAb targeted against EC2 of CD63 (named D545) was found to alter the spreading of platelet (Israels and McMillan-Ward, 2005).

In vivo, CD63 knockout mouse showed a mild phenotype where there was altered water balance by the kidney and gastrointestinal tract, while the lamellar inclusions were abnormally accumulated in the principle cells of the collecting duct (Schröder et al. 2009). However, a strong phenotype displayed in the CD63 gene knockout zebrafish which the embryos treated with morpholinos targeting CD63 were failed to hatch (Trikić et al. 2011). This impairment might due to the deficient of secreted proteolytic enzymes in the hatching gland which are required for chorion-softening (Trikić et al. 2011). In addition, there was cellular morphology disorganisation of the hatching glands in this in vivo model at the cellular and intracellular levels (Trikić et al. 2011).

  1. CD63 in the FcᶓRI-mediated mast cell degranulation

The association of tetraspanin CD63 with the high affinity IgE receptor, Fc epsilon receptor I (FcᶓRI) on the mast cell or basophil has been proven in a number of studies (Kitani et al. 1991; Smith et al. 1995). To study the signalling via this receptor, the rat basophilic leukaemia cell line (RBL-2H3) is extensively used as in vitro model because of the similar characteristics share with the mast cells. A study by Kitani et al. (1991) has demonstrated that anti-rat CD63 mAb effectively inhibit IgE-mediated degranulation, however, in the other study, hCD63 antibodies are able to activate secretion in the CD63 transfected RBL-2H3 cells (Smith et al. 1995).

Antibodies against the tetraspanins including CD63 are capable in modulating the degranulation because the tetraspanins are thought to be interacting then regulating the signalling cluster which involve in cell adhesion and cell spread mechanism that enhance the IgE-mediated secretion in the RBL-2H3 cell line (Higginbottom et al. 2000). Later, this was confirmed by a study in 2005 which mAb directed against CD63 inhibits mast cell adhesion to fibronectin and vitronectin and inhibits the FcεRI-mediated degranulation (Kraft et al. 2005). Additionally, the inhibition of the degranulation only happened in the cells that adhere to the ECM proteins (Kraft et al. 2005). To support this, Gab2-PI3K-PKCδ signaling pathway that is known to be vital for degranulation as well as adhesion also found to be suppressed by anti-CD63 (Kraft et al. 2005).

In 2010, a novel granular variant of tetraspanin CD63 was found to be an exclusive marker of degranulated human mast cell (Schäfer et al. 2010). In vitro, the human mast cell was found to be underwent repeated cycles of FcεRI-mediated degranulation and interestingly, antibodies against this CD63 variant impaired these cycles (Schäfer et al. 2010). Thus, it is thought that this variant might involves in piecemeal degranulation, a process which sustains and aggravates allergic responses, because it is assumed that partly degranulated human mast cells in vivo can stay at the activation site and amplify subsequent response in the presence of another antigen (Schäfer et al. 2010).

Recently, in vivo study using CD63 knockout mouse model (MC-deficient Kitw/w-v) found that there was a significant decrease in FcεRI-mediated degranulation in the mast cell derived from the bone marrow of this knockout mouse (Kraft et al. 2013). Hence, this reflects that in the absence or suppression of CD63, the in vivo acute allergic reactions can be reduced and made the tetraspanin CD63 as a crucial component in the allergic inflammation (Kraft et al. 2013).

  1. Project background

A mutated form of CD63 which lacks N-linked glycosylation at amino acid 172 in the EC2 of the protein named CD63N172A (Figure 1.2) has been previously generated by the lab (Gorakh Mal, PhD Thesis, University of Sheffield, 2005).

Figure 1.2: Potential glycosylation sites of CD63. There are three possible glycosylation sites at the large extracellular loop, EC2. Variant form of tetraspanin CD63, CD63N172A which has been generated previously is lacks of N-linked glycosylation site at the amino acid 172.

We believe that this CD63 variant probably tally to the recently described variant form by Schäfer et al. 2010. CD63N172A displays unusual expression compared to the wild type of CD63 (CD63wt) (Figure 1.3 and 1.4). The preliminary data that have been obtained so far also suggested that the IgE-mediated degranulation response is altered in RBL-2H3 cells transfected with this mutated CD63. To provide the clear understanding of the role of CD63 in the degranulation response with relevance to allergy, this project aims to further compare the differences of IgE-mediated degranulation between CD63N172A and CD63wt. The surface expression of CD63N172A, CD63wt and FcᶓRI will also be monitored and the possible differences in the trafficking will be investigated. Finally, this project aims to study the associations of both, CD63N172A and CD63wt with the, FcᶓRI.



Figure 1.3 Preliminary data - cell surface immunofluorescence staining. Cell surface expression of CD63wt and CD63N172A viewed by fluorescence microscope. Due to the C-terminal lysosomal targeting motif, tetraspanin CD63 abundantly present in late endosomal compartment. However, mutation of N-linked glycosylation site at the amino acid 172 cause the protein to expressed more exclusively on the cell surface.

Figure 1.4 Preliminary data - intracellular immunofluorescence staining. Using the unpaired t-test, this preliminary data shows that there was significant different of intracellular expression between the wild-type of CD63 and the mutant form of CD63 (p=0.0001). CD63wt is widely expressed inside the cell while only a few per cents intracellular expression of CD63N172A.

  1. Hypothesis

N172A mutant form of CD63 is hypothesized to be corresponds functionally to a variant of this protein which occurs naturally. We also hypothesized that over expression of CD63N172A alters the mast cell degranulation response upon IgE cross-linking, perhaps by modifying the trafficking of the FcᶓRI.

  1. Plan of investigation

The RBL-2H3 cell line is used as a mast cell model in this project. Transfectants that stably express wild type CD63 and N172A mutant forms of hCD63 have already been generated by the lab previously (Gorakh Mal, PhD Thesis, University of Sheffield, 2005). IgE-mediated degranulation response will be assessed by β-hexosaminidase release assay using colorimetric technique. The addition of the IgE will stimulated the cells and different concentration DNP-HSA will be added to make the IgE cross-linking. The surface expression of CD63wt, CD63N172A and the FcᶓRI will be monitored using specific antibodies and flow cytometry (FACS) analysis. The possible differences between CD63wt and CD63N172A in FcᶓRI trafficking also will be investigated using specific antibodies and FACS analysis. To investigate the associations of the FcᶓRI with CD63wt and CD63N172A, “pull-down” experiments will be carried out by immunoblotting.