|Note|| C4.4A is a GPI-anchored, multi-domain member of the Ly6/uPAR/alpha-neurotoxin (LU) protein domain family. C4.4A was identified by two independent groups seeking to identify cancer-related genes, the first observing that C4.4A was expressed in a metastasizing rat pancreatic adenocarcinoma cell line, but not on its non-metastasizing counterpart (Matzku et al., 1989), and the second showing the upregulation of C4.4A in an in vitro model system for wound healing in the urothelium, mimicking the progression of urothelial cancer (Smith et al., 2001). These findings suggested a putative role of C4.4A in cancer invasion and metastasis.|
|Description|| C4.4A consists of 346 amino acid residues, including a 30 residues signal peptide at the N-terminal and a C-terminal signal sequence for GPI anchorage (38 residues) that are cleaved post-translationally, yielding a mature protein of 278 residues, anchored to the cell membrane via GPI (Figure 2A). It contains two LU domains (domains I and II), each of about 90 amino acids, and a serine-, threonine-, proline-rich (STP-rich) region. LU domains adopt a "three-fingered" folding topology, that is characterized by 4 consensus disulfide bonds and an invariant C-terminal asparagine (Figure 2B). Intriguingly, domain I of C4.4A lacks one consensus cysteine bond, which is crucial to the proper folding of the single domain LU proteins. The STP-rich region is highly O-glycosylated, with 17 potential O-glycosylation sites. None of the 6 potential N-glycosylation sites of C4.4A are, however, located in this region. Differential degrees of glycosylation can probably explain the large variation in molecular weight observed in C4.4A from different sources (Hansen et al., 2004), deviating from the theoretical value of 36 kDa.|
| || Figure 2: Protein structure of C4.4A.|
A - Structural representation of the two LU domains and the STP-rich region of C4.4A (modified from Hansen et al., 2004). Insert: Ribbon diagram of the three-finger fold of a single LU domain (made in PyMOL™(DeLano Scientific), using PDB coordinates 1NEA).
B - Disulfide connectivity in C4.4A, with LU consensus cysteine bonds highlighted in yellow.
|Expression|| C4.4A is expressed in the suprabasal cells of squamous epithelia found in e.g. esophagus and skin, the basal layer being devoid of C4.4A (Figure 3A), and in the amnion membrane in human term placenta (Figure 3C) (Hansen et al., 2004). In mouse skin wound healing, which is a tissue remodelling process often used as a surrogate model for cancer invasion, C4.4A is upregulated by the migrating keratinocytes. C4.4A expression is also increased in phorbolester-induced hyperplasia of murine skin (Hansen et al., 2004), in the progression to melanoma (Seiter et al., 2001) and in urothelial transitional cell carcinomas (Smith et al., 2001).|
The preferential expression of C4.4A in normal epithelia of the squamous type is paralleled in cancer, where it is expressed in the tumour component of primarily squamous cell carcinomas (SCC) and only to a lesser extent in adenocarcinomas (AC) (Wang et al., 2006), as demonstrated in non-small cell lung cancer (NSCLC) (Figure 3D) (Hansen et al., 2007).
In esophageal squamous cell carcinomas (ESCC), C4.4A expression as present in the normal mucosa is lost upon transition to dysplasia and carcinoma in situ, but reappears at the invasive front of the tumour and in lymph node metastases (Figure 3B) (Hansen et al., 2008).
| || Figure 3: Expression of C4.4A in normal and malignant human tissue.|
Staining of human tissue sections with a polyclonal rabbit anti-C4.4A antibody produced at the Finsen Laboratory (Copenhagen, Denmark). C4.4A-negative basal cells are indicated by an arrow in panels A and B. (A and B, reproduced from Hansen et al., 2008; C and D, from Jacobsen, unpublished).
|Localisation|| C4.4A is tethered to the cell membrane via a GPI-anchor, but can under certain conditions also be found intracellularly. A soluble fragment of C4.4A, termed C4.4A', resulting from cleavage in the protease-sensitive region between domain II and the STP-rich region, releasing the two N-terminal LU domains, has been described in esophageal tissue (Hansen et al., 2008).|
|Function|| Structural homology of C4.4A to the urokinase receptor, uPAR, is not reflected at the functional level, the function of C4.4A still being unknown. Circumstancial evidence, nevertheless, points to a role of C4.4A in the modulation of cell/cell and/or cell/matrix interactions:|
1) The carbohydrate-binding protein galectin-3, which has been reported to be involved in cell/cell interactions, cell adhesion, migration, invasion and metastasis, has been identified as a ligand for C4.4A (Paret et al., 2005).
2) C4.4A and the cell adhesion molecule E-cadherin are co-expressed in the normal esophageal mucosa, and both are down-regulated in the progression to dysplasia (Hansen et al., 2008).
3) C4.4A-positive and not C4.4A-negative tumour cells are capable of penetrating a matrigel, and this process can be inhibited by a monoclonal anti-C4.4A antibody (Rosel et al., 1998).
4) Encapsulation of lung metastases in rats, arising after an intrafootpad injection with pancreatic tumour cells, disappears, when these tumour cells are transfected with C4.4A (Rosel et al., 1998).
5) C4.4A has recently been reported to be a novel substrate for the extracellular matrix-degrading metalloproteases ADAM10 and ADAM17 (A Disintegrin And Metalloprotease domain), which have been implicated in cell migration and proliferation, with a bearing on tumour invasion and metastasis (Esselens et al., 2008).
|Homology|| C4.4A shows homology to uPAR and other multi-domain proteins of the Ly6/uPAR/alpha-neurotoxin protein domain family (PRV-1/CD177, TEX101, PRO4356, GPQH2552).|
| Developmentally regulated expression of metastasis-associated antigens in the rat.|
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| Cell Growth Differ. 1996 May; 7(5): 663-78.|
| Metastasis-associated C4.4A, a GPI-anchored protein cleaved by ADAM10 and ADAM17.|
| Esselens CW, Malapeira J, Colome N, Moss M, Canals F, Arribas J.|
| Biol Chem. 2008 Aug 8. [Epub ahead of print]|
| CEBPbeta, JunD and c-Jun contribute to the transcriptional activation of the metastasis-associated C4.4A gene.|
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| Ly6 family member C4.4A binds laminins 1 and 5, associates with galectin-3 and supports cell migration.|
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