Tenascin-C: Form versus function

Abstract

Tenascin-C is a large, multimodular, extracellular matrix glycoprotein that exhibits a very restricted pattern of expression but an enormously diverse range of functions. Here, we discuss the importance of deciphering the expression pattern of, and effects mediated by, different forms of this molecule in order to fully understand tenascin-C biology. We focus on both post transcriptional and post translational events such as splicing, glycosylation, assembly into a 3D matrix and proteolytic cleavage, highlighting how these modifications are key to defining tenascin-C function.

Keywords: AD1/AD2, additional domain 1/ additional domain 2; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; ASMCs, aortic smooth muscle cells; BDNF, brain derived neurotrophic factor; BHKs, baby hamster kidney cells; BMP, bone morphogenetic protein; CA19–9, carbohydrate antigen 19–9; CALEB, chicken acidic leucine-rich EGF-like domain containing brain protein; CEA, carcinoembryonic antigen; CNS, central nervous system; CRC, colorectal carcinomas; CTGF, connective tissue growth factor; DCIS, ductal carcinoma in-situ; ECM, extracellular matrix; EDA-FN, extra domain A containing fibronectin; EDB-FN, extra domain B containing fibronectin; EGF-L, epidermal growth factor-like; EGF-R, epidermal growth factor receptor; ELISPOT, enzyme-linked immunospot assay; FBG, fibrinogen-like globe; FGF2, fibroblast growth factor 2; FGF4, fibroblast growth factor 4; FN, fibronectin; FNIII, fibronectin type III-like repeat; GMEM, glioma-mesenchymal extracellular matrix antigen; GPI, glycosylphosphatidylinositol; HB-EGF, heparin-binding EGF-like growth factor; HCEs, immortalized human corneal epithelial cell line; HGF, hepatocyte growth factor; HNK-1, human natural killer-1; HSPGs, heparan sulfate proteoglycans; HUVECs, human umbilical vein endothelial cells; ICC, immunocytochemistry; IF, immunofluorescence; IFNγ, interferon gamma; IGF, insulin-like growth factor; IGF-BP, insulin-like growth factor-binding protein; IHC, immunohistochemistry; IL, interleukin; ISH, in situ hybridization; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; MPNSTs, malignant peripheral nerve sheath tumors; Mr, molecular mass; NB, northern blot; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; NK, natural killer cells; NSCLC, non-small cell lung carcinoma; NSCs, neural stem cells; NT, neurotrophin; PAMPs, pathogen-associated molecular patterns; PDGF, platelet derived growth factor; PDGF-Rβ, platelet derived growth factor receptor β; PIGF, phosphatidylinositol-glycan biosynthesis class F protein; PLCγ, phospholipase-C gamma; PNS, peripheral nervous system; PTPRζ1, receptor-type tyrosine-protein phosphatase zeta; RA, rheumatoid arthritis; RCC, renal cell carcinoma; RD, rhabdomyosarcoma; RGD, arginylglycylaspartic acid; RT-PCR, real-time polymerase chain reaction; SB, Southern blot; SCC, squamous cell carcinoma; SMCs, smooth muscle cells; SVZ, sub-ventricular zone; TA, tenascin assembly domain; TGFβ, transforming growth factor β; TIMP, tissue inhibitor of metalloproteinases; TLR4, toll-like receptor 4; TNFα, tumor necrosis factor α; TSS, transcription start site; UBC, urothelial bladder cancer; UCC, urothelial cell carcinoma; VEGF, vascular endothelial growth factor; VSMCs, vascular smooth muscle cells; VZ, ventricular zone; WB, immunoblot/ western blot; bFGF, basic fibroblast growth factor; biosynthesis; c, charged; cancer; ccRCC, clear cell renal cell carcinoma; chRCC, chromophobe-primary renal cell carcinoma; development; glycosylation; mAb, monoclonal antibody; matrix assembly; mitogen-activated protein kinase, MAPK; pHo, extracellular pH; pRCC, papillary renal cell carcinoma; proteolytic cleavage; siRNA, small interfering RNA; splicing; tenascin-C; therapeutics; transcription.

Figure 1.

The exon structure of TNC with corresponding protein domains in tenascin-C. The human tenascin-C protein comprises 4 domains: a TA domain, 14.5 EGF-L repeats, up to 17 FNIII like repeats and an FBG domain. Eight of the FNIII repeats are constitutively expressed (FNIII 1–8 (gray), and 9 can be alternatively spliced (FNIIIA1-D (white). The TNC gene comprises 30 exons (1–28, plus AD1 and AD2). All exons are translated excluding the first. Exon 2 encodes the start sequence for translation of mRNA, and together exons 2 and 3 code for the signal peptide, the TA domain and all the EGF-L repeats. The 8 constitutively expressed FNIII repeats are coded for between exons 4–10 and 18–23, and the 9 alternatively spliced FNIII from exons 11–17. Each alternatively spliced FNIII repeat is encoded by its own exon, In contrast only the constitutive FNIII repeats 1 and 3 are encoded by a single exon; the remainder of the modules FNIII 2 and 4–8 are encoded by 2 exons each. Alternative splicing of FNIII domains within the tenascin-C pre-mRNA transcript means that the human TNC exon sequence varies in size from a maximum of 9154 bp to a minimum of 6251 bp. The FBG domain is coded for by exons 24–28.

Figure 2.

Schematic representation of human, rat, mouse and chicken tenascin-C. While each species contains 8 constitutively expressed FNIII repeats, the number and content of alternatively spliced FNIII repeats varies. Human tenascin-C contains 9 alternatively spliced FNIII repeats, rat 7, and mouse and chicken 6 each. Alternatively spliced repeats are typically more homologous than constitutive repeats. For example, constitutive mouse FNIII repeats share on average 44% nucleotide sequence identity to each other, in contrast to the alternatively spliced FNIII which share 52% identity. Of the mouse alternatively spliced FNIII, A2 and D share the lowest nucleotide identity at 41%, while A1 and A4 share 80%. Analysis of human tenascin-C also noted 80% amino acid sequence homology between the first 4 alternatively spliced modules (A1, A2, A3 and A4), in contrast to the other alternatively spliced FNIII repeats raising the possibility that these domains are the result of gene duplication of an ancestral FNIII module. The absence of any comparable homology between avian alternatively spliced FNIII repeats, allows for speculation that any such duplication occurred after the divergence of avian and mammalian lineages.

Figure 3.

Hexabrachion assembly is a 2-step process. Multimerization of the N-terminal region of tenascin-C during hexabrachion assembly. 1. The N-terminal region of 3 tenascin-C monomers. Black cylinders represent the N-terminal heptad repeat residues 118–145, and gray circles represent TA domains. 2. The N-terminal heptads contain 3 cysteine-rich heptad repeats with hydrophobic (h) and charged (c) amino acid residues arranged in the conformation hxxhcxc. These monomers form an intermediary trimer which is stabilized by α-helical coiled-coil interactions between the N-terminal domains of the monomers. Three. The oligomerization of the adjacent TA domains increases homophillic binding affinity between the 2 trimers, which bind to form the hexabrachion. Di-sulfide bonds stabilize the hexabrachion but are not required for its formation (Adapted from Kammerer et al.¹⁷⁰).

Figure 4.

Predicted N- and O-glycosylation sites within tenascin-C. Tenascin-C possesses 26 putative N- glycosylation sites, and 34 putative O-linked glycosylation sites; the locations of which are represented on the tenascin-C stick diagram by triangles and circles respectively.