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- Materials and methods
- Conflict of interest
- Supporting Information
The testicans are a three-member family of secreted proteoglycans structurally related to the BM-40/secreted protein acidic and rich in cystein (SPARC) osteonectin family of extracellular calcium-binding proteins. In vitro studies have indicated that testicans are involved in the regulation of extracellular protease cascades and in neuronal function. Here, we describe the biochemical characterization and tissue distribution of mouse testican-3 as well as the inactivation of the corresponding gene. The expression of testican-3 in adult mice is restricted to the brain, where it is located diffusely within the extracellular matrix, as well as associated with cells. Brain-derived testican-3 is a heparan sulphate proteoglycan. In cell culture, the core protein is detected in the supernatant and the extracellular matrix, whereas the proteoglycan form is restricted to the supernatant. This indicates possible interactions of the testican-3 core protein with components of the extracellular matrix which are blocked by addition of the glycosaminoglycan chains. Mice deficient in testican-3 are viable and fertile and do not show an obvious phenotype. This points to a functional redundancy among the different members of the testican family or between testican-3 and other brain heparan sulphate proteoglycans.
The testicans form a subgroup within the BM-40/SPARC/osteonectin family of modular extracellular proteins (Hartmann and Maurer 2001) and have also been referred to as the SPOCKs [secreted protein acidic and rich in cystein (SPARC)/Osteonectin CWCV and Kazal-like domains proteoglycan; Charbonnier et al. 1998]. Their modular structure is characterized by an N-terminal testican-specific domain followed by the follistatin-like (FS) and extracellular calcium-binding (EC) domains characteristic of the BM-40 family. Towards the C-terminus they contain a thyroglobulin-like domain (TY) and a novel sequence (domain V), which includes two potential glycosaminoglycan attachment sites.
Testican-1 was originally isolated as a proteolytic fragment from human seminal plasma carrying chondroitin sulphate and heparan sulphate chains (Bonnet et al. 1993). In the mouse, testican-1 is predominantly expressed in the nervous system during embryonic development and its expression correlates with periods of neuronal migration and axonal growth (Charbonnier et al. 2000). In adult mice, expression is restricted to the brain where it is located at post-synaptic densities (Bonnet et al. 1996). In search for homologous members of the BM-40 family, we identified cDNAs that were most similar to testican-1 and designated the corresponding proteins testican-2 and -3 (Vannahme et al. 1999; Hartmann and Maurer 2001).
In adult mice, testican-2 shows a broader tissue distribution than testican-1. Although the proteoglycan can be found primarily in the nervous system, it is also deposited in lung and especially in endocrine glands. In most organs, testican-2 is present throughout the extracellular space or localized at the cell surface, indicating that it may interact with membrane-bound molecules. (Schnepp et al. 2005). In contrast to testican-1, which carries both heparan and chondroitin sulphate chains, testican-2 is a pure heparan sulphate proteoglycan (Schnepp et al. 2005).
Functional information on testicans is still scarce, but there are clear indications that these may serve in the regulation of extracellular protease cascades. When expression cloning was used to identify regulators of pro-matrix metalloproteinase (MMP)-2 processing, mediated by membrane-type (MT) 1-MMP, a splice variant of testican-3, called N-Tes, was found (Nakada et al. 2001). Indeed, it was shown that testican-1 and testican-3 inhibit pro-MMP-2 activation by either MT1-MMP or MT3-MMP and that testican-2 abrogates the inhibition by the other family members (Nakada et al. 2003). Similarly, testican-1, but not testican-2, inhibits cathepsin L, (Bocock et al. 2003; Pavsic et al. 2008). Testican-1 was also found to inhibit attachment and neurite outgrowth in cultures of N2A neuroblastoma cells (Marr and Edgell 2003), While testican-2 is able to inhibit neurite outgrowth from primary cerebellar cells (Schnepp et al. 2005). Yet, inactivation of testican-1 in mice did not lead to any obvious phenotype (Röll et al. 2006).
In this study, we describe the biochemical characterization of mouse testican-3, its tissue distribution and the localization of the secreted protein in cell culture. Further, we describe the generation of mice deficient in testican-3 which, however, are viable and fertile and do not show any obvious morphological phenotype.
- Top of page
- Materials and methods
- Conflict of interest
- Supporting Information
We have identified and cloned the cDNA for murine testican-3. The mature protein shows the same domain organization as testican-1 and testican-2. An N-terminal domain unique for testicans is followed by paired follistatin-like (FS) and extracellular calcium-binding (EC) domains, a thyroglobulin-like (TY) domain and a C-terminal domain with two glycosaminoglycan attachment sites. The paired FS and EC domains are the signature of the BM-40/SPARC/osteonectin family. Testicans comprise the only members of that family which form proteoglycans as shown earlier for testican-1 (Bonnet et al. 1993) and -2 (Vannahme et al. 1999). The gene coding for testican-3 spans 406 kb and consists of 12 exons. The exon-intron structure with 12 exons is similar to that of Ticn-1 (Röll et al. 2006), whereas the Ticn-2 gene is composed of 11 exons (own unpublished data).
Exon 3 of Ticn-3 is a microexon coding for the three amino acids EVE, conserved among the testicans. We could earlier show that in Ticn-1 this exon is alternatively spliced and that transcripts with and without EVE are present in brain of adult mice (Röll et al. 2006). In the EST database 26 ESTs covering the region surrounding exon 3 of the murine and 100 ESTs covering that part of the human testican-3 gene are present. Although most of these (22/94) contain exon 3, some (4/6) lack the sequence, indicating that alternative splicing also occurs in testican-3 from both species. Microexons are present in a variety of eucaryotic genes and the preferred length is a multiple of three bp (Volfovsky et al. 2003). Examples include the gene for the neural cell adhesion protein NCAM where the insertion of a 30 bp exon is associated with a developmentally regulated loss of the stimulation of neurite outgrowth (Saffell et al. 1994).
The tissue distribution of testican-3 in mouse is similar to that of testican-1 (Bonnet et al. 1996) in that it is mainly restricted to the nervous system. In humans both proteoglycans are more widely expressed (Marr et al. 1997; NCBI Unigene EST profile viewer). After immunoprecipitation and subsequent western blot, testican-3 derived from brain extracts of newborn or adult mice was detected as a polydisperse band with an apparent molecular weight of 70–130 kDa. In contrast, in samples of recombinant testican-3 an additional band of an apparent molecular weight of about 60 kDa is present, representing the core protein. Treatment of brain-derived testican-3 with heparanase I and III led to a shift of the polydisperse band to the size of the core protein, whereas chontroitinase ABC had no effect. The results reveal that native testican-3 is a pure heparan sulphate proteoglycan. The value of about 60 kDa for the core protein is considerably higher than the 46790 Da calculated from the amino acid sequence of the recombinant protein. This may be because of irregular migration behaviour upon SDS-PAGE or post-translational modifications. For testican-2, we have already demonstrated the usage of an N-glycosylation site (Schnepp et al. 2005). For testican-3, neither N-glycosylation nor oligosaccharide O-glycosylation sites are predicted by the NetNGlyc 1.0 or NetOGlyc 3.1 (Julenius et al. 2005) software. We could confirm the lack of N-liked glycans in testican-3 by digestion with peptide N-glycosidase F. In contrast, despite the lack of predicted attachment sites, several mucin-type O-glycans could be demonstrated by mass spectrometry, which may account for the difference between the calculated and the apparent molecular mass.
On tissue sections a diffuse immunostaining for testican-3 was seen in some regions of the brain, indicating that testican-3 is a component of the brain extracellular matrix. Further, some but not all cells were stained for testican-3. The brain extracellular matrix, contributing 20% of the brain volume, is unique in its composition. It mainly consists of hyaluronan, chondroitin sulphate proteoglycans of the lectican family, tenascins and link proteins, whereas fibrous components like collagens or fibronectin are missing (reviewed in Rauch 2004). The components are secreted both by neurons and glia cells (Faissner et al. 2010) and are located either in the interstitial zones of the neuropil or in specialized structures of dense organized matrix, called perineuronal nets, which surround neurons and ensheath synapses (Celio et al. 1998). The diffuse immunostaining for testican-3 indicates that the proteoglycan is in part deposited dispersed in the neuropil. The more discrete cellular staining could indicate that it forms part of the perineuronal nets or that it binds to cells. We can also not exclude that the cellular staining reflects not yet secreted intracellular pools of testican-3. Testican-3 was mainly restricted to the grey matter, whereas in the main fibre tracts that contain glial but not neuronal cell somata, no testican-3 was detected.
Our results on the distribution of testican-3 protein are in agreement with situ hybridization data available from public resources.1 The ‘Allen Mouse Brain Atlas’ (http://mouse.brain-map.org/) contains in situ hybridization for testican-3 on adult brains. It shows a widespread expression by brain neurons. Further, a heat map representing the expression levels in different brain regions reflects our immunofluorescence results with the highest expression of testican-3 in the striatum and the thalamus and a lower expression in the cortex, the CA4 region of the hippocampus and the Purkinje cells of the cerebellum. Generally, low level of expression in agreement with our results at the protein level indicates that testican-3 is of low abundance. This is also reflected by that testican-3 cannot be detected by direct western blot analysis but requires prior concentration by immunoprecipitation. Similar in situ hybridization data are available from the ‘Brain Gene Expression Map’ (http://www.stjudebgem.org). This database also provides results from brains of younger mice (E11, E15, P7), revealing a higher expression in the CNS at earlier timepoints. This is also seen in western blots of immunoprecipitated testican-3 from mouse brains of different ages, with a more intense signal from brains of newborn mice (Fig. 4). The restriction to nervous tissues is also reflected in data from E14.5 mice in the ‘EMAGE gene expression database’ (http://www.emouseatlas.org/emage/).
In transfected HT1080 cells recombinant testican-3 was produced in two forms: a proteoglycan form and one representing the core protein. Analysis of the supernatant and extracellular matrix fractions of the cell cultures revealed that the core protein was present in both compartments, whereas the proteoglycan was restricted to the supernatant. This indicates that the glycosaminoglycan chains block the integration into the matrix. Related experiments showed that the integration of the human testican-3 core protein into the extracellular matrix is mediated by the N-terminal part of the protein. (Nakada et al. 2001).
Several members of the BM-40/SPARC/osteonectin family have been implicated in the establishment of neuronal connectivity. Testican-1 and testican-2 were shown to modulate attachment of neuronal cells and inhibit neurite outgrowth in vitro, respectively (Marr and Edgell 2003; Schnepp et al. 2005). SC1/hevin contributes to the correct neuronal positioning in the brain (Gongidi et al. 2004). Recently, it was shown that SPARC/BM-40/osteonectin regulates synaptic functions by preventing the maturation of cholinergic terminals (Albrecht et al. 2012). Yet, mice deficient in testican-1 or SC1 do not show an obvious phenotype (McKinnon et al. 2000; Röll et al. 2006). To elucidate the in vivo function of testican-3 we inactivated the gene in mouse. Testican-3 deficient mice developed normally were viable and fertile, and it did not show any obvious morphological or behavioural abnormalities. This may be explained by a functional redundancy within the BM-40/SPARC/osteonectin family. It can also not be excluded that the function of testican-3 is not because of the core protein but to the glycosaminoglycan chains. In that case also other brain heparan sulphate proteoglycans could account for a redundancy. Considering the pronounced homology between the three testicans and the functional similarities between testican-1 and testican-3, we expect that in vivo evidence for the role of these proteoglycans may be obtained through the generation of mice with double or triple deficiencies.