Mouse testican-2 was cloned, sequenced, and shown to be a proteoglycan with a multidomain structure closely similar to that of the human ortholog, previously described as a calcium binding extracellular matrix molecule of the BM-40/SPARC/osteonectin family (Vannahme, C., Schü bel, S., Herud, M., Gö sling, S., Hü lsmann, H., Paulsson, M., Hartmann, U., and Maurer, P. (1999). J. Neurochem. 73, 12-20). Recombinant mouse testican-2 was used to prepare specific antibodies that allowed the detection of testican-2 in various brain structures but also in lung, testis, and in several endocrine glands. Although the testican-2 expressed in EBNA-293 cells carried both heparan sulfate and chondroitin/dermatan sulfate glycosaminoglycan chains, the tissue form always contained only heparan sulfate. Both tissue-derived and recombinant testican-2 carried Nlinked glycans. Tissue-derived forms of testican-2 were detected as proteoglycans of varying size, whereas a portion of the molecules produced by EBNA-293 cells were core proteins, lacking glycosaminoglycans. Both the proteoglycan and core protein forms of testican-2 inhibited neurite extension from cultured primary cerebellar neurons and may play regulatory roles in the development of the central nervous system.

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. 1998]. Their modular structure is characterized by an N-terminal testican-specific domain followed by the follistatin-like (FS) and extracellular calciumbinding (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

Abstract:We have screened a human cDNA library using an expressed sequence tag related to the BM-40/secreted protein, acidic and rich in cysteine (SPARC)/osteonectin family of proteins and isolated a novel cDNA. It encodes a protein precursor of 424 amino acids that consists of a signal peptide, a follistatin-like domain, a Ca 2ϩ -binding domain, a thyroglobulin-like domain, and a C-terminal region with two putative glycosaminoglycan attachment sites. The protein is homologous to testican-1 and was termed testican-2. Testican-1 is a proteoglycan originally isolated from human seminal plasma that is also expressed in brain. Northern blot hybridization of testican-2 showed a 6.1-kb mRNA expressed mainly in CNS but also found in lung and testis. A widespread expression in multiple neuronal cell types in olfactory bulb, cerebral cortex, thalamus, hippocampus, cerebellum, and medulla was detected by in situ hybridization. A recombinant fragment consisting of the Ca 2ϩ -binding EF-hand domain and the thyroglobulin-like domain of testican-2 showed a reversible Ca 2ϩ -dependent conformational change in circular dichroism studies. Testican-1 and -2 form a novel Ca 2ϩ -binding proteoglycan family built of modular domains with the potential to participate in diverse steps of neurogenesis. Key Words: TesticanModular protein-Proteoglycan-Follistatin-CalciumCentral nervous system.

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