ABSTRACT
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.
Subject(s)
Osteonectin/metabolism , Proteoglycans/genetics , Proteoglycans/metabolism , Amino Acid Sequence , Animals , Brain/anatomy & histology , Brain/metabolism , Cell Line, Tumor , Cloning, Molecular , Extracellular Matrix/metabolism , Fibrosarcoma/pathology , Gene Expression Regulation, Developmental/genetics , Humans , Immunoprecipitation , Mice , Mice, Inbred C57BL , Mice, Knockout , Proteoglycans/deficiency , TransfectionABSTRACT
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., Schübel, S., Herud, M., Gösling, S., Hülsmann, 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 N-linked 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.
