The degradation of SPARC by proteases has been shown as a valid mechanism for the liberation of biologically active peptides from the parent sequence (for review, see Ref

The degradation of SPARC by proteases has been shown as a valid mechanism for the liberation of biologically active peptides from the parent sequence (for review, see Ref.32). (for review, see Ref.1). SPARC2(secreted protein, acidic and rich in cysteine), a prototypic matricellular protein, functions broadly in the assembly of extracellular matrix (ECM) and modulates cell cycle and cellular differentiation (for review, see Ref.2). However, less is known about its family member hevin, discovered in tonsillar tissue of adult humans (3). Initial work in murine models showed significant transcription of hevin mRNA in a diverse group of tissues, with brain consistently exhibiting the highest levels among hevin-positive tissues in unchallenged mice. Subsequent studies revealed hevin protein within the central nervous system, most notably the Purkinje cell-associated Bergmann glial cells, as well as astrocytes within the white matter of the cerebellum and throughout the murine brain (4,5). Recent work has shown increased expression of hevin in activated astrocytes during central nervous system stress and/or injury and in the excitatory synapses in induced-seizure models in rats (68). The shared homology and distribution of SPARC and hevin Rabbit Polyclonal to POU4F3 have lent credence to the concept of compensatory functions for these two matricellular proteins. Although the proteins are significantly different in size, the functional domains of CP-673451 SPARC are highly conserved within the C-terminal region of hevin. SPARC has been shown to be the target of several proteases that liberate specific domains, such as the follistatin and EF-hand motifs, or peptides within CP-673451 these domains, with functions different from those of the parent protein. Because these peptides are presentin vivo(911), it is not unexpected that proteolytic cleavage could act as a mechanism regulating the functions of SPARC and many other secreted, ECM-associated proteins (12). It is, therefore, likely that proteolysis of hevin would affect both its function and tissue distribution. ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs) cleaves the proteoglycans aggrecan and brevican (13). ADAMTS4 activity has been correlated with arthritis and the remodeling of cartilage, of which aggrecan is usually a major component, and with glioma in the brain, a tissue in which cleavage of brevican has been exhibited (1417). The deposition and breakdown of proteoglycans is CP-673451 usually important not only during neural development but also in central nervous system injury and neurodegenerative disease (for review, see Ref.18). Recent studies have revealed additional substrates for ADAMTS4 and have thereby broadened the potential role of this protease in the brain. Herein we demonstrate the cleavage of hevin by ADAMTS4in vivoandin vitro. This proteolysis liberates a hevin peptide, termed SPARC-like fragment (SLF), with significant homology to SPARC. Moreover, the cleavage is usually tissue-dependent, with predominance in the mouse brain. A monoclonal antibody was developed that selectively detects the ADAMTS4-derived peptide and differentiates it from full-length hevin as well as SPARC and its related peptides. The lack of hevin expression and hevin cleavage, as seen in hevin-null and ADAMTS4-null mice, respectively, results in altered Purkinje-cell morphology and a reactive gliosis phenotype in the brain of these adult animals. == EXPERIMENTAL PROCEDURES == == == == == == Antibodies == For immunoblotting and immunostaining procedures, the following antibodies (IgG) were used: polyclonal goat anti-hevin (specific for murine hevin) (R&D Systems, Minneapolis, MN), polyclonal rabbit anti-calbindin (Cell Signaling Technology, Danvers, MA), polyclonal goat anti-ADAMTS4 (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rabbit anti-glial fibrillary acidic protein (GFAP) (Lab Vision, Fremont, CA), monoclonal mouse anti-FLAG M2 (Sigma), normal IgG isotype controls (goat, rabbit, and mouse) (R&D Systems), monoclonal rat antibodies 12-155 and 12-54 against human and murine hevin, and a polyclonal rabbit antibody from our laboratory that was produced against murine hevin, affinity-purified against its immunogen, and cleared of SPARC-reactive species by a pass through SPARC-conjugated resin. == Cell Culture and Transfection == The expression vector for murine hevin with a C-terminal His6tag was kindly provided by Dr. Cagla Eroglu (Duke University, Durham, NC). The expression vector for human ADAMTS4 with a C-terminal FLAG epitope (19) was the nice gift of Dr. Duanqing Pei (University of Minnesota, Minneapolis, MN). HEK.