We have isolated the novel gene SMOC-2, which encodes a secreted modular protein containing an EF-hand calcium-binding domain homologous to that in BM-40. It further consists of two thyroglobulin-like domains, a follistatin-like domain and a novel domain found only in the homologous SMOC-1. Phylogenetic analysis of the calcium-binding domain sequences showed that SMOC-1 and -2 form a separate group within the BM-40 family. The human and mouse SMOC-2 sequences are coded for by genes consisting of 13 exons located on chromosomes 6 and 17, respectively. Analysis of recombinantly expressed protein showed that SMOC-2 is a glycoprotein with a calcium-dependent conformation. Results from Northern blots and reverse transcription PCR revealed a widespread expression in many tissues.
We have isolated the novel gene SMOC-1 that encodes a secreted modular protein containing an EF-hand calcium-binding domain homologous to that in BM-40. It further consists of two thyroglobulin-like domains, a follistatin-like domain and a novel domain. Recombinant expression in human cells showed that SMOC-1 is a glycoprotein with a calcium-dependent conformation. Results from Northern blots, reverse transcriptase-PCR, and immunoblots revealed a widespread expression in many tissues. Immunofluorescence studies with an antiserum directed against recombinant human SMOC-1 demonstrated a basement membrane localization of the protein and additionally its presence in other extracellular matrices. Immunogold electron microscopy confirmed the localization of SMOC-1 within basement membranes in kidney and skeletal muscle as well as its expression in the zona pellucida surrounding the oocyte.
The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its gly- Collagen IX is a heterotrimer of ␣1(IX), ␣2(IX), and ␣3(IX) polypeptide chains that fold into the triple helix characteristic of the members of the collagen family of extracellular matrix proteins (1). This helix consists in the case of collagen IX of COL1, COL2, and COL3 domains, numbered from the carboxyl terminus, which are flanked by short noncollagenous segments, domains NC1-NC4. The domain NC4 is formed by the 245 extreme N-terminal amino acid residues of the ␣1(IX) chain, since a corresponding region is absent from the ␣2(IX) and ␣3(IX) polypeptides (2).The function of collagen IX remains elusive. It is a minor component of the collagen fibrils of cartilage extracellular matrix and is also found in several other tissues. Collagen IX molecules are not present within the fibril body in cartilage but are instead associated with the surface of the collagen fibril and become covalently cross-linked to other collagen IX molecules and to collagen II, the main constituent of the fibril (2, 3). Collagen IX is not required for the assembly of the heterotypic collagen fibrils, but it is important for preservation of the long term stability of the cartilage extracellular matrix (4, 5). The molecular mechanism involved is not understood, however. The NC4 domain of collagen IX is seen in electron microscopy as a compact globulus projecting away from the fibril body, with the COL3 domain acting as a spacer arm (6, 7). This location and the high theoretical pI of the NC4 domain implicate collagen IX as a potential docking molecule, possibly connecting the host fibril to adjacent collagen fibrils or to other macromolecules of the extracellular matrix (8). Proteoglycans of the cartilage extracellular matrix may serve an intermediary purpose in these processes. A proteolytic fragment of collagen IX, lacking the NC4 domain and some other parts of the molecule, is indeed known to bind heparin with high affinity in vitro (9). The NC4 domain reportedly shows homology to the heparin-binding Nterminal domain of thrombospondin, but the residues believed to be crucial for heparin-binding potential of thrombospondin are not conserved in the NC4 domain (10). No research has yet been reported, however, on the glycosaminoglycan binding properties of the NC4 domain or full-length collagen IX.Studies in vitro have demonstrated that cartilage oligomeric
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.
Short-term restimulation assays combined with the analysis of effector function, in particular the detection of cytokine production, are useful tools for the analysis and isolation of antigen-specific T cells. Until now, restimulations with soluble protein antigens failed to efficiently reactivate CD8+ T cells. We have developed a recombinant protein of the immunodominant cytomegalovirus (CMV) matrix protein pp65 for in vitro restimulation of pp65-specific CD4+ as well as CD8+ T cells. The efficiency of the CMV pp65 - Recombinant Protein to reactivate pp65-experienced CD4+ and CD8+ T cells and the specificity of the restimulated T cells were analysed. PBMC from CMV seropositive donors were restimulated with CMV pp65 - Recombinant Protein or a complete pool of overlapping pp65 peptides. Afterwards T cells were analysed for intracellular IFN-γ production by flow cytometry. Interestingly, we observed that stimulation with CMV pp65 - Recombinant Protein results in IFN-γ production in CD4+ as well as CD8+ T cells with frequencies comparable to that using the peptide pool as antigen (n=17). In contrast, upon stimulation of PBMC from CMV seronegative donors with CMV pp65 - Recombinant Protein neither IFN-γ nor TNF-α were detectable in T cells (n=6). Furthermore, we tested the specificity of CMV pp65 - Recombinant Protein-reactive CD4+ and CD8+ T cells. Therefore, IFN-γ-producing T cells were magnetically isolated after short-term stimulation with pp65 using the IFN-γ cytokine secretion assay and expanded for 7 days. Subsequently, the isolated and expanded CD4+ and CD8+ T cells were restimulated with pp65 peptide pool. More than 80 % of the CD4+ and CD8+ T cells produced IFN-γ and more than 80 % of the CD8+ T cells were positively stained with MHC class I/pp65 tetramers. These results demonstrate that CMV pp65 - Recombinant Protein efficiently and specifically reactivates pp65-experienced CD4+ as well as CD8+ T cells. Therefore, CMV pp65 - Recombinant Protein is a useful antigen for the detection and isolation of pp65-experienced CD4+ and CD8+ effector/memory T cells.
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