Mouse Testican-2

Abstract

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. 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. The testicans form a subgroup within the BM-40/SPARC/osteonectin family of modular extracellular proteins (1Hartmann U. Maurer P. Matrix Biol. 2001; 20: 23-35Crossref PubMed Scopus (108) Google Scholar) and have also been referred to as the SPOCKs (SPARC/osteonectin, CWCV and Kazal-like domains) (2Charbonnier F. Perin J.P. Mattei M.G. Camuzat A. Bonnet F. Gressin L. Alliel P.M. Genomics. 1998; 48: 377-380Crossref PubMed Scopus (29) Google Scholar). Their modular structure is characterized by an N-terminal testican-specific domain followed by the follistatin-like- and extracellular calcium-binding (EC) 1The abbreviations used are: EC, extracellular calcium binding; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MMP, matrix metalloproteinase.1The abbreviations used are: EC, extracellular calcium binding; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MMP, matrix metalloproteinase. domains that define the BM-40 family. Toward the C terminus, in contrast to most other members of this family, the testicans carry a thyroglobulin-like domain and a novel domain with two potential glycosaminoglycan attachment sites. For testican-1 a substitution with both chondroitin and heparan sulfate was shown (3Bonnet F. Perin J.P. Maillet P. Jolles P. Alliel P.M. Biochem. J. 1992; 288: 565-569Crossref PubMed Scopus (39) Google Scholar, 4Alliel P.M. Perin J.P. Jolles P. Bonnet F.J. Eur. J. Biochem. 1993; 214: 347-350Crossref PubMed Scopus (106) Google Scholar); for testican-1 and -2 it has been demonstrated that the EC domain is functional and can bind calcium (5Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (201) Google Scholar, 6Vannahme C. Schübel S. Herud M. Gösling S. Hülsmann H. Paulsson M. Hartmann U. Maurer P. J. Neurochem. 1999; 73: 12-20Crossref PubMed Scopus (54) Google Scholar). Testican-1 was originally isolated as a proteolytic fragment from human seminal plasma (3Bonnet F. Perin J.P. Maillet P. Jolles P. Alliel P.M. Biochem. J. 1992; 288: 565-569Crossref PubMed Scopus (39) Google Scholar). 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 (7Charbonnier F. Chanoine C. Cifuentes-Diaz C. Gallien C.L. Rieger F. Alliel P.M. Perin J.P. Mech. Dev. 2000; 90: 317-321Crossref PubMed Scopus (18) Google Scholar). In adult mice, expression is restricted to the brain where it is located at post-synaptic densities (8Bonnet F. Perin J.P. Charbonnier F. Camuzat A. Roussel G. Nussbaum J.L. Alliel P.M. J. Biol. Chem. 1996; 271: 4373-4380Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In search for homologous members of the BM-40 family we identified cDNAs that were most similar to testican-1 and were hence termed testican-2 and -3 (6, 1). Testican-2 mRNA was detected in mouse brain, lung, and testis, and within the brain testican-2 was much more widely expressed than testican-1 (6Vannahme C. Schübel S. Herud M. Gösling S. Hülsmann H. Paulsson M. Hartmann U. Maurer P. J. Neurochem. 1999; 73: 12-20Crossref PubMed Scopus (54) Google Scholar). 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 (9Nakada M. Yamada A. Takino T. Miyamori H. Takahashi T. Yamashita J. Sato H. Cancer Res. 2001; 61: 8896-8902PubMed Google Scholar). 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 (10Nakada M. Miyamori H. Yamashita J. Sato H. Cancer Res. 2003; 63: 3364-3369PubMed Google Scholar). Similarly, testican-1 has been shown to inhibit cathepsin L, but not cathepsin B (11Bocock J.P. Edgell C.J. Marr H.S. Erickson A.H. Eur. J. Biochem. 2003; 270: 4008-4015Crossref PubMed Scopus (43) Google Scholar). Testican-1 was also studied for a potential regulation of neuronal functions and was found to inhibit attachment and neurite outgrowth in cultures of N2a neuroblastoma cells (12Marr H.S. Edgell C.J. Matrix Biol. 2003; 22: 259-266Crossref PubMed Scopus (29) Google Scholar). In the present work we cloned mouse testican-2 and expressed it as a recombinant protein. This allowed us to study its structure, tissue distribution, and interactions with neurons. Isolation of Mouse Testican-2 cDNA Clones—A mouse brain cDNA library (ICR outbred strain, Harlan Sprague-Dawley, newborns; Stratagene) was screened with a 1.4-kb 32P-labeled PstI-BamHI fragment derived from the human testican-2 full-length cDNA (6Vannahme C. Schübel S. Herud M. Gösling S. Hülsmann H. Paulsson M. Hartmann U. Maurer P. J. Neurochem. 1999; 73: 12-20Crossref PubMed Scopus (54) Google Scholar). Hybridization was carried out at 42 °C in 50% formamide, 5 × Denhardt′s solution (0.1% bovine serum albumin, 0.1% Ficoll 400, and 0.1% polyvinylpyrrolidone), 5 × SSPE (0.75 m sodium chloride, 50 mm sodium phosphate, pH 7.6, and 5 mm EDTA), 1.5% SDS, and 0.2 mg/ml salmon sperm DNA. Filters were washed twice for 15 min in 0.1 × SSC (15 mm sodium chloride, 1.5 mm sodium citrate, pH 7.5) and 0.1% SDS at 65 °C and exposed to an x-ray film. Positive plaques were excised and rescreened. Several positive plaques from the final rescreen were in vivo excised, yielding cDNA in the pBluescript vector. The plasmids were sequenced on both strands with flanking and internal primers using an ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit, and the products were resolved on an ABI Prism 377 Automated Sequencer (PE Biosystems). Nucleotide sequence analysis and homology searches were performed with the programs of the GCG package. Recombinant Expression of the Full-length Testican-2 Protein—A cDNA fragment spanning the full-length cDNA of murine testican-2 was generated by PCR using the forward primer 5′-GCC CGC TAG CCG AAG GCG ACG CCA AGG-3′ and the reverse primer 5′-CAA TGA CTG CGG CCG CTA TCT ACC AGA TGT AGC C-3′. Primers introduced the new restriction sites NheI and NotI, respectively. PCR-amplified cDNA was cloned in the pCRII vector (Invitrogen) and sequenced on both strands by cycle sequencing. A NheI/NotI restriction fragment of the pCRII/testican-2 plasmid was purified and cloned into the eukaryotic expression vector pCEP-Pu-His6-Myc-factor X (13Wuttke M. Müller S. Nitsche D.P. Paulsson M. Hanisch F.-G. Maurer P. J. Biol. Chem. 2001; 276: 36839-36848Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Correct insertion of the fragment in the pCEP-Pu/testican-2 construct was verified by sequencing. Plasmids were transfected into the human embryonic kidney cell line EBNA-293 (Invitrogen) using an electroporator (Bio-Rad) according to the instructions of the manufacturer. Growth and selection of transfected cells were carried out as described (5Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (201) Google Scholar). Conditioned serum-free medium of EBNA-293 cells (2.6 liters) was collected and passed over a nickel-nitrilotriacetic acid column (Qiagen). Testican-2 was eluted with a linear gradient of 0.005–0.25 m imidazole in 50 mm sodium phosphate, pH 8.0, containing 0.3 m sodium chloride. The testican-2-containing fractions were pooled and, after dialysis against 50 mm Tris-HCl, pH 8.6, submitted to ion exchange chromatography on a Resource Q column (Amersham Biosciences). The column was eluted with a linear NaCl gradient (0–1 m) and testican-2 detected in two peaks at 0.4 and 0.6 m NaCl. Preparation of Specific Antibodies against Mouse Testican-2—A mixture of equal amounts of the proteoglycan and core protein form of recombinant mouse testican-2, obtained after chromatography on a Resource Q column, was used to immunize a rabbit. The resulting antiserum was adsorbed by repeated passage through a column of CNBr-Sepharose (Amersham Biosciences) to which proteins extracted from EBNA-293 cells with 0.15 m NaCl, 50 mm Tris-HCl, 10 mm EDTA, pH 7.4, had been coupled. The adsorbed antiserum was then passed through a second affinity column with recombinant testican-2 coupled to CNBr-Sepharose and the bound antibodies to testican-2 eluted with 3 m KSCN, 50 mm Tris-HCl, pH 7.4, followed by rapid dialysis against 0.15 m NaCl, 50 mm Tris-HCl, pH 7.4. To block a remaining cross-reactivity with testican-1, 1 μg of purified recombinant testican-1/10 μg of antibody was added before use. Indirect Immunofluorescence Microscopy—Tissues from adult (older than 8 weeks) C57/BL6 mice were embedded in Tissue Tek, and 7-μm cryosections were prepared and stored at –20 °C. Sections were air dried for 1 h and fixed with 2% paraformaldehyde/phosphate-buffered saline for 20 min. After washing with 0.1% Triton X-100 in Tris-buffered saline and 0.1 m glycine in Triton/Tris-buffered saline, sections were blocked for 1 h with 10% bovine serum albumin in Triton/Tris-buffered saline. The affinity-purified antibodies against testican-2 were diluted in Triton/Tris-buffered saline containing 1% fetal calf serum and 1% bovine serum albumin and incubated on the sections at 4 °C overnight. Detection was done with Cy3-labeled goat anti-rabbit IgG (Jackson ImmunoResearch), and the were a of sections was done by Preparation of Tissue from adult than 8 C57/BL6 mice were in 5 0.15 m NaCl, 50 mm Tris-HCl, pH 7.4, containing 10 mm and a protease mixture and for h at 4 °C. For of lung, 4 m was in the were prepared by at × for min at 4 °C. and or recombinant forms of testican-2 were with specific or to the of the In were performed on to of recombinant protein or from of sulfate was by with 0.1 and in 50 mm NaCl, 50 mm Tris-HCl, 4 mm pH at °C overnight. sulfate and chondroitin sulfate were by with in 50 mm NaCl, 50 mm sodium 50 mm Tris-HCl, pH at °C for 4 h with an equal of added after were performed in the for the For analysis of the were with as described were added to a final of 0.1% SDS and and the were with 1 or of at °C. was done in and proteins were to Testican-2 was detected with the affinity-purified primary antibodies followed by a anti-rabbit were in 5 of 0.1% was carried out in linear on a with a a laser and a 1 of the solution was on the and 1 of a prepared solution of acid in with 0.1% acid was The was then by of 1 of that in a For of the an of 20 was and the was to laser were into an was carried out using the and ion of bovine serum albumin for cells were prepared from C57/BL6 mice as previously described E. J. Biol. PubMed Scopus Google Scholar) and in 10% mm 10 were with 15 and incubated at °C for 1 The were washed twice with and from the mouse M. M. R. Timpl R. J. Eur. J. Biochem. PubMed Scopus Google Scholar) was added at and incubated at °C overnight. were at a of × either in medium or in medium with 20 recombinant testican-2 core protein or proteoglycan and in at °C for After in for 10 min at the neuronal cells were by with a antiserum to mouse by C. and a Cy3-labeled antibody and by and Sequencing of Mouse Testican-2—A mouse brain cDNA library was screened with a 1.4-kb derived from the human full-length cDNA (6Vannahme C. Schübel S. Herud M. Gösling S. Hülsmann H. Paulsson M. Hartmann U. Maurer P. J. Neurochem. 1999; 73: 12-20Crossref PubMed Scopus (54) Google and were isolated of these an of whereas the other two were but and the sequence with to the only in the of the and A M. 1996; PubMed Scopus Google Scholar) the sequence is found within the isolated an mRNA of The cDNA a protein sequence of that this protein is to human testican-2 (6Vannahme C. Schübel S. Herud M. Gösling S. Hülsmann H. Paulsson M. Hartmann U. Maurer P. J. Neurochem. 1999; 73: 12-20Crossref PubMed Scopus (54) Google Scholar) over its that the mouse had been The domain structure is to human The N-terminal portion is to as a a of with a H. J. S. G. 1997; PubMed Scopus Google Scholar). The protein of with a of can be into The N-terminal not homology to proteins testican-1 and -3 1). is followed by a domain homologous to A domain is homologous to the EC domain of BM-40 E. Maurer P. Timpl R. J. Biol. 1996; PubMed Scopus Google characterized by two that were shown to be functional in human testican-2 (6Vannahme C. Schübel S. Herud M. Gösling S. Hülsmann H. Paulsson M. Hartmann U. Maurer P. J. Neurochem. 1999; 73: 12-20Crossref PubMed Scopus (54) Google Scholar). the EC a thyroglobulin-like domain and a domain to is The two glycosaminoglycan attachment sites at and Recombinant Expression and of Mouse Testican-2—A cDNA fragment spanning the full-length cDNA was generated by cloned in the eukaryotic expression vector pCEP-Pu-His6-Myc-factor X (13Wuttke M. Müller S. Nitsche D.P. Paulsson M. Hanisch F.-G. Maurer P. J. Biol. Chem. 2001; 276: 36839-36848Abstract Full Text Full Text PDF PubMed Scopus (81) Google and expressed in the human embryonic kidney cell line The recombinant testican-2 an N-terminal was from serum-free cell medium and purified by affinity chromatography on a column with When the testican-2 to the medium and the purified by affinity was by and two were a at and a in the from to the of the a substitution with glycosaminoglycan chains, the affinity-purified was on a Resource Q ion the the testican-2 core was eluted at 0.4 m NaCl and the proteoglycan form at 0.6 m In a from of a of of testican-2 core protein and of testican-2 proteoglycan was but the of the two forms of testican-2 medium Expression of Testican-2 in Mouse mixture of equal amounts of testican-2 core protein and proteoglycan was used to immunize a rabbit. The antiserum obtained had a but both with other members of the BM-40 protein family and with The antibodies be only by of a for affinity an of the serum by repeated passage through a column to which proteins from a of EBNA-293 cells had been followed by binding of the specific testican-2 antibodies to recombinant testican-2 coupled to The from the still a cross-reactivity with testican-1, but this be blocked by the of recombinant testican-1 to the antibody to use. and were performed with antibody The antibodies were used to the tissue of testican-2 in adult In we have shown that testican-2 mRNA is present in mouse brain, lung, and testis, with most of the brain positive in in (6Vannahme C. Schübel S. Herud M. Gösling S. Hülsmann H. Paulsson M. Hartmann U. Maurer P. J. Neurochem. 1999; 73: 12-20Crossref PubMed Scopus (54) Google Scholar). be by that testican-2 is and In brain sections testican-2 was with the cell and also of neurons as the cells in the the cells in and and the cells in the In were in of the brain, and there was a that not be found in other a for testican-2 was detected in the and were with the in the from the This with the expression in testis, us for the of testican-2 in other endocrine glands. Indeed, testican-2 is expressed in the whereas the is and In the the were but not the and the testican-2 is and it that both and are In testis, testican-2 was in a with A may be with and analysis of only a in the in of Testican-2 by and in Mouse cell were for expression of testican-2 by of medium cell N2a and a cell line derived from and a cell line clear positive and a whereas cells were that testican-2 expression was to the neuronal derived from kidney cells and were clear in testican-2 The were and cell In a of molecules was testican-2 but the in the of these a were also and 50 either core proteins or proteolytic The of these to with the of the an that the analysis of mouse tissue the proteoglycan form of testican-2, with obtained in of brain, and the and of the and were also not with that of recombinant testican-2 that the in cell present also in To that the in testican-2 structure is to the of the bound to recombinant and tissue-derived testican-2 was by the more form of recombinant testican-2 was with or a mixture of and with a of the to a a whereas a portion of the to the of the core protein. When both were testican-2 as a core protein recombinant testican-2 is a proteoglycan both chondroitin/dermatan sulfate and heparan sulfate A of the testican-2 core protein was with an A clear in was both in and in with the the before and after are and the of is that only attachment located in the EC was from the sequence by the The of for the core protein of and is still than the from the acid sequence of the recombinant protein. potential binding sites for were identified by present in the Their substitution with this remaining in were performed on testican-2 extracted from mouse brain, and with in a core protein whereas had that the tissue forms of testican-2 are heparan sulfate proteoglycans of testican-2 from with and a in the of of Testican-2 on proteoglycans have been shown to neuronal neurite and axonal to the of the nervous system (1Hartmann U. Maurer P. Matrix Biol. 2001; 20: 23-35Crossref PubMed Scopus (108) Google Scholar, A. R. E. F. Res. 20: PubMed Scopus Google Scholar, E. 1996; PubMed Scopus Google Scholar, Tissue Res. 1997; PubMed Scopus Google Scholar). To for of testican-2 on neurite mouse primary cells were on with mouse a to neurite outgrowth Timpl R. H. J. PubMed Scopus Google in the or of To the glycosaminoglycan play a either the core protein or the proteoglycan was After a the neuronal cells were by using antibodies to the neuronal in In a similar of cells to the but were with to neurite cultures on neurite outgrowth and extension whereas both the core protein and proteoglycan form of testican-2 a in neurite growth added in that the core protein is the Mouse testican-2 cDNA was cloned and the full-length protein expressed in human embryonic kidney EBNA-293 This allowed the of the antiserum against testican-2 and of the tissue of testican-2 protein to information on the expression of the mRNA that we previously obtained by and in (6Vannahme C. Schübel S. Herud M. Gösling S. Hülsmann H. Paulsson M. Hartmann U. Maurer P. J. Neurochem. 1999; 73: 12-20Crossref PubMed Scopus (54) Google Scholar). This is an in the of the extracellular structures in which testican-2 In brain we a with testican-2 antibodies the cell of of neurons. In a and expression in the and in other endocrine as the the and the with the of its in the lung, testican-2 is only expressed in or endocrine in adult present the functional of this restricted expression are but the to the central nervous system is with testican-1, which may be a of structures (8Bonnet F. Perin J.P. Charbonnier F. Camuzat A. Roussel G. Nussbaum J.L. Alliel P.M. J. Biol. Chem. 1996; 271: 4373-4380Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In most testican-2 to be present the extracellular or at the cell that the proteoglycan may with of proteoglycans with neuronal cell were shown for and which both bind to members of the P. T. L. M. M. J. Biol. PubMed Scopus Google Scholar, P. L. M. J. Biol. PubMed Scopus Google Scholar). This binding is to cell neurite and during the development of the nervous system. an testican-2 and neurons that in an inhibition of neurite growth testican-2 was added to primary cerebellar neurons cultured on as a were obtained with both proteoglycan and core protein a to the protein the The by which neurons the of testican-2 has not been but we to the of the testican-2 as as the of the in vivo The inhibition of neurite growth may be a more of the family, this can also be testican-1 is added to cultured N2a neuroblastoma cells (12Marr H.S. Edgell C.J. Matrix Biol. 2003; 22: 259-266Crossref PubMed Scopus (29) Google Scholar). A of testican-2 be in with demonstrated that the as previously shown for other members of the BM-40 family M. Paulsson M. M. Timpl R. Eur. J. Biochem. PubMed Scopus Google Scholar, C. Gösling S. C. Paulsson M. Maurer P. Hartmann U. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google can also be with the also a to be The of a of at the of testican-2, as a and the of a second domain that the protein is from the we have shown that the is functional and testican-2 is present in the of neuroblastoma cell as as cell derived from or cells expressed testican-2 in two with of these glycosaminoglycan and the other This may that testican-2 is a but the analysis of testican-2 by cell or extracted from mouse to this for the in vivo form of The of a form of testican-2 produced in EBNA-293 cells may be to a of the protein and a of the this was not testican-2 was expressed in other cell that amounts of protein not forms of testican-2 were also detected in to the proteoglycan in cell where may be products of proteolytic Similarly, the proteoglycan form of testican-1 is to forms in of human plasma Marr H.S. Edgell C.J. Biochem. Res. 2001; PubMed Scopus (18) Google Scholar). present the of is not but for testican-1 it was that may be for activation or Testican-2 from EBNA-293 cells and from mouse in that the contained both chondroitin/dermatan sulfate and heparan sulfate whereas the are heparan sulfate This not the for the EBNA-293 testican-2 on neurite outgrowth as the in the core protein. work the of testican-2 with cells and the of this proteoglycan in the development of the nervous system and of endocrine glands.