Structural Characterization of TSC-36/Flik

Abstract

Recombinant forms of the glycoprotein TSC-36/Flik were expressed in human cells and used to compare their structural and functional properties with those described for other members of the BM-40/SPARC/osteonectin protein family. TSC-36 was found to occur in two charge isoforms that differ in the extent of sialylation of otherwise identical N-linked, complex type oligosaccharides. Conformational analysis with both circular dichroism and intrinsic fluorescence spectroscopy showed a lack of significant structural changes upon calcium addition or depletion. This finding is in contrast to results obtained for several other BM-40 family members and indicates that the extracellular calcium-binding domain in TSC-36 is non-functional. The lack of conservation of important functional features common to several other members of the BM-40 family indicates that TSC-36, despite its sequence homology to BM-40, has evolved clearly distinct properties. Recombinant forms of the glycoprotein TSC-36/Flik were expressed in human cells and used to compare their structural and functional properties with those described for other members of the BM-40/SPARC/osteonectin protein family. TSC-36 was found to occur in two charge isoforms that differ in the extent of sialylation of otherwise identical N-linked, complex type oligosaccharides. Conformational analysis with both circular dichroism and intrinsic fluorescence spectroscopy showed a lack of significant structural changes upon calcium addition or depletion. This finding is in contrast to results obtained for several other BM-40 family members and indicates that the extracellular calcium-binding domain in TSC-36 is non-functional. The lack of conservation of important functional features common to several other members of the BM-40 family indicates that TSC-36, despite its sequence homology to BM-40, has evolved clearly distinct properties. TSC-36 is an extracellular glycoprotein belonging to the BM-40/SPARC/osteonectin family of proteins containing both extracellular calcium-binding (EC) 1The abbreviations used are: EC, extracellular calcium-binding; FS, follistatin-like; fuc, fucosidase; hex, β-N-acetylhexosaminidase; HPLC, high-performance liquid chromatography; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionisation-time of flight; neur, neuraminidase; RFU, relative fluorescence unit(s); TGF-β, transforming growth factor β; VWC, von Willebrand factor type C-like; CD, circular dichroism; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. and follistatin (FS)-like domains (1Shibanuma M. Mashimo J. Mita A. Kuroki T. Nose K. Eur. J. Biochem. 1993; 217: 13-19Crossref PubMed Scopus (229) Google Scholar). The other members of this family are the glycoproteins BM-40/SPARC/osteonectin (2Termine J.D. Kleinman H.K. Whitson S.W. Conn K.M. McGarvey M.L Martin G.R. Cell. 1981; 26: 99-105Abstract Full Text PDF PubMed Scopus (863) Google Scholar), SC1/hevin (3Johnston I.G. Paladino T. Gurd J.W. Brown I.R. Neuron. 1990; 4: 165-176Abstract Full Text PDF PubMed Scopus (124) Google Scholar), QR1 (4Guermah M. Crisanti P. Laugier D. Dezelee P Bidou L. Pessac B. Calothy G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4503-4507Crossref PubMed Scopus (63) Google Scholar), and SMOC-1 (5Vannahme C. Smyth N. Miosge N. Gösling S. Frie C. Paulsson M. Maurer P. Hartmann U. J. Biol. Chem. 2002; 277: 37977-37986Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and -2 (6Vannahme C. Gösling S. Paulsson M. Maurer P. Hartmann U. Biochem. J. 2003; 373: 805-814Crossref PubMed Scopus (93) Google Scholar) as well as the proteoglycans testican-1 (7Alliel P.M. Perin J.P. Jolles P. Bonnet F.J. Eur. J. Biochem. 1993; 214: 347-350Crossref PubMed Scopus (107) Google Scholar), -2 (8Vannahme C. Schübel S. Herud M. Gösling S. Hülsmann H. Paulsson M. Hartmann U. Maurer P. J. Neurochem. 1999; 73: 12-20Crossref PubMed Scopus (56) Google Scholar), and -3 (9Hartmann U. Maurer P. Matrix Biol. 2001; 20: 23-35Crossref PubMed Scopus (108) Google Scholar) (Fig. 1). For several of the members the calcium-binding domain, comprising two EF-hands, has been shown to be functional and for BM-40 and SC-1 an affinity for collagens was shown, leading to an anchorage of these proteins in extracellular matrix structures (10Sasaki T. Hohenester E. Göhring W. Timpl R. EMBO J. 1998; 17: 1625-1634Crossref PubMed Scopus (124) Google Scholar, 11Hambrock H.O. Nitsche D.P. Hansen U. Bruckner P. Maurer P. Hartmann U. J. Biol. Chem. 2003; 278: 11351-11358Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). For BM-40 the binding was shown to be calcium-dependent, and a collagen-binding surface could be mapped on the EC domain. The many proposed functions of this protein family include inhibition of cell adhesion, influencing tumor invasion and metastasis, growth factor binding, and protease inhibition (12Bradshaw A.D. Sage E H. J. Clin. Invest. 2001; 107: 1049-1054Crossref PubMed Scopus (521) Google Scholar, 13Nakada M. Yamada A. Takino T. Miyamori H. Takahashi T. Yamashita J. Sato H. Cancer Res. 2001; 61: 8896-8902PubMed Google Scholar). TSC-36 is one of the family members with the least similarity to BM-40. The N-terminal region consists of only 12 amino acid residues, and the FS and EC domains are followed by a C-terminal domain with homology to the von Willebrand factor type C-like (VWC) domain. TSC-36 (TGF-β-stimulated clone 36) was first cloned as a TGF-β-induced protein from a mouse osteoblast cell line (1Shibanuma M. Mashimo J. Mita A. Kuroki T. Nose K. Eur. J. Biochem. 1993; 217: 13-19Crossref PubMed Scopus (229) Google Scholar). It was shown that the expression of TSC-36 in fibroblasts could be suppressed through transfection with the oncogenes v-ras and v-myc, whereas v-src, v-raf, and v-abl did not have any influence (14Mashimo J. Maniwa R. Sugino H. Nose K. Cancer Lett. 1997; 113: 213-219Crossref PubMed Scopus (42) Google Scholar). In aggressive, strongly proliferating lung cancer cells TSC-36 mRNA could not be demonstrated, and overexpression of TSC-36 in such cells had an antiproliferative effect (15Sumimoto K. Kurisaki A. Yamakawa N. Tsuchida K. Shimizu E. Sone S. Sugino H. Cancer Lett. 2000; 155: 37-46Crossref PubMed Scopus (64) Google Scholar). By use of a subtractive cDNA library of non-transformed versus v-fos-transformed fibroblasts (FBR-v-fos), TSC-36 was shown to be down-regulated by v-fos and transfection of FBR-v-fos cells with TSC-36 inhibited their migration into Matrigel in an in vitro assay for tumor invasion (16Johnston I.M.P. Spence H.J. Winnie J.N. McGarry L. Vass J.K. Meagher L. Stapleton G. Ozanne B.W. Oncogene. 2000; 19: 5348-5358Crossref PubMed Scopus (62) Google Scholar). The orthologous proteins from rat, human, chick, Xenopus, and macaque have been cloned and sequenced (17Zwijsen A. Blockx H. van Arnheim W. Willems J. Fransen L. Devos K. Raymackers J. Van de Voorde A. Slegers H. Eur. J. Biochem. 1994; 225: 937-946Crossref PubMed Scopus (60) Google Scholar, 18Patel K. Connolly D.J. Amthor H. Nose K. Cooke J. Dev. Biol. 1996; 178: 327-342Crossref PubMed Scopus (53) Google Scholar, 19Okabayashi K. Shoji H. Onuma Y. Nakamura T. Nose K. Sugino H. Asahima M. Biochem. Biophys. Res. Commun. 1999; 254: 42-48Crossref PubMed Scopus (27) Google Scholar, 20Tochitani S. Liang F. Watakabe A. Hashikawa T. Yamamori T. Eur. J. Neurosci. 2001; 13: 297-307Crossref PubMed Scopus (51) Google Scholar). The chick TSC-36 orthologue, also called Flik for follistatin-like, appears to play a role in mesodermal dorsalization, neural induction (18Patel K. Connolly D.J. Amthor H. Nose K. Cooke J. Dev. Biol. 1996; 178: 327-342Crossref PubMed Scopus (53) Google Scholar), and in subsequent maintenance of midline sonic hedgehog signaling thus influencing axial patterning and forebrain development (21Towers P. Patel K. Withington S. Isaac A. Cooke J. Dev. Biol. 1999; 214: 298-317Crossref PubMed Scopus (34) Google Scholar) in the chick embryo. It further influences somite compartmentalization and myogenesis (22Amthor H. Connolly D. Patel K. Brand-Saberi B. Wilkinson D.G. Cooke J. Christ B. Dev. Biol. 1996; 178: 343-362Crossref PubMed Scopus (83) Google Scholar). We have recombinantly expressed and characterized mouse TSC-36 with the purpose of comparing its properties to those of other members of the BM-40 protein family. In doing so we identified two charge isoforms that could be shown to differ in their sialylation pattern. Our work provides a molecular basis for further studies of TSC-36 function. Construction of Expression Vectors for Full-length and Truncated forms of TSC-36—The plasmid pGEM/36-A1 (1Shibanuma M. Mashimo J. Mita A. Kuroki T. Nose K. Eur. J. Biochem. 1993; 217: 13-19Crossref PubMed Scopus (229) Google Scholar) was used to generate a full-length mouse TSC-36 cDNA, which encompasses the entire open reading frame but lacks the signal peptide, by PCR using the forward primer 1 (5′-GCCCCGCTAGCCCACGGCGAGGAGGAACC) and reverse primer 2 (5′-CAATGACTGCGGCCGCTTAGATCTCTTTGGTGTTCAC). The primers introduced new restriction sites (NheI and NotI) and a stop codon. The NheI/NotI-restricted PCR products were purified and inserted between the same restriction sites of pCEP-Pu-BM40 (23Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (203) Google Scholar). Sequencing revealed a 9-bp deletion (nucleotides 573-581) coding for the amino acids FLK. The deletion was corrected by replacing a EcoRI/NotI fragment with a corresponding 2-kb fragment from the pRcCMV/36 c1-2 containing the missing 9 bp. The corrected vector was subjected to a second PCR using the above-mentioned primers. This vector also served as a template to generate cDNA fragments representing the pair of EC-VWC domains and the EC domain using the following primers: forward primer 3 (5′-GCCCCGCTAGCCGTTGTCTGCTATCAAGCTAAC) and reverse primer 2 (see above) were used for the pair of EC-VWC domains and forward primer 3 (see above) and reverse primer 4 (5′-CAATGACTGCGGCCGCTTACTCAGGAGGGTTGAAGGAT) for the EC domain. The NheI/NotI-restricted PCR products were purified and, in the case of full-length TSC-36, inserted between the same restriction sites of pCEP-Pu-BM40 (23Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (203) Google Scholar), to obtain the final expression vector pCEP-Pu-TSC-36. The cDNA fragments of EC-VWC and EC were cloned in the modified expression vector pCEP-Pu with an N-terminal tag consisting of six histidines, a myc epitope, and an enterokinase recognition site for cleavage of the tag (24Wuttke 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 (82) Google Scholar), to obtain expression vectors pCEP-Pu-TSC-36-EC-VWC and pCEP-Pu-TSC-36-EC, respectively. The correct insertion and sequence of the inserts was verified by cycle sequencing of both strands using an ABI Prism 377 Automated Sequencer (PE Biosystems). Cell Culture and Transfection—The human embryonic kidney cell line EBNA-293 (Invitrogen), which constitutively expresses the EBNA-1 protein from Epstein-Barr virus, was used for transfection with pCEP-Pu-TSC-36, pCEP-Pu-TSC-36-EC-VWC, and pCEP-Pu-TSC-36-EC and selected with puromycin as described previously (23Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (203) Google Scholar). Conditioned serum-free media were collected 48 h after replacement of the serum-containing medium and stored at -80 °C. Purification of Full-length TSC-36 and Separation of TSC-36 A and B—Conditioned serum-free medium (4 liters) was dialyzed against 50 mm Tris-HCl, pH 8.6, and passed over a DEAE-Sepharose FF (Amersham Biosciences) column equilibrated in the same buffer at 4 °C. The column was eluted with a linear NaCl gradient, and the recombinant protein eluted at 0.1-0.2 m NaCl. The were dialyzed against m in mm pH and to a FF (Amersham Biosciences) column that several TSC-36 was found in the and was dialyzed against 50 mm Tris-HCl, pH 8.6, on a DEAE-Sepharose FF and eluted with m NaCl. was by on in mm Tris-HCl, pH The correct N-terminal sequence was by on a protein following the Separation of the two charge isoforms of TSC-36 was by on a (Amersham Biosciences) column eluted with a NaCl in 50 mm Tris-HCl, pH A was eluted at m and at m NaCl. Purification of the EC-VWC serum-free medium was by dialyzed against m 50 mm pH and to a column with Matrix The EC-VWC protein eluted at a of mm and was dialyzed against 50 mm BisTris, pH and to a column eluted with a NaCl in the same A was eluted at m and at mm NaCl. Purification of the EC serum-free medium was dialyzed against 50 mm Tris-HCl, pH to a column with FF (Amersham Biosciences) for eluted with a linear NaCl in the same buffer and was dialyzed against 50 mm pH The protein was to a column with Matrix in this buffer and eluted at a of mm the showed two of which the was to to The EC domain was from this by on a column of (Amersham Biosciences) equilibrated in 50 mm pH was to the of PubMed Scopus Google Scholar), and proteins were by with were in a at in cells of 1 The in was on the basis of a molecular mass of The of the was by addition of 2 mm of the was by subsequent addition of 4 mm A with buffer mm Tris-HCl, pH was and from were using of for TSC-36, for the EC-VWC domain and for the EC domain to and von von Biochem. PubMed Scopus Google Scholar). fluorescence was with a in cells at with at for the full-length TSC-36, the EC-VWC domain and the EC domain 1 were in mm Tris-HCl, pH were also with 2 mm in the same buffer and after addition of 4 mm of protein mass and purified proteins were with and by to for the proteins were with of in 50 mm Tris-HCl, pH at °C. and were by mass For analysis of and proteins were further with in 50 mm pH for h at and, after with and with subjected to mass For mass the were in of was in linear on a with a a laser and a 1 of the was on the and 1 of a of acid in with acid was The was by addition of 1 of which in a For of the an of was used and the was to laser were into an was using the and signal of for of were by with with with and other and by or as previously described (24Wuttke 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 (82) Google Scholar). TSC-36 was used to TSC-36 was to (Amersham and were by of the over the affinity matrix and with 3 m in 50 mm Tris-HCl, pH were dialyzed against m 50 mm Tris-HCl, pH of Cell Culture of mouse embryonic and of several cell were to 48 h after replacement of the serum-containing were to on were and with to TSC-36, followed by a to (Amersham Biosciences) was used to Recombinant of Full-length and Truncated TSC-36 expressed TSC-36 in human embryonic kidney cells to and In addition to the full-length protein we two forms corresponding to the EC domain and the domain pair EC and VWC, respectively. For of these proteins were with an N-terminal tag (24Wuttke 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 (82) Google Scholar). a in the of full-length TSC-36 from cell the protein was to a in 50 mm Tris-HCl, pH those the eluted in two distinct at and m a (Fig. of from the first and second in their in identical as in the (Fig. and that of the protein EC-VWC eluted in two by on after by affinity on a that the between the two A and in the domain pair of the recombinant EC domain was not for in this of the purified proteins (Fig. both and showed a for with TSC-36 A with an molecular mass of TSC-36 with EC-VWC A with and EC-VWC with A molecular mass in the of EC-VWC was by shown to a The EC domain the other proteins and was as a in the region of In case the migration was from the molecular mass from amino acid which the of an and domain of TSC-36 of the purified recombinant proteins was on a or with with The were full-length TSC-36 A 1 and and 2 and the EC-VWC domain pair A 3 and and 4 and and the EC domain and domain structures of the recombinant of of the recombinant circular dichroism were in the For full-length TSC-36 and the EC-VWC domain the were on containing both significant could be between the in the of 2 mm calcium and after addition of (Fig. The for the full-length TSC-36 was for a protein with with a at and a at The EC domain and the EC-VWC domain pair showed but with of that in the domain of recombinant TSC-36 proteins from circular dichroism in a new The lack of changes upon of calcium that the EC domain in TSC-36 not be functional in calcium binding, but could also be to that a is or to be by circular dichroism We also intrinsic fluorescence at calcium or depletion. The amino acid sequence of TSC-36 and that are between domains so that recombinant proteins are to in fluorescence changes in fluorescence were a lack of changes (Fig. of TSC-36 and mass of of the recombinant proteins was by mass spectroscopy and with the mass from the amino acid sequence For protein the mass was at least the the of The of was by of proteins with and analysis of the The results showed that the of the mass was to and that both for the full-length TSC-36 and the EC-VWC domain pair the mass in was in the of sites using the of in and which in the EC domain. of were found in the full-length TSC-36 and the EC-VWC domain pair in with this the EC fragment a the to the that is only amino acid from the of this fragment (Fig. This site be used to the in a protein containing also a domain. In case the mass after was the the of forms of of with the a of the in and of the recombinant TSC-36 proteins and their were by mass and used to the mass of were by mass and used to the mass of were by mass and used to the mass of were by mass and used to the mass of were by mass and used to the mass of mass after were by mass and used to the mass of were by mass and used to the mass of were by mass and used to the mass of were by mass and used to the mass of were by mass and used to the mass of mass of were by mass and used to the mass of in a new the of the and two sites both isoforms of full-length TSC-36 were with and the were by mass The identified of the amino acid in TSC-36 A and of those in TSC-36 (Fig. The C-terminal could not be of the of cleavage sites in this region of the to in the of the protein corresponding to and were missing in both that these sites are the other were found containing and and This not that these sites are used in of the protein The was only from the A but the two at and in this in and are to be and of TSC-36 were from the A and isoforms of TSC-36 and with the for were by (Fig. which revealed with to acid The from the TSC-36 had a acid that those from the A The acid could be from the by (Fig. the was to the full-length TSC-36 A and these eluted at the same upon protein liquid on a that in acid is the of the two charge isoforms acid of the structures of TSC-36 isoforms A and in a new The obtained after with were on a and the structures were identified by with (Fig. The same structures were obtained for both isoforms of were complex type a isoforms the same of and complex type oligosaccharides. the between the two isoforms is not to structures but to in acid structures in TSC-36 in a new of TSC-36 by and by Cell in against the full-length TSC-36 was by binding to the same to The did not with any of the BM-40 family -2 and and SMOC-1 and in or in after This was used to the expression and of TSC-36 in from and fibroblasts and from cell of and and to compare TSC-36 from these cells with the recombinant protein (Fig. TSC-36 from these with but not identical to that of recombinant were to with the of but the proteins showed as one or two with the cell and are both of human and the TSC-36 showed clearly after a that introduced by A of cell was to of TSC-36 (Fig. TSC-36 was in the medium of and cell of the results with and and The cell line and the cell line were strongly The cell and whereas and were A of other or cell were also the cell were those of rat, and human that the TSC-36 from these The TSC-36 forms by the cell in the of for recombinant TSC-36 expressed in with a in A of was to the homology in sequence and domain between TSC-36 and BM-40 is in a similarity between the two proteins in structural and functional properties. A of the two showed the of two in the TSC-36 sequence P. C. Hohenester E. Göhring W. Timpl R. J. J. Biol. PubMed Scopus Google Scholar). The first in TSC-36 a deletion of one amino acid with the a that not a calcium affinity as by the of C. 1994; PubMed Scopus Google Scholar). The second to the In an to the calcium binding, recombinant TSC-36 and the proteins representing the EC-VWC domain pair and the EC domain were by circular dichroism spectroscopy with and addition of calcium and in the of For proteins calcium addition in only changes in the and addition of in a not significant (Fig. circular dichroism spectroscopy did not any of a the was to of fluorescence at calcium and (Fig. of the of and in the TSC-36 this was to of could be in of the recombinant we that calcium binding to TSC-36 this not have any for the of the For the same were with human and BM-40 calcium addition significant in the of as by circular dichroism and a in the of the fluorescence J. W. Paulsson M. Sage H. B. 26: PubMed Scopus Google Scholar, P. U. M. P. K. R. J. Timpl R. Eur. J. Biochem. PubMed Scopus Google Scholar, J. Maurer P. U. R. K. Timpl R. T. J. J. Biol. 1994; PubMed Scopus (53) Google Scholar). In in the case of BM-40 of the any of calcium C. BM-40 lacks amino acid in the EC domain, which the B. It also an which is to an the EC domain in a P. Göhring W. T. K. Timpl R. R. Cell Sci. 1997; PubMed Scopus Google Scholar). TSC-36 a deletion of amino acid residues, which two and in the EC domain. the of full-length TSC-36 and the EC-VWC domain pair by we found that the recombinant proteins occur in two In studies of TSC-36 by a was by TSC-36 showed a of from to (17Zwijsen A. Blockx H. van Arnheim W. Willems J. Fransen L. Devos K. Raymackers J. Van de Voorde A. Slegers H. Eur. J. Biochem. 1994; 225: 937-946Crossref PubMed Scopus (60) Google Scholar). We could that this is to sialylation and that the two isoforms have an identical protein (Fig. and the same in the same Expression of TSC-36 was in and fibroblasts and in a of cell of and of these showed molecular for TSC-36 in the of by that the of for the recombinant proteins is to be for forms of The functional role of the and sialylation of TSC-36 but is to the protein strongly and well its We are to of for and mass