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Isolation of a cDNA encoding a putative SPARC from the brine shrimp, Artemiafranciscana
Gene 268 (2001) 53±58
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Isolation of a cDNA encoding a putative SPARC from the brine shrimp, Artemia franciscana q Shin Tanaka, Fumiko Nambu, Ziro Nambu* Biology, Department of Medical Technology, School of Health Sciences, University of Occupational and Environmental Health, Japan, Yahatanishi-ku, Kitakyushu, 807-8555, Japan Received 3 November 2000; received in revised form 13 February 2001; accepted 1 March 2001 Received by A.J. van Wijnen
Abstract SPARC (Secreted protein, acidic, rich in cysteine) is an extracellular matrix-associated and anti-adhesive glycoprotein extensively studied in vertebrates. Its presence among invertebrates has been reported in nematodes and ¯ies. We cloned a cDNA containing a complete open reading frame for SPARC from the brine shrimp, Artemia franciscana. The amino acid sequence identity between the Artemia and the ¯y SPARCs was 55%, whereas that of the Artemia and the nematode proteins was 45%. Artemia and vertebrates exhibited a sequence identity of 30% in the predicted aa sequences. The SPARC consisted of four domains commonly found among reported SPARCs. The protein comprised 291 amino acids, having a signal peptide, a follistatin-like domain, one N-glycosylation site and one calcium-binding EF-hand motif. Fourteen cysteine residues conserved among all the secreted forms of SPARCs were present in the Artemia SPARC, and four extra cysteine residues were also found in it. The extra residues were conserved among SPARCs of the arthropods and the nematode. Phylogenetic analyses showed that the sequences of SPARCs were grouped into those of vertebrates and invertebrates. Though the structural organization of SPARC was conserved among all the species studied, SPARC within a group was highly conserved within that group, but divergent between the two. Northern blots revealed the presence of a 1.1 kb mRNA, which was faintly expressed in embryos and considerably detected in prenauplii and nauplii. The isolation of a SPARC cDNA from Artemia franciscana provides intriguing features of the divergent protein, SPARC. q 2001 Elsevier Science B.V. All rights reserved. Keywords: BM-40; Calcium-binding; Development; Extracellular matrix; Osteonectin
1. Introduction SPARC, also termed osteonectin and BM-40, is a Ca 21binding glycoprotein expressed in extracellular matrices of various cell types undergoing morphogenesis, development, remodeling and wound healing (Lane and Sage, 1994; Yan and Sage, 1999). SPARC cDNA was ®rst cloned by Mason et al. (1986) from mouse embryo parietal endoderm cells which synthesized a thick basement membrane known as Reichert's membrane. The protein of SPARC (osteonectin) Abbreviations: aa, amino acid; BM-40, basement membrane-40; bp, base pair(s); cDNA, DNA complementary to RNA; Cys, cysteine; DIG labeled, digoxigenin labeled; EC domain, extracellular calcium-binding domain; EF-hand, a convenient mnemonic for the helix-loop-helix motif; FS-like domain, follistatin-like domain; kb, kilobase(s); ORF, open reading frame; rRNA, ribosomal RNA; S, sedimentation constant; SPARC, secreted protein, acidic, rich in cysteine q Accession number of Artemia SPARC is AB052961. * Corresponding author. Iseigaoka 1-1, Yahatanishi-ku, Kitakyushu, 8078555, Japan. Tel.: 181-93-691-7227; fax: 181-93-691-7142. E-mail address: [email protected] (Z. Nambu).
was ®rst isolated by Termine et al. (1981) as a bone-speci®c protein that bound selectively to both hydroxyapatite and collagen. BM-40 was isolated by Dziadek et al. (1986) from the basement-membrane-producing Engelbreth± Holm±Sw-arm mouse tumor. SPARC is grouped as one of the `matricellular proteins' termed by Bornstein (1995), that is, extracellular regulatory proteins that interact with a wide range of both matrix proteins and cell surface receptors as well as with other molecules such as growth factors. These proteins do not contribute to the structural integrity of the extracellular matrix. SPARC has an anti-adhesive function that leads to cell rounding and inhibition of cell spreading (Murphy-Ullrich et al., 1991; Sage and Bornstein, 1991; Lane and Sage, 1994). Antiproliferation is also a major function of SPARC (Yan and Sage, 1999). The protein is a potent cell cycle inhibitor that arrests cells in mid-G1. SPARC inhibits the proliferation of several kinds of cell types. Primary mesenchymal cells isolated from SPARC-null animals display accelerated rates of proliferation (Bradshaw et al., 1999). SPARC-null mice revealed a speci®c role of SPARC
0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00419-X
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S. Tanaka et al. / Gene 268 (2001) 53±58
(Gilmour et al., 1998; Norose et al., 1998). SPARC-de®cient mice develop normally but show severe age-onset cataract formation and disruption of the lens. This suggests that SPARC is essential for maintenance of lens transparency in mice. The disruption of the Sparc locus in mice results in the alteration of two fundamental processes of lens development: differentiation of epithelial cells and maturation of ®ber cells (Bassuk et al., 1999). The protein properties of SPARCs and the molecular nature of the genes and their transcripts have been thoroughly studied in vertebrates and C. elegans (Schwarzbauer and Spencer, 1993; Lane and Sage, 1994; Maurer et al., 1997; Yan and Sage, 1999). SPARC is a single copy gene (Mason et al., 1986; Young et al., 1986; Schwarzbauer and Spencer, 1993). The deduced aa sequences of SPARCs from the cDNAs have been found to be highly conserved in vertebrates (mouse, Mason et al., 1986; bovine, Bolander et al., 1988; human, Swaroop et al., 1988; Villarreal et al., 1989; frog, Xenopus laevis, Damjanovski et al., 1992; chicken, Bassuk et al., 1993), resulting in more than 78% aa sequence identities among them (Lane and Sage, 1994). SPARCs encoded by mouse, human and C. elegans genes are about 38% identical (Schwarzbauer and Spencer, 1993). As described above, SPARCs have mainly been investigated in mammals. Important and comparative information regarding the molecular nature of SPARC has also been obtained from Aves, Amphibia and Nematoda. The sequences of rat, ¯y and trout SPARCs were deposited on databases. While searching genes expressed speci®cally in a developmental stage of Artemia (Tanaka et al., 1999), we isolated an unknown cDNA. Sequencing of the cDNA revealed it to be a crustacean SPARC cDNA, which provided us with new aspects of the protein. We ®rst describe several features of SPARC cDNA from crustacean A. franciscana, then the close relationship of the protein with those of the ¯y and the nematode, and ®nally, its expression during post-dormant development. 2. Materials and methods 2.1. Isolation and sequencing of SPARC cDNA from prenauplii of A. franciscana. Cloning and sequencing of cDNAs were done according
to the method described by Tanaka et al. (1999). Total RNA was extracted from 12 h cultured prenauplii of A. franciscana, a natural product of the Great Salt Lake, Utah, USA (Japan Pet Drugs Co., Tokyo and Los Angeles, CA, USA). After purifying Poly(A) 1 RNA, double stranded cDNA was synthesized and ligated with EcoR I ± Not I adaptor. A cDNA library was constructed in lgt10 vector (Stratagene) using gigapack w III Gold (Stratagene). When we intended to isolate cDNAs speci®cally expressed in one stage of developing Artemia (Tanaka et al., 1999), one unknown cDNA connected with an incomplete trehalase cDNA by EcoR I ± Not I adaptor (Fig. 1) was isolated. The sequence was subcloned into the Not I site of pBluescript SK(2) vector and named N3L. It was revealed to be SPARC cDNA by nucleotide sequencing. 2.2. Northern blot analysis The N3L cDNA was DIG-labeled according to the random priming method using a DIG DNA Labeling Kit (Roche). Methods of electrophoresis and hybridization are described elsewhere (Tanaka et al., 1999). An aliquot of total RNA, weighing 20 mg, was separated by electrophoresis in a 1% agarose gel containing formaldehyde, and blotted on to Hybond-N 1 membrane (Amersham). Hybridization was performed at 688C followed by washing at 658C. 3. Results and discussion 3.1. cDNA and deduced amino acid sequence of Artemia SPARC The N3L cDNA comprised 1083 bp containing an ORF of 873 bp (Fig. 2). The start codon was found to be 52 bp from the 5 0 end of the cDNA. The 3 0 end of the cDNA included a possible polyadenylation signal region, AATTAAA, and an oligo(A) tail. The protein deduced from the ORF consisted of 291 aa. A computer-assisted search of the Artemia protein revealed it as a SPARC. Vertebrate SPARC cDNAs encode proteins of 298±304 aa and nematode SPARC cDNA comprises 264 aa (Lane and Sage, 1994). Fly SPARC deduced from its cDNA is composed of 281 aa (Fig. 3). The Artemia and the ¯y SPARCs were 55% identical. The genome
Fig. 1. SPARC cDNA connected with a cDNA fragment of trehalase by EcoR I ± Not I adaptor. Artemia SPARC cDNA was ®rst isolated with an incomplete trehalase cDNA, as shown in the ®gure. EcoR I and Not I sites are represented as E and N, respectively. Dotted box means N3L SPARC cDNA. Open box shows the trehalase cDNA. EcoR I ± Not I adaptor regions are vertically bold faced.
S. Tanaka et al. / Gene 268 (2001) 53±58
Fig. 2. Nucleotide and deduced aa sequences of the Artemia SPARC cDNA, N3L. The nucleotide and aa sequences are numbered from the ®rst nucleotide and methionine codon. Asterisks indicate stop codons. A possible polyadenylation signal region is heavily underlined. Cleavable signal peptide is outlined. Potential N-glycosylation site is dotted. The dotted box indicates EF-hand Ca 21-binding motif. Potential cyclic AMP-dependent phosphorylation site is boxed. Conserved 14 Cys residues on SPARCs are circled. Four extra Cys residues found on the Artemia protein are double-circled.
sequence of D. melanogaster has been reported elsewhere (Adams et al., 2000). Sequence identity between the SPARC deduced from the ¯y's genome and the Artemia SPARC was also 55%, though the two sequences of the ¯y SPARCs were somewhat different from each other. SPARCs encoded by Artemia and nematode genes were 45% identical. Percent aa identity between SPARCs of Artemia and vertebrate was much lower, being 30%. The fundamental structural homology of the Artemia SPARC to the reported SPARCs was considerable, as shown in Fig. 3. This strongly demonstrates that the Artemia protein is indeed a homologue of the reported SPARCs. The Artemia protein was similar to COOH-terminal regions of SC1 from rat brain (Johnston et al., 1990) and QR1 from quail retina (Guermah et al., 1991). These proteins are called
55
SPARC-related proteins and they also have longer SPARC-unrelated sequences in their NH2-terminal regions (Maurer et al., 1995). The weight matrix method (von Heijne, 1986) showed the presence of a cleavable signal sequence consisting of 20 aa followed by a secreted form of 271 aa (Fig. 2). Domain I (Ala 21±Asp 68) of the Artemia protein contained 11 acidic aa residues (aspartic and glutamic acids) and 11 basic aa residues (lysines, arginines and a histidine). The very acidic character and Ca 21-binding ability of domain I of the human protein has been reported elsewhere (Maurer et al., 1992). This domain contains the major immunological epitopes of SPARC (Yan and Sage, 1999). The nematode protein contains 11 acidic and 7 basic aa residues. A truncated sequence containing this domain of the nematode protein shows Ca 21-binding ability (Schwarzbauer and Spencer, 1993). Domain II (Asp 69±Glu 157) contained ten Cys residues which were characteristic of the FS-like domain. Moreover, the Artemia protein had two extra Cys residues (Cys 129 and Cys 136) in this domain. The nematode and the ¯y SPARCs also contained two extra Cys residues which could be aligned with that of the Artemia protein (Fig. 3). Domain II of vertebrate SPARC contains a Cu 21-binding motif, but such a motif was lacking in the arthropod and the nematode proteins. One potential N-glycosylation site was conserved in domain II in all types of SPARCs. Several gaps of aa sequence were required in domain III (Cys 158±His 220) of the Artemia protein to ®t the optimal alignment among these proteins. EF-hand motif and an endogenous protease-sensitive site which were conserved among the vertebrate proteins were absent in this domain. The nematode and the ¯y protein also lack the motif and the site. The arthropod and nematode proteins contained one extra Cys residue in this domain. Domain IV(Pro 221±Ile 291) contained an EF-hand Ca 21binding motif (Asp 253±Trp 265). The arthropod and the nematode proteins contained one additional Cys residue. A potential cyclic AMP-dependent phosphorylation site (Arg 224± Ser 227) was found in the Artemia protein. Domains III and IV of the human SPARC are currently considered to be not independent but to represent one domain, in the sense of a single folding unit, called C-terminal EC domain (Maurer et al., 1995; Hohenester et al., 1996). The recombinant form of the EC domain from the nematode SPARC showed somewhat different results from those of human SPARC. These results could be interpreted as being due to the extra disul®de bonds and two deletions/ insertions in its structure (Maurer et al., 1997). 3.2. Phylogenetic analyses of SPARCs A phylogenetic tree was drawn by the neighbor-joining method (Saitou and Nei, 1987) (Fig. 4). The analyses showed that the mammalian proteins were very close to each other and that the avian and the amphibian proteins
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S. Tanaka et al. / Gene 268 (2001) 53±58
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Fig. 4. A phylogenetic tree of SPARCs. Unrooted neighbor-joining tree among SPARCs is represented. Scale bar indicates 0.1 of branch length. Values on each branch represent percent of bootstrap probability. Sequence information is the same as in Fig. 3.
were close to them. The piscine protein was relatively far within the vertebrates. The arthropod and the nematode proteins were distinctively grouped into another branch. These results showed that the sequences of SPARCs were grouped into vertebrates and invertebrates composed of arthropod and nematode. This strongly suggests that additional or peculiar functions of the protein might be expected for the invertebrates. 3.3. Developmental expression of Artemia SPARC A 1.1 kb mRNA was detected in all the stages tested in Artemia development (Fig. 5). The 1.1 kb transcript was observed faintly in the dormant cysts (0 h) and embryos at 3 h of culture. The transcript became abundant in the E2 prenauplius stage (12 h) in which Artemia morphogenesis progressed. More expression was observed in the nauplius stage (30 h) in which growth and metamorphosis proceeded. 4. Conclusion Isolation and sequencing of the Artemia SPARC cDNA
Fig. 5. Developmental expression of Artemia SPARC. Total RNA samples were extracted from A. franciscana at indicated periods of post-dormant development, and 20 mg of each RNA sample was electrophoresed, transferred to nylon membrane, and hybridized with the DIG-labeled PCR probe. Positions of molecular weight markers are indicated. Developmental changes of 2.6 kb transcript of the Artemia trehalase (Tanaka et al., 1999) which were detected in the same specimens are shown as comparators. 18S rRNA stained by ethidium bromide is also shown at the bottom.
provided the ®rst description of a putative SPARC sequence for a crustacean species. The results of sequence analyses on the cDNA and the deduced protein of Artemia SPARC suggested a close relation among SPARCs of the arthropods and the nematode. The aa sequences of the Artemia and the ¯y SPARCs were 55% identical and those of the Artemia and the nematode proteins were 45% identical. The sequence identity of the protein between vertebrates and the Artemia was 30%. We believe that the difference in the proteins between vertebrates and invertebrates is a key in searching for new characteristics of SPARC. The developmental study of the Artemia SPARC mRNA showed a suggestive correlation between its expression and embryo-
Fig. 3. Structural similarity of the Artemia SPARC to the reported SPARCs. The aa sequences of secreted forms of SPARCs were aligned by the Clustal W program (Thompson et al., 1994). A part of the alignment in the C-terminal region of the nematode protein was modi®ed to ®t the location of the C-terminal cysteine for those of the arthropods. Numbering of aa is the same as in Fig. 2. Identical aa (ten or eight cases out of ten SPARCs) are reversely printed in black. Green color indicates identical aa among vertebrate SPARCs. Blue color shows identical aa between the arthropod and the nematode SPARCs. Magenta color means identical aa between the arthropods. Fourteen Cys residues conserved among all the SPARCs are printed in red. Four extra Cys residues found on the arthropod and the nematode SPARCs are arrowheaded. Sequence information was obtained from databases, and accession numbers are as follows: SWISSPROT/PIR, P36378 (frog); P36377 (chicken); P13213 (bovine); P16975 (rat); P07214 (mouse); P09486 (human); P34714 (C. elegans); GenBank/EMBL, AJ133736 (D. melanogaster); U25721 (trout).
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nic morphogenesis or larval development. Further molecular studies will have to be undertaken to characterize new functions and localization of Artemia SPARC. References Adams, M.D., et al., 2000. The genome sequence of Drosophila melanogaster. Science 287, 2185±2195. Bassuk, J.A., Iruela-Arispe, M.L., Lane, T.F., Benson, J.M., Berg, R.A., Sage, E.H., 1993. Molecular analysis of chicken embryo SPARC (osteonectin). Eur. J. Biochem. 218, 117±127. Bassuk, J.A., Birkebak, T., Rothmier, J.D., Clark, J.M., Bradshaw, A., Muchowski, P.J., Howe, C.C., Clark, J.I., Sage, E.H., 1999. Disruption of the Sparc locus in mice alters the differentiation of lenticular epithelial cells and leads to cataract formation. Exp. Eye Res. 68, 321±331. Bolander, M.E., Young, M.F., Fisher, L.W., Yamada, Y., Termine, J.D., 1988. Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid). Proc. Natl. Acad. Sci. USA 85, 2919±2923. Bornstein, P., 1995. Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1. J. Cell Biol. 130, 503±506. Bradshaw, A.D., Francki, A., Motamed, K., Howe, C., Sage, E.H., 1999. Primary mesenchymal cells isolated from SPARC-null animals display altered morphology and rates of proliferation. Mol. Biol. Cell 10, 1569± 1579. Damjanovski, S., Liu, F., Ringuette, M., 1992. Molecular analysis of Xenopus laevis SPARC (Secreted Protein, Acidic, Rich in Cysteine). A highly conserved acidic calcium-binding extracellular-matrix protein. Biochem. J. 281, 513±517. Dziadek, M., Paulsson, M., Aumailley, M., Timpl, R., 1986. Puri®cation and tissue distribution of a small protein (BM-40) extracted from a basement membrane tumor. Eur. J. Biochem. 161, 455±464. Gilmour, D.T., Lyon, G.J., Carlton, M.B.L., Sanes, J.R., Cunningham, J.M., Anderson, J.R., Hogan, B.L.M., Evans, M.J., Colledge, W.H., 1998. Mice de®cient for the secreted glycoprotein SPARC/osteonectin/ BM40 develop normally but show severe age-onset cataract formation and disruption of the lens. EMBO J. 17, 1860±1870. Guermah, M., Crisanti, P., Laugier, D., Dezelee, P., Bidou, L., Pessac, B., Calothy, G., 1991. Transcription of a quail gene expressed in embryonic retinal cells is shut off sharply at hatching. Proc. Natl. Acad. Sci. USA 88, 4503±4507. Hohenester, E., Maurer, P., Hohenadl, C., Timpl, R., Jansonius, J.N., Engel, J., 1996. Structure of a novel extracellular Ca 21-binding module in BM40. Nature Struct. Biol. 3, 67±73. Johnston, I.G., Paladino, T., Gurd, J.W., Brown, I.R., 1990. Molecular cloning of SC1: a putative brain extracellular matrix glycoprotein showing partial similarity to osteonectin/ BM40/SPARC. Neuron 2, 165±176. Lane, T.F., Sage, E.H., 1994. The biology of SPARC, a protein that modulates cell-matrix interactions. FASEB J. 8, 163±173. Mason, I.J., Taylor, A., Williams, J.G., Sage, H., Hogan, B.L.M., 1986. Evidence from molecular cloning that SPARC, a major product of mouse embryo parietal endoderm, is related to an endothelial cell `culture shock' glycoprotein of Mr 43 000. EMBO J. 5, 1465±1472.
Maurer, P., Mayer, U., Bruch, M., JenoÈ, P., Mann, K., Landwehr, R., Engel, J., Timpl, R., 1992. High-af®nity and low-af®nity calcium binding and stability of the multidomain extracellular 40-kDa basement membrane glycoprotein (BM-40/SPARC/osteonectin). Eur. J. Biochem. 205, 233± 240. Maurer, P., Hohenadl, C., Hohenester, E., GoÈhring, W., Timpl, R., Engel, J., 1995. The C-terminal portion of BM-40 (SPARC/osteonectin) is an autonomously folding and crystallizable domain that binds calcium and collagen IV. J. Mol. Biol. 253, 347±357. Maurer, P., Sasaki, T., Mann, K., GoÈhring, W., Schwarzbauer, J.E., Timpl, R., 1997. Structural and functional characterization of the extracellular calcium-binding protein BM-40/secreted protein, acidic, rich in cysteine/osteonectin from the nematode Caenorhabditis elegans. Eur. J. Biochem. 248, 209±216. Murphy-Ullrich, J.E., Lightner, V.A., Aukhil, I., Yan, Y.Z., Erickson, H.P., HoÈoÈk, M., 1991. Focal adhesion integrity is downregulated by the alternatively spliced domain of human tenascin. J. Cell Biol. 115, 1127± 1136. Norose, K., Clark, J.I., Syed, N.A., Basu, A., Heber-Katz, E., Sage, E.H., Howe, C.C., 1998. SPARC de®ciency leads to early-onset cataractogenesis. Invest. Ophthalmol. Vis. Sci. 39, 2674±2680. Sage, E.H., Bornstein, P., 1991. Extracellular proteins that modulate cellmatrix interactions. J. Biol. Chem. 266, 14831±14834. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406±425. Schwarzbauer, J.E., Spencer, C.S., 1993. The Caenorhabditis elegans homologue of the extracellular calcium binding protein SPARC/osteonectin affects nematode body morphology and mobility. Mol. Biol. Cell 4, 941±952. Swaroop, A., Hogan, B.L.M., Francke, U., 1988. Molecular analysis of the cDNA for human SPARC/osteonectin/BM-40: sequence, expression, and localization of the gene to chromosome 5q31-q33. Genomics 2, 37±47. Tanaka, S., Nambu, F., Nambu, Z., 1999. Cloning and characterization of cDNAs encoding trehalase from post-dormant embryos of the brine shrimp, Artemia franciscana. Zool. Sci. 16, 269±277. Termine, J.D., Kleinman, H.K., Whitson, S.W., Conn, K.M., McGarvey, M.L., Martin, G.R., 1981. Osteonectin, a bone-speci®c protein linking mineral to collagen. Cell 26, 99±105. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-speci®c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673±4680. Villarreal, X.C., Mann, K.G., Long, G.L., 1989. Structure of human osteonectin based upon analysis of cDNA and genomic sequences. Biochemistry 28, 6483±6491. von Heijne, G., 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683±4690. Yan, Q., Sage, E.H., 1999. SPARC, a matricellular glycoprotein with important biological functions. J. Histochem. Cytochem. 47, 1495± 1505. Young, M.F., Bolander, M.E., Day, A.A., Ramis, C.I., Robey, P.G., Yamada, Y., Termine, J.D., 1986. Osteonectin mRNA: distribution in normal and transformed cells. Nucleic Acids Res. 14, 4483±4497.
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Isolation of a cDNA encoding a putative SPARC from the brine shrimp, Artemia franciscana q Shin Tanaka, Fumiko Nambu, Ziro Nambu* Biology, Department of Medical Technology, School of Health Sciences, University of Occupational and Environmental Health, Japan, Yahatanishi-ku, Kitakyushu, 807-8555, Japan Received 3 November 2000; received in revised form 13 February 2001; accepted 1 March 2001 Received by A.J. van Wijnen
Abstract SPARC (Secreted protein, acidic, rich in cysteine) is an extracellular matrix-associated and anti-adhesive glycoprotein extensively studied in vertebrates. Its presence among invertebrates has been reported in nematodes and ¯ies. We cloned a cDNA containing a complete open reading frame for SPARC from the brine shrimp, Artemia franciscana. The amino acid sequence identity between the Artemia and the ¯y SPARCs was 55%, whereas that of the Artemia and the nematode proteins was 45%. Artemia and vertebrates exhibited a sequence identity of 30% in the predicted aa sequences. The SPARC consisted of four domains commonly found among reported SPARCs. The protein comprised 291 amino acids, having a signal peptide, a follistatin-like domain, one N-glycosylation site and one calcium-binding EF-hand motif. Fourteen cysteine residues conserved among all the secreted forms of SPARCs were present in the Artemia SPARC, and four extra cysteine residues were also found in it. The extra residues were conserved among SPARCs of the arthropods and the nematode. Phylogenetic analyses showed that the sequences of SPARCs were grouped into those of vertebrates and invertebrates. Though the structural organization of SPARC was conserved among all the species studied, SPARC within a group was highly conserved within that group, but divergent between the two. Northern blots revealed the presence of a 1.1 kb mRNA, which was faintly expressed in embryos and considerably detected in prenauplii and nauplii. The isolation of a SPARC cDNA from Artemia franciscana provides intriguing features of the divergent protein, SPARC. q 2001 Elsevier Science B.V. All rights reserved. Keywords: BM-40; Calcium-binding; Development; Extracellular matrix; Osteonectin
1. Introduction SPARC, also termed osteonectin and BM-40, is a Ca 21binding glycoprotein expressed in extracellular matrices of various cell types undergoing morphogenesis, development, remodeling and wound healing (Lane and Sage, 1994; Yan and Sage, 1999). SPARC cDNA was ®rst cloned by Mason et al. (1986) from mouse embryo parietal endoderm cells which synthesized a thick basement membrane known as Reichert's membrane. The protein of SPARC (osteonectin) Abbreviations: aa, amino acid; BM-40, basement membrane-40; bp, base pair(s); cDNA, DNA complementary to RNA; Cys, cysteine; DIG labeled, digoxigenin labeled; EC domain, extracellular calcium-binding domain; EF-hand, a convenient mnemonic for the helix-loop-helix motif; FS-like domain, follistatin-like domain; kb, kilobase(s); ORF, open reading frame; rRNA, ribosomal RNA; S, sedimentation constant; SPARC, secreted protein, acidic, rich in cysteine q Accession number of Artemia SPARC is AB052961. * Corresponding author. Iseigaoka 1-1, Yahatanishi-ku, Kitakyushu, 8078555, Japan. Tel.: 181-93-691-7227; fax: 181-93-691-7142. E-mail address: [email protected] (Z. Nambu).
was ®rst isolated by Termine et al. (1981) as a bone-speci®c protein that bound selectively to both hydroxyapatite and collagen. BM-40 was isolated by Dziadek et al. (1986) from the basement-membrane-producing Engelbreth± Holm±Sw-arm mouse tumor. SPARC is grouped as one of the `matricellular proteins' termed by Bornstein (1995), that is, extracellular regulatory proteins that interact with a wide range of both matrix proteins and cell surface receptors as well as with other molecules such as growth factors. These proteins do not contribute to the structural integrity of the extracellular matrix. SPARC has an anti-adhesive function that leads to cell rounding and inhibition of cell spreading (Murphy-Ullrich et al., 1991; Sage and Bornstein, 1991; Lane and Sage, 1994). Antiproliferation is also a major function of SPARC (Yan and Sage, 1999). The protein is a potent cell cycle inhibitor that arrests cells in mid-G1. SPARC inhibits the proliferation of several kinds of cell types. Primary mesenchymal cells isolated from SPARC-null animals display accelerated rates of proliferation (Bradshaw et al., 1999). SPARC-null mice revealed a speci®c role of SPARC
0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00419-X
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(Gilmour et al., 1998; Norose et al., 1998). SPARC-de®cient mice develop normally but show severe age-onset cataract formation and disruption of the lens. This suggests that SPARC is essential for maintenance of lens transparency in mice. The disruption of the Sparc locus in mice results in the alteration of two fundamental processes of lens development: differentiation of epithelial cells and maturation of ®ber cells (Bassuk et al., 1999). The protein properties of SPARCs and the molecular nature of the genes and their transcripts have been thoroughly studied in vertebrates and C. elegans (Schwarzbauer and Spencer, 1993; Lane and Sage, 1994; Maurer et al., 1997; Yan and Sage, 1999). SPARC is a single copy gene (Mason et al., 1986; Young et al., 1986; Schwarzbauer and Spencer, 1993). The deduced aa sequences of SPARCs from the cDNAs have been found to be highly conserved in vertebrates (mouse, Mason et al., 1986; bovine, Bolander et al., 1988; human, Swaroop et al., 1988; Villarreal et al., 1989; frog, Xenopus laevis, Damjanovski et al., 1992; chicken, Bassuk et al., 1993), resulting in more than 78% aa sequence identities among them (Lane and Sage, 1994). SPARCs encoded by mouse, human and C. elegans genes are about 38% identical (Schwarzbauer and Spencer, 1993). As described above, SPARCs have mainly been investigated in mammals. Important and comparative information regarding the molecular nature of SPARC has also been obtained from Aves, Amphibia and Nematoda. The sequences of rat, ¯y and trout SPARCs were deposited on databases. While searching genes expressed speci®cally in a developmental stage of Artemia (Tanaka et al., 1999), we isolated an unknown cDNA. Sequencing of the cDNA revealed it to be a crustacean SPARC cDNA, which provided us with new aspects of the protein. We ®rst describe several features of SPARC cDNA from crustacean A. franciscana, then the close relationship of the protein with those of the ¯y and the nematode, and ®nally, its expression during post-dormant development. 2. Materials and methods 2.1. Isolation and sequencing of SPARC cDNA from prenauplii of A. franciscana. Cloning and sequencing of cDNAs were done according
to the method described by Tanaka et al. (1999). Total RNA was extracted from 12 h cultured prenauplii of A. franciscana, a natural product of the Great Salt Lake, Utah, USA (Japan Pet Drugs Co., Tokyo and Los Angeles, CA, USA). After purifying Poly(A) 1 RNA, double stranded cDNA was synthesized and ligated with EcoR I ± Not I adaptor. A cDNA library was constructed in lgt10 vector (Stratagene) using gigapack w III Gold (Stratagene). When we intended to isolate cDNAs speci®cally expressed in one stage of developing Artemia (Tanaka et al., 1999), one unknown cDNA connected with an incomplete trehalase cDNA by EcoR I ± Not I adaptor (Fig. 1) was isolated. The sequence was subcloned into the Not I site of pBluescript SK(2) vector and named N3L. It was revealed to be SPARC cDNA by nucleotide sequencing. 2.2. Northern blot analysis The N3L cDNA was DIG-labeled according to the random priming method using a DIG DNA Labeling Kit (Roche). Methods of electrophoresis and hybridization are described elsewhere (Tanaka et al., 1999). An aliquot of total RNA, weighing 20 mg, was separated by electrophoresis in a 1% agarose gel containing formaldehyde, and blotted on to Hybond-N 1 membrane (Amersham). Hybridization was performed at 688C followed by washing at 658C. 3. Results and discussion 3.1. cDNA and deduced amino acid sequence of Artemia SPARC The N3L cDNA comprised 1083 bp containing an ORF of 873 bp (Fig. 2). The start codon was found to be 52 bp from the 5 0 end of the cDNA. The 3 0 end of the cDNA included a possible polyadenylation signal region, AATTAAA, and an oligo(A) tail. The protein deduced from the ORF consisted of 291 aa. A computer-assisted search of the Artemia protein revealed it as a SPARC. Vertebrate SPARC cDNAs encode proteins of 298±304 aa and nematode SPARC cDNA comprises 264 aa (Lane and Sage, 1994). Fly SPARC deduced from its cDNA is composed of 281 aa (Fig. 3). The Artemia and the ¯y SPARCs were 55% identical. The genome
Fig. 1. SPARC cDNA connected with a cDNA fragment of trehalase by EcoR I ± Not I adaptor. Artemia SPARC cDNA was ®rst isolated with an incomplete trehalase cDNA, as shown in the ®gure. EcoR I and Not I sites are represented as E and N, respectively. Dotted box means N3L SPARC cDNA. Open box shows the trehalase cDNA. EcoR I ± Not I adaptor regions are vertically bold faced.
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Fig. 2. Nucleotide and deduced aa sequences of the Artemia SPARC cDNA, N3L. The nucleotide and aa sequences are numbered from the ®rst nucleotide and methionine codon. Asterisks indicate stop codons. A possible polyadenylation signal region is heavily underlined. Cleavable signal peptide is outlined. Potential N-glycosylation site is dotted. The dotted box indicates EF-hand Ca 21-binding motif. Potential cyclic AMP-dependent phosphorylation site is boxed. Conserved 14 Cys residues on SPARCs are circled. Four extra Cys residues found on the Artemia protein are double-circled.
sequence of D. melanogaster has been reported elsewhere (Adams et al., 2000). Sequence identity between the SPARC deduced from the ¯y's genome and the Artemia SPARC was also 55%, though the two sequences of the ¯y SPARCs were somewhat different from each other. SPARCs encoded by Artemia and nematode genes were 45% identical. Percent aa identity between SPARCs of Artemia and vertebrate was much lower, being 30%. The fundamental structural homology of the Artemia SPARC to the reported SPARCs was considerable, as shown in Fig. 3. This strongly demonstrates that the Artemia protein is indeed a homologue of the reported SPARCs. The Artemia protein was similar to COOH-terminal regions of SC1 from rat brain (Johnston et al., 1990) and QR1 from quail retina (Guermah et al., 1991). These proteins are called
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SPARC-related proteins and they also have longer SPARC-unrelated sequences in their NH2-terminal regions (Maurer et al., 1995). The weight matrix method (von Heijne, 1986) showed the presence of a cleavable signal sequence consisting of 20 aa followed by a secreted form of 271 aa (Fig. 2). Domain I (Ala 21±Asp 68) of the Artemia protein contained 11 acidic aa residues (aspartic and glutamic acids) and 11 basic aa residues (lysines, arginines and a histidine). The very acidic character and Ca 21-binding ability of domain I of the human protein has been reported elsewhere (Maurer et al., 1992). This domain contains the major immunological epitopes of SPARC (Yan and Sage, 1999). The nematode protein contains 11 acidic and 7 basic aa residues. A truncated sequence containing this domain of the nematode protein shows Ca 21-binding ability (Schwarzbauer and Spencer, 1993). Domain II (Asp 69±Glu 157) contained ten Cys residues which were characteristic of the FS-like domain. Moreover, the Artemia protein had two extra Cys residues (Cys 129 and Cys 136) in this domain. The nematode and the ¯y SPARCs also contained two extra Cys residues which could be aligned with that of the Artemia protein (Fig. 3). Domain II of vertebrate SPARC contains a Cu 21-binding motif, but such a motif was lacking in the arthropod and the nematode proteins. One potential N-glycosylation site was conserved in domain II in all types of SPARCs. Several gaps of aa sequence were required in domain III (Cys 158±His 220) of the Artemia protein to ®t the optimal alignment among these proteins. EF-hand motif and an endogenous protease-sensitive site which were conserved among the vertebrate proteins were absent in this domain. The nematode and the ¯y protein also lack the motif and the site. The arthropod and nematode proteins contained one extra Cys residue in this domain. Domain IV(Pro 221±Ile 291) contained an EF-hand Ca 21binding motif (Asp 253±Trp 265). The arthropod and the nematode proteins contained one additional Cys residue. A potential cyclic AMP-dependent phosphorylation site (Arg 224± Ser 227) was found in the Artemia protein. Domains III and IV of the human SPARC are currently considered to be not independent but to represent one domain, in the sense of a single folding unit, called C-terminal EC domain (Maurer et al., 1995; Hohenester et al., 1996). The recombinant form of the EC domain from the nematode SPARC showed somewhat different results from those of human SPARC. These results could be interpreted as being due to the extra disul®de bonds and two deletions/ insertions in its structure (Maurer et al., 1997). 3.2. Phylogenetic analyses of SPARCs A phylogenetic tree was drawn by the neighbor-joining method (Saitou and Nei, 1987) (Fig. 4). The analyses showed that the mammalian proteins were very close to each other and that the avian and the amphibian proteins
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Fig. 4. A phylogenetic tree of SPARCs. Unrooted neighbor-joining tree among SPARCs is represented. Scale bar indicates 0.1 of branch length. Values on each branch represent percent of bootstrap probability. Sequence information is the same as in Fig. 3.
were close to them. The piscine protein was relatively far within the vertebrates. The arthropod and the nematode proteins were distinctively grouped into another branch. These results showed that the sequences of SPARCs were grouped into vertebrates and invertebrates composed of arthropod and nematode. This strongly suggests that additional or peculiar functions of the protein might be expected for the invertebrates. 3.3. Developmental expression of Artemia SPARC A 1.1 kb mRNA was detected in all the stages tested in Artemia development (Fig. 5). The 1.1 kb transcript was observed faintly in the dormant cysts (0 h) and embryos at 3 h of culture. The transcript became abundant in the E2 prenauplius stage (12 h) in which Artemia morphogenesis progressed. More expression was observed in the nauplius stage (30 h) in which growth and metamorphosis proceeded. 4. Conclusion Isolation and sequencing of the Artemia SPARC cDNA
Fig. 5. Developmental expression of Artemia SPARC. Total RNA samples were extracted from A. franciscana at indicated periods of post-dormant development, and 20 mg of each RNA sample was electrophoresed, transferred to nylon membrane, and hybridized with the DIG-labeled PCR probe. Positions of molecular weight markers are indicated. Developmental changes of 2.6 kb transcript of the Artemia trehalase (Tanaka et al., 1999) which were detected in the same specimens are shown as comparators. 18S rRNA stained by ethidium bromide is also shown at the bottom.
provided the ®rst description of a putative SPARC sequence for a crustacean species. The results of sequence analyses on the cDNA and the deduced protein of Artemia SPARC suggested a close relation among SPARCs of the arthropods and the nematode. The aa sequences of the Artemia and the ¯y SPARCs were 55% identical and those of the Artemia and the nematode proteins were 45% identical. The sequence identity of the protein between vertebrates and the Artemia was 30%. We believe that the difference in the proteins between vertebrates and invertebrates is a key in searching for new characteristics of SPARC. The developmental study of the Artemia SPARC mRNA showed a suggestive correlation between its expression and embryo-
Fig. 3. Structural similarity of the Artemia SPARC to the reported SPARCs. The aa sequences of secreted forms of SPARCs were aligned by the Clustal W program (Thompson et al., 1994). A part of the alignment in the C-terminal region of the nematode protein was modi®ed to ®t the location of the C-terminal cysteine for those of the arthropods. Numbering of aa is the same as in Fig. 2. Identical aa (ten or eight cases out of ten SPARCs) are reversely printed in black. Green color indicates identical aa among vertebrate SPARCs. Blue color shows identical aa between the arthropod and the nematode SPARCs. Magenta color means identical aa between the arthropods. Fourteen Cys residues conserved among all the SPARCs are printed in red. Four extra Cys residues found on the arthropod and the nematode SPARCs are arrowheaded. Sequence information was obtained from databases, and accession numbers are as follows: SWISSPROT/PIR, P36378 (frog); P36377 (chicken); P13213 (bovine); P16975 (rat); P07214 (mouse); P09486 (human); P34714 (C. elegans); GenBank/EMBL, AJ133736 (D. melanogaster); U25721 (trout).
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nic morphogenesis or larval development. Further molecular studies will have to be undertaken to characterize new functions and localization of Artemia SPARC. References Adams, M.D., et al., 2000. The genome sequence of Drosophila melanogaster. Science 287, 2185±2195. Bassuk, J.A., Iruela-Arispe, M.L., Lane, T.F., Benson, J.M., Berg, R.A., Sage, E.H., 1993. Molecular analysis of chicken embryo SPARC (osteonectin). Eur. J. Biochem. 218, 117±127. Bassuk, J.A., Birkebak, T., Rothmier, J.D., Clark, J.M., Bradshaw, A., Muchowski, P.J., Howe, C.C., Clark, J.I., Sage, E.H., 1999. Disruption of the Sparc locus in mice alters the differentiation of lenticular epithelial cells and leads to cataract formation. Exp. Eye Res. 68, 321±331. Bolander, M.E., Young, M.F., Fisher, L.W., Yamada, Y., Termine, J.D., 1988. Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid). Proc. Natl. Acad. Sci. USA 85, 2919±2923. Bornstein, P., 1995. Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1. J. Cell Biol. 130, 503±506. Bradshaw, A.D., Francki, A., Motamed, K., Howe, C., Sage, E.H., 1999. Primary mesenchymal cells isolated from SPARC-null animals display altered morphology and rates of proliferation. Mol. Biol. Cell 10, 1569± 1579. Damjanovski, S., Liu, F., Ringuette, M., 1992. Molecular analysis of Xenopus laevis SPARC (Secreted Protein, Acidic, Rich in Cysteine). A highly conserved acidic calcium-binding extracellular-matrix protein. Biochem. J. 281, 513±517. Dziadek, M., Paulsson, M., Aumailley, M., Timpl, R., 1986. Puri®cation and tissue distribution of a small protein (BM-40) extracted from a basement membrane tumor. Eur. J. Biochem. 161, 455±464. Gilmour, D.T., Lyon, G.J., Carlton, M.B.L., Sanes, J.R., Cunningham, J.M., Anderson, J.R., Hogan, B.L.M., Evans, M.J., Colledge, W.H., 1998. Mice de®cient for the secreted glycoprotein SPARC/osteonectin/ BM40 develop normally but show severe age-onset cataract formation and disruption of the lens. EMBO J. 17, 1860±1870. Guermah, M., Crisanti, P., Laugier, D., Dezelee, P., Bidou, L., Pessac, B., Calothy, G., 1991. Transcription of a quail gene expressed in embryonic retinal cells is shut off sharply at hatching. Proc. Natl. Acad. Sci. USA 88, 4503±4507. Hohenester, E., Maurer, P., Hohenadl, C., Timpl, R., Jansonius, J.N., Engel, J., 1996. Structure of a novel extracellular Ca 21-binding module in BM40. Nature Struct. Biol. 3, 67±73. Johnston, I.G., Paladino, T., Gurd, J.W., Brown, I.R., 1990. Molecular cloning of SC1: a putative brain extracellular matrix glycoprotein showing partial similarity to osteonectin/ BM40/SPARC. Neuron 2, 165±176. Lane, T.F., Sage, E.H., 1994. The biology of SPARC, a protein that modulates cell-matrix interactions. FASEB J. 8, 163±173. Mason, I.J., Taylor, A., Williams, J.G., Sage, H., Hogan, B.L.M., 1986. Evidence from molecular cloning that SPARC, a major product of mouse embryo parietal endoderm, is related to an endothelial cell `culture shock' glycoprotein of Mr 43 000. EMBO J. 5, 1465±1472.
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