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The complex multidomain organization of SCO-spondin protein is highly conserved in mammals
B RA I N RE SE A R CH RE V I EW S 53 ( 20 0 7 ) 3 2 1–3 2 7
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s r e v
Review
The complex multidomain organization of SCO-spondin protein is highly conserved in mammals☆ Olivier Meiniel, Annie Meiniel⁎ INSERM UMR 384, Faculté de Médecine, 28 Place Henri Dunant, 63001 Clermont-Ferrand Cedex, France
A R T I C LE I N FO
AB S T R A C T
Article history:
The multidomain organization of SCO-spondin protein is a special feature of the chordate
Accepted 20 September 2006
phylum. This protein is expressed in the central nervous system (CNS) from the time a
Available online 27 November 2006
dorsal neural tube appears in the course of phylogenetical evolution. With the advance of the systematic whole genomes sequencing, we were able to determine the SCO-spondin
Keywords:
amino acid sequence in four mammalian species using the Wise2 software. From the
Mammalian SCO-spondin
ClustalW alignment of bovine (Bos taurus), human (Homo sapiens), murine (Mus musculus) and
Protein domain
rat (Rattus norvegicus) proteins, a consensus sequence for mammalian SCO-spondin was
TSR superfamily
determined and further validated with the dog (Canis familiaris) SCO-spondin sequence. The
Central nervous system
analysis of this consensus sequence is consistent with a very high degree of conservation in
Development
the amino acids composition and multidomain organization of SCO-spondin in mammals. In addition, the identification of conserved domains, namely, Emilin (EMI), von Willebrand
Abbreviations:
factor D (vWD), low-density lipoprotein receptor type A (LDLrA) domains, SCO repeats
CNS, central nervous system
(SCOR), thrombospondin type 1 repeats (TSR), a coagulation factor 5/8 type C (FA5-8C) or
CTCK, C-terminal cystine knot
discoidin motif and a C-terminal cystine knot (CTCK) domain, provides a greater insight into
EMI, Emilin
the putative function of this multidomain protein. SCO-spondin belongs to the TSR
FA5-8C, coagulation factor 5/8 type C
superfamily given the presence of a great number of TSR (26). A finer classification of the
LDLrA, low-density lipoprotein
TSR motifs in groups 1, 2 and 3 is proposed on the basis of different cysteine patterns.
receptor type A
Interestingly, group 2 TSR are present in a number of CNS developmental proteins including
SCO, subcommissural organ
R-spondins, F-spondins and Mindins.
SCOR, SCO-spondin repeat SP, signal peptide TIL, trypsin inhibitor-like TILa, trypsin inhibitor-like associated TSP-1, thrombospondin 1 TSR, thrombospondin type 1 repeat vWD, von Willebrand factor D vWDa, von Willebrand factor D associated
☆
Confidential accession numbers: Homo sapiens: BN000852; Canis familiaris: BN000732. ⁎ Corresponding author. Fax: +33 4 73 27 61 32. E-mail address: [email protected] (A. Meiniel).
0165-0173/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainresrev.2006.09.007
© 2006 Elsevier B.V. All rights reserved.
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Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . 2.1. Amino acids conservation . . . . . . . 2.2. Conservation of the modular structure 3. Experimental procedure. . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . .
1.
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Introduction
The complete sequence and modular organization of SCOspondin were first characterized in Bos taurus (Gobron et al., 2000). This extra cellular matrix-like protein, a member of the thrombospondin type 1 repeat (TSR) superfamily (Adams and Tucker, 2000), has been shown to be expressed in the subcommissural organ (SCO), a derivative of the dorsal diencephalon, both in early ontogenetical development and during adult life (see Meiniel et al., 2003, for a review). SCO-spondin protein is also expressed transitory during embryonic stages in the floor plate (Richter et al., 2001; Meiniel, unpublished results). This ependymal secretion may well be involved in developmental processes, including commissural axon pathfinding (Lehmann and Naumann, 2005), similarly to other proteins of the TSR superfamily expressed early in the CNS (see Adams and Tucker, 2000, for a review). A biochemical conservation of the material secreted by the subcommissural organ was first revealed using immunological probes directed against Reissner's fibre (RF), a structure present all along the central canal of the spinal cord (Sterba et al., 1982; Rodriguez et al., 1984). Antibodies raised against bovine RF were able to specifically recognize the secretory material present in the SCO of all vertebrate species examined except anthropoid apes and human (Rodriguez et al., 1992; Oksche et al., 1993), and in the floor plate (Rodriguez et al., 1996; Lopez-Avalos et al., 1997; Yulis et al., 1998; Lichtenfeld et al., 1999). A high conservation of the sugar moities was also revealed using lectins. N-linked high-mannose chains were associated with the precursor form of the secretion located in the endoplasmic reticulum of the SCO secretory ependymal cells, and complex carbohydrate chains to the mature form in the secretory vacuoles and Reissner's fibre (Meiniel and Meiniel, 1985; Rodriguez et al., 1986; Meiniel et al., 1988). From this time, SCO-spondin was identified as the major and specific component of the SCO/RF complex, in the bovine (Gobron et al., 1996, 2000; Nualart et al., 1998; Didier et al., 2000). Recent progress in the systematic whole genome sequencing in several mammalian species permitted to analyze the degree of conservation of this protein. The ortholog to the bovine SCO-spondin gene was found in Homo sapiens on chromosome 7, in Rattus norvegicus on chromosome 4, in Canis familiaris on chromosome 16, and in Mus musculus on chromosome 6 (Gonçalves-Mendes et al., 2003). Based on these genomic data, SCO-spondin proteins could be predicted for these mammalian species
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using the Wise2 software. The remarkable conservation resulting from the comparison of these proteins led us to determine a consensus amino acid sequence for mammalian SCO-spondin. A general scheme of the multidomain organization could be drawn from this consensus and an improved description of the structural modules could be established. In addition, we propose a subdivision into three groups of the SCO-spondin TSR motifs on the basis of particular cysteine patterns. SCO-spondin, F-spondin, Mindin (Feinstein and Klar, 2004) and R-spondin (Kamata et al., 2004), which were shown to be expressed early in the CNS, actually all share group 2 TSRs and could therefore be involved in related developmental mechanisms.
2.
Results and discussion
2.1.
Amino acids conservation
A consensus sequence was established from the alignment of the M. musculus (mouse), R. norvegicus (rat), H. sapiens (human) and B. taurus (bovine) SCO-spondin proteins in order to investigate variations in the amino acids (AA) composition in mammals. Only 66 AA, from a total number of 5166 AA for the whole consensus sequence, were found to be different in the four species and thus were replaced by an X for “any amino acid” (Fig. 1A). This result is consistent with a very high degree of conservation between the four sequences of mammalian species. It was confirmed by comparing pairs of species using ClustalW software: this gave 87% identity between R. norvegicus and M. musculus, 79% between H. sapiens and B. taurus, 75% between H. sapiens and R. norvegicus, 72% between H. sapiens and M. musculus, 70% between B. taurus and M. musculus, 73% between B. taurus and R. norvegicus, for a final 62% identity between all four species. Furthermore, the informative interest of the consensus sequence given in Fig. 1A was validated by aligning it with the most recently predicted SCO-spondin homolog: the C. familiaris (dog) protein. This sequence actually showed 80.7% identity with the SCO-spondin consensus sequence determined in this study, whereas by comparing C. familiaris sequence to the other species, we obtained only 71% identity with M. musculus, 73% with R. norvegicus, 78% with H. sapiens and 79% with B. taurus. Thus, this consensus can be held as a reference to evaluate the conservation of further mammalian SCO-spondin sequences.
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2.2.
Conservation of the modular structure
In parallel with the high degree of conservation in the AA composition, we observed the same modular structure in the
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four species which allowed us to define a general scheme for mammalian SCO-spondin protein (Fig. 1B). The description of the original mosaic organization of SCOspondin (Gobron et al., 2000, in the bovine) was improved by
Fig. 1 – (A) Consensus amino acid sequence of the mammalian SCO-spondin protein. The signal peptide (SP) is boxed in brown and the Emilin (EMI) module is boxed in black. The von Willebrand D (vWD) domains are printed in white and boxed in pink and the von Willebrand D-associated (vWDa) regions are printed in black and boxed in pink. The SCO-spondin repeat (SCOR) modules are boxed in red with the core of these repeats printed in white. The low-density lipoprotein receptor type A (LDLrA) domains are boxed in green, the coagulation factor 5/8 type C (FA5-8C) motif is boxed in dark blue and the C-terminal cystine knot (CTCK) domain is boxed in yellow. The von Willebrand C (vWC) domain is boxed in yellow but printed in red. The thrombospondin type 1 repeat (TSR) motifs are boxed in blue, with group 1 TSRs printed in white, group 2 TSRs printed in black, and group 3 TSRs printed in red. (B) Scheme of the mosaic organization of the various structural domains.
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directly analyzing the consensus sequence given in Fig. 1A using SMART sequence analysis software. In fact, a newly defined motif called Emilin (EMI) was identified as a single motif at the NH2-terminal of the protein (Figs. 1A and B). The EMI domain (PFAM accession number PF07546) was first characterized by Doliana et al. (2000) and was shown to play a role in the protein multimerization, a function which is shared with the von Willebrand factor D (vWD) domains (PFAM accession number PF00094) also located in the NH2-terminal of SCO-spondin but in three repeated copies (Figs. 1A and B, Fig. 2A). Analysis of the association of EMI and vWD domains showed it to be specific to the SCO-spondin protein. In this paper, we also propose a so-called von Willebrand factor D-associated (vWDa) domain which is conserved in the COOH end of each of the three vWD motifs (Fig. 1A and B). However, vWDa domain has no accession number in databases: it was defined by hand from the ClustalW multiple sequence alignment. This vWDa region, together with the vWD itself, was also found four times in human mucins and von Willebrand factor precursors and may also be involved in protein multimerization. These EMI and vWD domains, together with another previously described motif named C-terminal cystine knot (CTCK), complete the description of the structural modules thought to be involved in polymerization. In fact, CTCK_2 (PROSITE accession number PS01225), a conserved domain located in the C-terminal end of SCO-spondin (Figs. 1A and B) and many other modular proteins, is required for the formation of dimers before the multimerization step. These structural motifs would partly explain the formation of Reissner's fibre, an aggregation of SCO-spondin proteins, present in the central canal of the spinal cord. We also identified another new module of an average length of 98 amino acid residues, named SCO-spondin repeat (SCOR), which was found to be repeated sixteen times in the SCO-spondin consensus sequence (Figs. 1A and B). There is no pattern referenced in any database for this particular conserved module; therefore, it was determined by hand on the basis of strong homologies with trypsin inhibitor-like (TIL) and trypsin inhibitor-like-associated (TILa) regions (PFAM accession numbers PF01826 and PF02345, respectively). The sixteen different SCORs found in SCO-spondin are particularly conserved in their middle part, called the core. The cores of SCOR motifs were aligned in order to show the particularly conserved cysteine positions which characterize this module (Fig. 2B). SCOR usually includes two additional conserved cysteines on the COOH side and four to six more on the NH2 side of its core. Fig. 1A shows the SCOR motifs boxed in red, with sides printed in black and cores printed in white. Only cores were aligned, since the conservation was much less remarkable on the NH2 and COOH sides. SCOR shares structural homologies with other protease inhibitors than TILs in the MEROPS peptidase database. For
instance, SCOR also displayed positive hits for serine protease inhibitors belonging to MEROPS family I19 (InterPro accession number IPR009041), i.e., vWC_out (SMART accession number SM00215) and the Pacifastin inhibitor. However, the best homology score obtained with the cores of SCOR motifs was the family of cysteine-rich protease inhibitor I8, clan IA (InterPro accession number IPR002919; PFAM accession number PF01826), to which TIL belongs. Inhibitors of family I8 inhibit serine peptidases which belong to family S1, and probably also M4. In the SCO-spondin consensus, there are several motif combinations involving SCORs: vWD-SCOR, SCOR-vWD, SCORLDLrA, TSR-SCOR, SCOR-TSR and SCOR-CTCK. Interestingly, we noticed that a similar type of arrangement was encountered in SCO-spondin, zonadhesins and immunoglobulin G (IgG) FC binding fragment. In fact, the combination of TIL-TILa-vWD was found several times in zonadhesins and the IgG FC binding region, while in SCO-spondin, both SCOR-vWD and vWD-SCOR arrangements were found. Furthermore, the presence of low-density lipoprotein receptor type A (LDLrA) domains (PROSITE accession number PS50068) repeated ten times in the consensus sequence (Figs. 1A and B, Fig. 2C) could provide a clue as to the function of SCORs. In fact, LDLrA are known to interact with proteases or protease inhibitors (Herz, 2001). Hence, there may be a functional link between LDLrAs and SCORs, which could both be involved in the regulation of either protease activation or protease inhibition. Therefore, maturation of a native and probably inactive form of the protein into active components might be processed with the cooperation of those motifs. Two types of motifs, coagulation factor 5/8 type C or discoidin (FA5-8C; PROSITE accession number PS50022 for FA5-8C_3) and thrombospondin type 1 repeat (TSR; PROSITE accession number PS50092) present in SCO-spondin consensus (Figs. 1A and B) were initially described in blood proteins, where they were shown to play a role in coagulation or platelet aggregation. Both motifs are found early in unicellular organisms: the FA5-8C module in bacteria as a sugar binding motif, and TSR in circumsporozoite (CS) protein of the parasite Plasmodium falciparum as an attachment module to hepatic cells. These motifs are recruited in several proteins to mediate various functions in diverse biological systems. For example, the FA5-8C motif is found twice in coagulation factors V and VIII while TSR is repeated three times in thrombospondin 1 (TSP-1). In mammalian SCO-spondin, FA5-8C was found once and twenty six TSRs were aligned on the basis of a conserved pattern of cysteine, tryptophan and arginine. Nevertheless, distinct patterns of cysteine residues led to a more accurate classification into 3 groups of TSRs (Fig. 2D): 16 group 1 TSRs are characterized by the absence of a cysteine in NH2 (xxxxxWxxW) and the presence of a cysteine in position +2 of the two conserved arginines (RxRxC); 8 group 2 TSRs show a cysteine in
Fig. 2 – Multiple sequence alignments of SCO-spondin conserved modules. Conserved cysteine residues are printed in red, whereas other conserved amino acids are printed in blue. X, for “any amino acid”, is printed in grey. (A) Alignment of von Willebrand D (vWD) domains. (B) Alignment of the cores of SCO-spondin repeats (SCORs). (C) Alignment of low-density lipoprotein receptor type A (LDLrA) domains. (D) Alignment of groups 1, 2 and 3 thrombospondin type 1 repeats (TSRs).
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amino terminus (CxxxxWxxW) and no cysteine in the central region of the TSR (RxRxx); 2 group 3 TSRs comprise a pattern of 8 conserved cysteines instead of 6 for groups 1 and 2. This variation in cysteine positioning and number probably results in changes in disulfide bond formation, and therefore in the 3D structure. Recently, Tan et al. (2002) described the crystal structure of TSRs in TSP-1. Each TSR is composed of three antiparallel strands and, compared to the TSRs of TSP-1, the addition of an extra cysteine at the first anti-parallel beta strand together with the absence of a cysteine at the second anti-parallel beta strand would define a second TSR group with a modified disulfide bond. Interestingly, several proteins containing TSRs of group 2 are expressed early in the course of neural development, namely R-spondin, F-spondin, Mindin and the relatives HB-GAM and Midkine (Kamata et al., 2004; Feinstein and Klar, 2004). SCO-spondin and F-spondin share a similar pattern of expression in the floor plate, flexural organ and subcommissural organ and could have a redundant activity (Feinstein and Klar, 2004). The biological function of F-spondin and SCO-spondin on the deflection of commissural axons in the neural tube was assessed respectively by experiments of gain and loss of function (Burstyn-Cohen et al., 1999) and by analyses of mutants with defective floor plate (Lehmann and Naumann, 2005). Furthermore, F-spondin and SCO-spondin were both shown to promote neurite outgrowth of various neuronal cell populations, in cell culture (see Meiniel et al., 2003, for a review). Thus, SCO-spondin is a complex modular protein which belongs to an emergent family of proteins containing group 2 TSRs. The exact functional significance of the various motifs remains to be analyzed experimentally but both the high degree of conservation and the pattern of expression of SCOspondin support the idea of a relevant role in the differentiation of the neural tube.
3.
Experimental procedure
M. musculus (CAD42654.1), R. norvegicus (CAF33425.1), H. sapiens (BN000852) and C. familiaris (BN000732) SCO-spondin proteins were predicted using Wise2 software (http://www.sanger.ac. uk/Software/Wise2/) versus B. taurus (CAC94914.1) protein sequence, which was previously determined by screening a bovine SCO cDNA library (Gobron et al., 2000). SSPO and Sspo gene symbols were respectively approved by HUGO Gene Nomenclature Committee (HGNC) and Mouse Genome Informatics (MGI). The consensus amino acid sequence of the SCO-spondin protein was determined from the multiple alignment of SCOspondin sequences of the following mammalian species: B. taurus, M. musculus, R. norvegicus and H. sapiens. Multiple sequence alignment was performed using ClustalW software (Thompson and Gibson, 1994; http://pbil.univ-lyon1.fr/). The consensus sequence generated by the ClustalW alignment was modified by hand. Three or four amino acids that were identical between the four species gave a capital letter within the consensus, whereas two identical amino acids gave a lower case letter. When all four amino acids were different, an X was used in the consensus for “any amino acid”.
The search for conserved motifs was performed directly on the mammalian SCO-spondin consensus using SMART sequence analysis software (http://smart.embl-heidelberg. de/). Conserved domains were labeled on the consensus sequence and further aligned using ClustalW software. Alignments were modified by hand. Information about conserved protein motifs was found in the description rubrics of the following databases: PFAM (http://pfam.cgb.ki.se/index.html), PROSITE (http://www. expasy.org/prosite/), MEROPS (Rawlings et al., 2004; http:// merops.sanger.ac.uk/), InterPro (http://www.ebi.ac.uk/interpro/ index.html) and SMART (http://smart.embl-heidelberg.de/).
Acknowledgments We thank Kirsty Bates, Senior Curator of the EMBL Nucleotide Database Curation Team, and Tam Paterson Sneddon, Gene Nomenclature Advisor of the HUGO Gene Nomenclature Committee (HGNC), for their contribution. Work in the laboratory of A. Meiniel is supported by INSERM. O. Meiniel is supported by a DRRT post-doctoral fellowship (Région Auvergne-Limousin).
REFERENCES
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s r e v
Review
The complex multidomain organization of SCO-spondin protein is highly conserved in mammals☆ Olivier Meiniel, Annie Meiniel⁎ INSERM UMR 384, Faculté de Médecine, 28 Place Henri Dunant, 63001 Clermont-Ferrand Cedex, France
A R T I C LE I N FO
AB S T R A C T
Article history:
The multidomain organization of SCO-spondin protein is a special feature of the chordate
Accepted 20 September 2006
phylum. This protein is expressed in the central nervous system (CNS) from the time a
Available online 27 November 2006
dorsal neural tube appears in the course of phylogenetical evolution. With the advance of the systematic whole genomes sequencing, we were able to determine the SCO-spondin
Keywords:
amino acid sequence in four mammalian species using the Wise2 software. From the
Mammalian SCO-spondin
ClustalW alignment of bovine (Bos taurus), human (Homo sapiens), murine (Mus musculus) and
Protein domain
rat (Rattus norvegicus) proteins, a consensus sequence for mammalian SCO-spondin was
TSR superfamily
determined and further validated with the dog (Canis familiaris) SCO-spondin sequence. The
Central nervous system
analysis of this consensus sequence is consistent with a very high degree of conservation in
Development
the amino acids composition and multidomain organization of SCO-spondin in mammals. In addition, the identification of conserved domains, namely, Emilin (EMI), von Willebrand
Abbreviations:
factor D (vWD), low-density lipoprotein receptor type A (LDLrA) domains, SCO repeats
CNS, central nervous system
(SCOR), thrombospondin type 1 repeats (TSR), a coagulation factor 5/8 type C (FA5-8C) or
CTCK, C-terminal cystine knot
discoidin motif and a C-terminal cystine knot (CTCK) domain, provides a greater insight into
EMI, Emilin
the putative function of this multidomain protein. SCO-spondin belongs to the TSR
FA5-8C, coagulation factor 5/8 type C
superfamily given the presence of a great number of TSR (26). A finer classification of the
LDLrA, low-density lipoprotein
TSR motifs in groups 1, 2 and 3 is proposed on the basis of different cysteine patterns.
receptor type A
Interestingly, group 2 TSR are present in a number of CNS developmental proteins including
SCO, subcommissural organ
R-spondins, F-spondins and Mindins.
SCOR, SCO-spondin repeat SP, signal peptide TIL, trypsin inhibitor-like TILa, trypsin inhibitor-like associated TSP-1, thrombospondin 1 TSR, thrombospondin type 1 repeat vWD, von Willebrand factor D vWDa, von Willebrand factor D associated
☆
Confidential accession numbers: Homo sapiens: BN000852; Canis familiaris: BN000732. ⁎ Corresponding author. Fax: +33 4 73 27 61 32. E-mail address: [email protected] (A. Meiniel).
0165-0173/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainresrev.2006.09.007
© 2006 Elsevier B.V. All rights reserved.
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Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . 2.1. Amino acids conservation . . . . . . . 2.2. Conservation of the modular structure 3. Experimental procedure. . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . .
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Introduction
The complete sequence and modular organization of SCOspondin were first characterized in Bos taurus (Gobron et al., 2000). This extra cellular matrix-like protein, a member of the thrombospondin type 1 repeat (TSR) superfamily (Adams and Tucker, 2000), has been shown to be expressed in the subcommissural organ (SCO), a derivative of the dorsal diencephalon, both in early ontogenetical development and during adult life (see Meiniel et al., 2003, for a review). SCO-spondin protein is also expressed transitory during embryonic stages in the floor plate (Richter et al., 2001; Meiniel, unpublished results). This ependymal secretion may well be involved in developmental processes, including commissural axon pathfinding (Lehmann and Naumann, 2005), similarly to other proteins of the TSR superfamily expressed early in the CNS (see Adams and Tucker, 2000, for a review). A biochemical conservation of the material secreted by the subcommissural organ was first revealed using immunological probes directed against Reissner's fibre (RF), a structure present all along the central canal of the spinal cord (Sterba et al., 1982; Rodriguez et al., 1984). Antibodies raised against bovine RF were able to specifically recognize the secretory material present in the SCO of all vertebrate species examined except anthropoid apes and human (Rodriguez et al., 1992; Oksche et al., 1993), and in the floor plate (Rodriguez et al., 1996; Lopez-Avalos et al., 1997; Yulis et al., 1998; Lichtenfeld et al., 1999). A high conservation of the sugar moities was also revealed using lectins. N-linked high-mannose chains were associated with the precursor form of the secretion located in the endoplasmic reticulum of the SCO secretory ependymal cells, and complex carbohydrate chains to the mature form in the secretory vacuoles and Reissner's fibre (Meiniel and Meiniel, 1985; Rodriguez et al., 1986; Meiniel et al., 1988). From this time, SCO-spondin was identified as the major and specific component of the SCO/RF complex, in the bovine (Gobron et al., 1996, 2000; Nualart et al., 1998; Didier et al., 2000). Recent progress in the systematic whole genome sequencing in several mammalian species permitted to analyze the degree of conservation of this protein. The ortholog to the bovine SCO-spondin gene was found in Homo sapiens on chromosome 7, in Rattus norvegicus on chromosome 4, in Canis familiaris on chromosome 16, and in Mus musculus on chromosome 6 (Gonçalves-Mendes et al., 2003). Based on these genomic data, SCO-spondin proteins could be predicted for these mammalian species
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using the Wise2 software. The remarkable conservation resulting from the comparison of these proteins led us to determine a consensus amino acid sequence for mammalian SCO-spondin. A general scheme of the multidomain organization could be drawn from this consensus and an improved description of the structural modules could be established. In addition, we propose a subdivision into three groups of the SCO-spondin TSR motifs on the basis of particular cysteine patterns. SCO-spondin, F-spondin, Mindin (Feinstein and Klar, 2004) and R-spondin (Kamata et al., 2004), which were shown to be expressed early in the CNS, actually all share group 2 TSRs and could therefore be involved in related developmental mechanisms.
2.
Results and discussion
2.1.
Amino acids conservation
A consensus sequence was established from the alignment of the M. musculus (mouse), R. norvegicus (rat), H. sapiens (human) and B. taurus (bovine) SCO-spondin proteins in order to investigate variations in the amino acids (AA) composition in mammals. Only 66 AA, from a total number of 5166 AA for the whole consensus sequence, were found to be different in the four species and thus were replaced by an X for “any amino acid” (Fig. 1A). This result is consistent with a very high degree of conservation between the four sequences of mammalian species. It was confirmed by comparing pairs of species using ClustalW software: this gave 87% identity between R. norvegicus and M. musculus, 79% between H. sapiens and B. taurus, 75% between H. sapiens and R. norvegicus, 72% between H. sapiens and M. musculus, 70% between B. taurus and M. musculus, 73% between B. taurus and R. norvegicus, for a final 62% identity between all four species. Furthermore, the informative interest of the consensus sequence given in Fig. 1A was validated by aligning it with the most recently predicted SCO-spondin homolog: the C. familiaris (dog) protein. This sequence actually showed 80.7% identity with the SCO-spondin consensus sequence determined in this study, whereas by comparing C. familiaris sequence to the other species, we obtained only 71% identity with M. musculus, 73% with R. norvegicus, 78% with H. sapiens and 79% with B. taurus. Thus, this consensus can be held as a reference to evaluate the conservation of further mammalian SCO-spondin sequences.
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2.2.
Conservation of the modular structure
In parallel with the high degree of conservation in the AA composition, we observed the same modular structure in the
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four species which allowed us to define a general scheme for mammalian SCO-spondin protein (Fig. 1B). The description of the original mosaic organization of SCOspondin (Gobron et al., 2000, in the bovine) was improved by
Fig. 1 – (A) Consensus amino acid sequence of the mammalian SCO-spondin protein. The signal peptide (SP) is boxed in brown and the Emilin (EMI) module is boxed in black. The von Willebrand D (vWD) domains are printed in white and boxed in pink and the von Willebrand D-associated (vWDa) regions are printed in black and boxed in pink. The SCO-spondin repeat (SCOR) modules are boxed in red with the core of these repeats printed in white. The low-density lipoprotein receptor type A (LDLrA) domains are boxed in green, the coagulation factor 5/8 type C (FA5-8C) motif is boxed in dark blue and the C-terminal cystine knot (CTCK) domain is boxed in yellow. The von Willebrand C (vWC) domain is boxed in yellow but printed in red. The thrombospondin type 1 repeat (TSR) motifs are boxed in blue, with group 1 TSRs printed in white, group 2 TSRs printed in black, and group 3 TSRs printed in red. (B) Scheme of the mosaic organization of the various structural domains.
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directly analyzing the consensus sequence given in Fig. 1A using SMART sequence analysis software. In fact, a newly defined motif called Emilin (EMI) was identified as a single motif at the NH2-terminal of the protein (Figs. 1A and B). The EMI domain (PFAM accession number PF07546) was first characterized by Doliana et al. (2000) and was shown to play a role in the protein multimerization, a function which is shared with the von Willebrand factor D (vWD) domains (PFAM accession number PF00094) also located in the NH2-terminal of SCO-spondin but in three repeated copies (Figs. 1A and B, Fig. 2A). Analysis of the association of EMI and vWD domains showed it to be specific to the SCO-spondin protein. In this paper, we also propose a so-called von Willebrand factor D-associated (vWDa) domain which is conserved in the COOH end of each of the three vWD motifs (Fig. 1A and B). However, vWDa domain has no accession number in databases: it was defined by hand from the ClustalW multiple sequence alignment. This vWDa region, together with the vWD itself, was also found four times in human mucins and von Willebrand factor precursors and may also be involved in protein multimerization. These EMI and vWD domains, together with another previously described motif named C-terminal cystine knot (CTCK), complete the description of the structural modules thought to be involved in polymerization. In fact, CTCK_2 (PROSITE accession number PS01225), a conserved domain located in the C-terminal end of SCO-spondin (Figs. 1A and B) and many other modular proteins, is required for the formation of dimers before the multimerization step. These structural motifs would partly explain the formation of Reissner's fibre, an aggregation of SCO-spondin proteins, present in the central canal of the spinal cord. We also identified another new module of an average length of 98 amino acid residues, named SCO-spondin repeat (SCOR), which was found to be repeated sixteen times in the SCO-spondin consensus sequence (Figs. 1A and B). There is no pattern referenced in any database for this particular conserved module; therefore, it was determined by hand on the basis of strong homologies with trypsin inhibitor-like (TIL) and trypsin inhibitor-like-associated (TILa) regions (PFAM accession numbers PF01826 and PF02345, respectively). The sixteen different SCORs found in SCO-spondin are particularly conserved in their middle part, called the core. The cores of SCOR motifs were aligned in order to show the particularly conserved cysteine positions which characterize this module (Fig. 2B). SCOR usually includes two additional conserved cysteines on the COOH side and four to six more on the NH2 side of its core. Fig. 1A shows the SCOR motifs boxed in red, with sides printed in black and cores printed in white. Only cores were aligned, since the conservation was much less remarkable on the NH2 and COOH sides. SCOR shares structural homologies with other protease inhibitors than TILs in the MEROPS peptidase database. For
instance, SCOR also displayed positive hits for serine protease inhibitors belonging to MEROPS family I19 (InterPro accession number IPR009041), i.e., vWC_out (SMART accession number SM00215) and the Pacifastin inhibitor. However, the best homology score obtained with the cores of SCOR motifs was the family of cysteine-rich protease inhibitor I8, clan IA (InterPro accession number IPR002919; PFAM accession number PF01826), to which TIL belongs. Inhibitors of family I8 inhibit serine peptidases which belong to family S1, and probably also M4. In the SCO-spondin consensus, there are several motif combinations involving SCORs: vWD-SCOR, SCOR-vWD, SCORLDLrA, TSR-SCOR, SCOR-TSR and SCOR-CTCK. Interestingly, we noticed that a similar type of arrangement was encountered in SCO-spondin, zonadhesins and immunoglobulin G (IgG) FC binding fragment. In fact, the combination of TIL-TILa-vWD was found several times in zonadhesins and the IgG FC binding region, while in SCO-spondin, both SCOR-vWD and vWD-SCOR arrangements were found. Furthermore, the presence of low-density lipoprotein receptor type A (LDLrA) domains (PROSITE accession number PS50068) repeated ten times in the consensus sequence (Figs. 1A and B, Fig. 2C) could provide a clue as to the function of SCORs. In fact, LDLrA are known to interact with proteases or protease inhibitors (Herz, 2001). Hence, there may be a functional link between LDLrAs and SCORs, which could both be involved in the regulation of either protease activation or protease inhibition. Therefore, maturation of a native and probably inactive form of the protein into active components might be processed with the cooperation of those motifs. Two types of motifs, coagulation factor 5/8 type C or discoidin (FA5-8C; PROSITE accession number PS50022 for FA5-8C_3) and thrombospondin type 1 repeat (TSR; PROSITE accession number PS50092) present in SCO-spondin consensus (Figs. 1A and B) were initially described in blood proteins, where they were shown to play a role in coagulation or platelet aggregation. Both motifs are found early in unicellular organisms: the FA5-8C module in bacteria as a sugar binding motif, and TSR in circumsporozoite (CS) protein of the parasite Plasmodium falciparum as an attachment module to hepatic cells. These motifs are recruited in several proteins to mediate various functions in diverse biological systems. For example, the FA5-8C motif is found twice in coagulation factors V and VIII while TSR is repeated three times in thrombospondin 1 (TSP-1). In mammalian SCO-spondin, FA5-8C was found once and twenty six TSRs were aligned on the basis of a conserved pattern of cysteine, tryptophan and arginine. Nevertheless, distinct patterns of cysteine residues led to a more accurate classification into 3 groups of TSRs (Fig. 2D): 16 group 1 TSRs are characterized by the absence of a cysteine in NH2 (xxxxxWxxW) and the presence of a cysteine in position +2 of the two conserved arginines (RxRxC); 8 group 2 TSRs show a cysteine in
Fig. 2 – Multiple sequence alignments of SCO-spondin conserved modules. Conserved cysteine residues are printed in red, whereas other conserved amino acids are printed in blue. X, for “any amino acid”, is printed in grey. (A) Alignment of von Willebrand D (vWD) domains. (B) Alignment of the cores of SCO-spondin repeats (SCORs). (C) Alignment of low-density lipoprotein receptor type A (LDLrA) domains. (D) Alignment of groups 1, 2 and 3 thrombospondin type 1 repeats (TSRs).
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amino terminus (CxxxxWxxW) and no cysteine in the central region of the TSR (RxRxx); 2 group 3 TSRs comprise a pattern of 8 conserved cysteines instead of 6 for groups 1 and 2. This variation in cysteine positioning and number probably results in changes in disulfide bond formation, and therefore in the 3D structure. Recently, Tan et al. (2002) described the crystal structure of TSRs in TSP-1. Each TSR is composed of three antiparallel strands and, compared to the TSRs of TSP-1, the addition of an extra cysteine at the first anti-parallel beta strand together with the absence of a cysteine at the second anti-parallel beta strand would define a second TSR group with a modified disulfide bond. Interestingly, several proteins containing TSRs of group 2 are expressed early in the course of neural development, namely R-spondin, F-spondin, Mindin and the relatives HB-GAM and Midkine (Kamata et al., 2004; Feinstein and Klar, 2004). SCO-spondin and F-spondin share a similar pattern of expression in the floor plate, flexural organ and subcommissural organ and could have a redundant activity (Feinstein and Klar, 2004). The biological function of F-spondin and SCO-spondin on the deflection of commissural axons in the neural tube was assessed respectively by experiments of gain and loss of function (Burstyn-Cohen et al., 1999) and by analyses of mutants with defective floor plate (Lehmann and Naumann, 2005). Furthermore, F-spondin and SCO-spondin were both shown to promote neurite outgrowth of various neuronal cell populations, in cell culture (see Meiniel et al., 2003, for a review). Thus, SCO-spondin is a complex modular protein which belongs to an emergent family of proteins containing group 2 TSRs. The exact functional significance of the various motifs remains to be analyzed experimentally but both the high degree of conservation and the pattern of expression of SCOspondin support the idea of a relevant role in the differentiation of the neural tube.
3.
Experimental procedure
M. musculus (CAD42654.1), R. norvegicus (CAF33425.1), H. sapiens (BN000852) and C. familiaris (BN000732) SCO-spondin proteins were predicted using Wise2 software (http://www.sanger.ac. uk/Software/Wise2/) versus B. taurus (CAC94914.1) protein sequence, which was previously determined by screening a bovine SCO cDNA library (Gobron et al., 2000). SSPO and Sspo gene symbols were respectively approved by HUGO Gene Nomenclature Committee (HGNC) and Mouse Genome Informatics (MGI). The consensus amino acid sequence of the SCO-spondin protein was determined from the multiple alignment of SCOspondin sequences of the following mammalian species: B. taurus, M. musculus, R. norvegicus and H. sapiens. Multiple sequence alignment was performed using ClustalW software (Thompson and Gibson, 1994; http://pbil.univ-lyon1.fr/). The consensus sequence generated by the ClustalW alignment was modified by hand. Three or four amino acids that were identical between the four species gave a capital letter within the consensus, whereas two identical amino acids gave a lower case letter. When all four amino acids were different, an X was used in the consensus for “any amino acid”.
The search for conserved motifs was performed directly on the mammalian SCO-spondin consensus using SMART sequence analysis software (http://smart.embl-heidelberg. de/). Conserved domains were labeled on the consensus sequence and further aligned using ClustalW software. Alignments were modified by hand. Information about conserved protein motifs was found in the description rubrics of the following databases: PFAM (http://pfam.cgb.ki.se/index.html), PROSITE (http://www. expasy.org/prosite/), MEROPS (Rawlings et al., 2004; http:// merops.sanger.ac.uk/), InterPro (http://www.ebi.ac.uk/interpro/ index.html) and SMART (http://smart.embl-heidelberg.de/).
Acknowledgments We thank Kirsty Bates, Senior Curator of the EMBL Nucleotide Database Curation Team, and Tam Paterson Sneddon, Gene Nomenclature Advisor of the HUGO Gene Nomenclature Committee (HGNC), for their contribution. Work in the laboratory of A. Meiniel is supported by INSERM. O. Meiniel is supported by a DRRT post-doctoral fellowship (Région Auvergne-Limousin).
REFERENCES
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