SCO-Spondin and RF-GlyI: TwoDesignations for the Same GlycoproteinSecreted by the Subcommissural Organ

Robert Didier, Isabelle Creveaux, Robert Meiniel, Alain Herbet, Bernard Dastugue,and Annie Meiniel*Laboratoire de Biochimie Medicale, INSERM U 384, Faculte de Medecine, Clermont-Ferrand, France

SCO-spondin and RF-GlyI are two designations forcDNAs strongly expressed in the bovine subcommissuralorgan (SCO), characterized, respectively, in 1996 and1998 by two different research groups. Because bothcDNAs were partial sequences and exhibited close sim-ilarities in their nucleotide and deduced amino acid se-quences, it was thought that they might be part of thesame encoding sequence. To find out, we performed39RACE using a SCO-spondin-specific upstream primer.From the RT-PCR product generated and by nested PCRtechniques, we amplified both SCO-spondin and RF-GlyIspecific products with the expected length. Also, probesgenerated from both PCR products hybridized to thesame major 14 kb transcript in Northern blot analyses,clearly showing that SCO-spondin and RF-GlyI cDNAsdo belong to the same encoding sequence. In addition,we amplified, cloned, and sequenced a PCR product of3 kb spanning both the known SCO-spondin and RF-GlyIsequences. The deduced amino acid sequence containsnine thrombospondin type 1 repeats that alternate withsequences sharing similarities with the D-domain of vonWillebrand factor. Taken together, these findings showthat SCO-spondin and RF-GlyI are two designations ofthe same gene encoding proteins secreted by the bovineSCO and forming Reissner’s fiber. In addition, comparedto the sequence provided by Nualart et al. (1998), weextended the reading frame and identified new con-served domains in the 39 end of SCO-spondin. The pu-tative function of SCO-spondin on axonal pathfinding isdiscussed regarding the presence of a great number ofthrombospondin type 1 repeats. J. Neurosci. Res. 61:500–507, 2000. © 2000 Wiley-Liss, Inc.

Key words: subcommissural organ; Reissner’s fiber; RF-GlyI; SCO-spondin; protein domains

The function of the subcommissural organ/Reissner’s fiber (SCO/RF) complex within the vertebratecentral nervous system (CNS) has long been a matter ofspeculation. RF is formed by aggregation of glycoproteinssecreted by the SCO; this thread-like structure runs fromthe edge of the SCO, located in the roof of the thirdventricle, to the caudal end of the central canal (Oksche,

1969; Oksche et al., 1993; Meiniel et al., 1996). Themolecular characterization of the glycoproteins secreted bythe SCO and giving rise to RF may help in assigning afunction to the SCO/RF complex. Recently, SCO-spondin has been defined as a large multidomain glyco-protein (Gobron et al., 1996) synthesized by the SCO andcontributing to the formation of Reissner’s fiber. Thepartial sequencing and characterization of SCO-spondinprovided evidence that this molecule shared conserveddomains called thrombospondin type 1 repeats with othermembers of the thrombospondin superfamily expressed inthe developing vertebrate nervous system, includingthrombospondins 1 and 2, F-spondins 1 and 2, mindins 1and 2, and semaphorins F and G. In all these molecules,the thrombospondin type 1 repeats is thought to be im-portant in neurite outgrowth by promoting cell-to-substratum adhesion in reference to the thrombospondinsactivity (Neugebauer et al., 1991; O’Shea et al., 1991;Osterhout et al., 1992; DeFreitas et al., 1995).

Using immunological screening of a bovine SCOcDNA library, a strategy similar to that employed to isolatethe first SCO-spondin cDNA clone (Meiniel et al., 1995),Nualart et al. (1998) have recently characterized a cDNAclone corresponding to the carboxy-terminal region of aprotein designated as RF-GlyI, which is also expressed inthe subcommissural organ. This cDNA displays a relativelyhigh degree of homology with the SCO-spondin partialcDNA, and the corresponding peptidic sequence containsthrombospondin type 1 repeats, as with SCO-spondin. Allthese data raised the question of whether several proteinsexhibiting close similarity could be synthesized in theSCO, or whether SCO-spondin and RF-GlyI sequencescould correspond to two different regions of a largercDNA encoding the same molecule.

Contract grant sponsor: IRME “Institut de Recherche sur la MoelleEpiniere.”

*Correspondence to: Annie Meiniel, Laboratoire de Biochimie Medicale,INSERM U 384, Faculte de Medecine, 28 Place Henri-Dunant 63001Clermont-Ferrand Cedex, France.E-mail: Annie.Meiniel@inserm.u-clermont1.fr

Received 11 October 1999; Revised 30 April 2000; Accepted 22 May 2000

Journal of Neuroscience Research 61:500–507 (2000)

© 2000 Wiley-Liss, Inc.

In this study, we show that, after extraction ofmRNAs from bovine SCO and 39 rapid amplification ofcDNA ends (RACE) using an SCO-spondin-specificprimer, both SCO-spondin and RF-GlyI sequences couldbe amplified by nested PCR. Used as probes, these PCRproducts labeled the same major 14 kb transcript in North-ern blot analysis. Using the same procedure, we alsoamplified a PCR product of 3 kb overlapping the SCO-spondin and RF-GlyI known sequences, which providesdirect proof that both sequences belong to the same en-coding sequence. Furthermore, the SCO-spondin readingframe was extended to the C-terminal cystine knot do-main.

MATERIALS AND METHODS

SCO of bovine fetuses were dissected out at the localslaughterhouse and immediately frozen in liquid nitrogen.Poly-A1 mRNAs were isolated using the QuickPrep MicromRNA purification kit (Pharmacia, Gaithersburg, MD).

3*RACE

To obtain the nucleotide sequence encoding the carboxy-terminal portion of SCO-spondin a 39RACE procedure wasemployed. Four hundred nanograms Poly-A1 mRNA was usedas a template for first-strand cDNA synthesis. Reverse transcrip-tion was performed in the presence of the Moloney murineleukemia virus reverse transcriptase (M-MuLV-RT; Boehr-inger, Mannheim, Germany) and using an oligo-(dT) anchorprimer, an oligonucleotide composed of an anchor primer fol-lowed by 17 thymidine residues at its 39 end (59-CTCGACTCGAGTCGACATCG-T17-39). One-tenth of thefirst-strand cDNA was used as a template for PCR amplification.Amplification was performed using the anchor primer that bindsto each cDNA at its 39 end and a forward primer SCO1(59-CTGCAGCCGCAGCTGTAACGTG-39) specific to theSCO-spondin gene that anneals to 1.8 kb upstream from the 39end of the known sequence (AJ 132106; see Fig. 1). Sampleswere amplified with the expanded long template PCR system(Boehringer) according to the supplier’s protocol. Conditionsfor first-round PCR were 30 cycles of denaturation for 30 sec at94°C, annealing for 30 sec at 60°C and elongation for 6 min at68°C.

Nested PCR

The product from the first round of PCR was purifiedusing a PCR purification kit (Qiagen, Chatsworth, CA) asspecified by the manufacturer, and 1/50 was used as a templatein a second round of PCR. With internal primers, three nestedPCRs were performed (Fig. 1). Primers SCO2 (59-CCCGTGGACTTCTCCACCTGTG-39) and SCO3 (59-GTGCCGCTCCCCAGTCAG-39), located downstream fromthe primer SCO1, were used for PCR amplification of aninternal 920 bp fragment on the SCO-spondin sequence. FromRF-GlyI sequence (AF 078930), primers RF1 (59-ATACCTGAGCCCTTGGCTCTGC-39) and RF2 (59-GCAGCTCCGATGTCTTTTGGTGG-39) were used to pro-duce a 1,052 bp amplification product. Amplification wasperformed under the following cycle conditions: 2 min at 94°Cand 30 cycles of 30 sec at 94°C (denaturation), 30 sec at 62°C

(annealing), and 2 min at 68°C (elongation). In the third PCR,primer SCO4 (59-GCAGGTGGCCAGGATCTTCTCC-39)was used against part of the existing SCO-spondin sequence,216 bp upstream from the 39 end, and primer RF3 (59-GCCTTGGCTGGTGCACTGTAGC-39) was situated 372 bpdownstream from the 59 end of the known sequence of RF-GlyI. The thermal cycling program was as described above,except for elongation at 68°C for 4 min.

The amplified products were electrophoresed on a 0.8%agarose gel. The bands were excised from the gels, and the DNAwas extracted using the QIAquick gel extraction kit (Qiagen)according to the manufacturer’s instructions. PCR productswere then subcloned into pGEM-T vector (Promega, Madison,WI) before DNA sequencing.

Sequencing

The subclones were entirely sequenced on both strandsusing SP6 and T7 primers and specific oligonucleotides. Se-quencing reactions were performed using the Big Dye-terminator cycle sequencing kit (Applied Biosystems, FosterCity, CA) on a Perkin Elmer GeneAmp 9600 thermocycler.Sequencing products were analyzed on an ABI 377 automatedsequencer with Sequencing Analysis v.2.1.2 software (AppliedBiosystems). Nucleotide and amino acid sequences were com-pared to the nonredundant sequence databases at the NationalCenter for Biotechnology Information (NCBI) using version 2.0 ofBLAST (Altschul et al., 1997). Protein similarity searches wereperformed with the ProfilScan program on the server available athttp://www.isrec.isb-sib.ch/software/PFSCAN_form.html.

Northern Blot Analysis

Two micrograms of poly-A1 mRNAs were fractionatedon 1% agarose gels containing 6% formaldehyde (Lehrach et al.,1977) and blotted onto Nytran membranes (Schleicher andSchnell, Keene, NH) overnight. The mRNAs were fixed bybaking at 80°C for 2 hr.

Prehybridization was performed at 65°C for 1 hr (40%formamide, 0.75 M NaCl, 5% dextran sulfate, 50 mg/ml hep-arin, 1% SDS, 10 mg/ml poly-A, 50 mg/ml salmon spermDNA). After random-primed labeling (Oncor-Appligene,Gaithersburg, MD), hybridization was carried out overnight at42°C in the same solution containing 2 3 106 cpm/ml cDNAprobe. Washes were performed at 42°C as follows: 23 SSC,20 min; 23 SSC, 20 min; 0.53 SSC, 0.1% SDS, 20 min. Finalwash was with 0.53 SSC, 0.1% SDS at 65°C. Autoradiographicexposure was usually for 2 days using one intensifying screen at280°C.

The filter was first hybridized with the RF-GlyI RT-PCR product. The probe was then removed using the followingstripping procedure: The filter was placed at 80°C in 50%formamide, 0.053 SSC, 0.05% SDS, 20 mM Tris-HCl, pH 7.5,for 1 hr. The filter was then hybridized with the SCO-spondinRT-PCR product.

RESULTSSCO-Spondin and RF-GlyI as Part of the SameEncoding Sequence

After 39RACE using the anchor and SCO1 primers,nested PCR performed with SCO2 and SCO3 internal

SCO-Spondin, A Brain-Secreted Glycoprotein 501

primers specific for the SCO-spondin sequence (Fig. 1)generated a single band whose size, 920 bp, correspondedto the predicted base pair length (Fig. 2). This PCRproduct was sequenced and perfectly matched the knownSCO-spondin sequence. On the same strand generated by39RACE, nested PCR with RF1 and RF2 primers (Fig. 1)derived from the RF-GlyI sequence, yielded the expectedproduct of 1,052 base pairs (Fig. 2). Sequencing analysisconfirmed the identity of this product with the known

RF-GlyI sequence, except for the presence of an addi-tional base leading to a modification and extension of theopen reading frame.

These two specific RT-PCR products, used asprobes in Northern blot analysis of embryonic subcom-missural organ poly-A1 mRNAs, revealed the presence ofa major transcript of 14 kb (Fig. 3). Because the same blotwas successively hybridized with both probes, the autora-diograms could be superimposed, clearly demonstratingthat they belong to the same transcript.

Amplification and Characterization of a SCO-Spondin/RF-GlyI Overlapping Clone

When pair primers (SCO4 and RF3; Fig. 1) wereboth used from the known SCO-spondin and RF-GlyIsequences, nested PCR enabled us to amplify a single bandof about 3 kb (Fig. 2). Nucleotide and deduced amino acidsequences of this PCR product are shown in Figure 4A.This contained 3,012 nucleotides, with a single openreading frame of 1,004 amino acids. As expected, thisproduct overlapped the 39 region of the known SCO-spondin sequence on 216 bp and the 59 region of theRF-GlyI sequence on 327 bp (Fig. 4A).

In using the ProfilScan program, database searchsimilarity indicated that this polypeptide chain of 1,004residues was arranged in different domains. The overallstructure of this region is illustrated in Figure 6. Ninethrombospondin (TSP) type 1 repeats made up of 54–59residues were found, accounting for approximately half ofthis sequence. On the basis of some conserved residues,these repeats can be aligned with repeats identified inthrombospondin (Fig. 4B). The conservation of nearly allthe cysteine residues and of several other amino acids, suchas tryptophane and arginine, was readily apparent. Thissequence also contained four domains rich in cysteineresidues, that displayed similarities with the D-domain ofVon Willebrand factor (Fig. 4C). Their homology with

Fig. 2. Agarose gel electrophoresis of nested PCR product. Lane M:DNA molecular weight marker (1 kb DNA ladder; Gibco-BRL, GrandIsland, NY). Lane 1: Amplification of the 920 bp fragment with theSCO-spondin pair primers. Lane 2: Amplification of the 1,052 bpfragment with the RF-GlyI pair primers. Lane 3: Amplification of the3,012 bp fragment spanning the SCO-spondin and RF-GlyI knownsequences.

Fig. 1. Schematic overview of the RT-PCR and nested PCR reactions with the position of the pairprimers and corresponding amplification products.

502 Didier et al.

the D-domain of von Willebrand factor (vWf) rangedbetween 36% and 46%, much of which was accounted forby the conservation of position of the 11 cysteine residues.These D-like domains were inserted between throm-bospondin repeats. Taken together, approximately 74% ofthe polypeptide chain consisted of one or the other ofthese repetitive structures. Finally, eight potential sites forasparagine-linked glycosylation (Asn-X-Ser/Thr) distrib-uted randomly were detected at positions 71, 98, 274, 397,429, 622, 832, and 903.

Extension of the C-Terminal End ofSCO-Spondin

Pair primers RF1 and RF2 yielded to a sequenceidentical to that published by Nualart et al. (1998), exceptfor the presence of an additional base (Fig. 5). This changein the nucleotide sequence led to a modification of theopen reading frame and an extention of the carboxy-terminal end of SCO-spondin. In this novel sequence of407 amino acids (Fig. 5), four additional domains, namely,one TSP type 1 repeat, one vWf D-domain, one vWfC-domain, and one carboxy-terminal cystine knot(CTCK) domain were characterized (Figs. 5, 6) comparedto the sequence published by Nualart et al. (1998). Thismodification of the stop codon position explains why aregion defined as untranslated could be screened usingantibodies (Nualart et al., 1998).

DISCUSSIONIn the present study, we provide several lines of

evidence that SCO-spondin and RF-GlyI cDNAs are partof the same sequence transcribed by the same gene. BothcDNAs were amplified from 39 extension of SCO-spondin and corresponded to a transcript of 14 kb previ-ously identified as the main SCO-spondin transcript spe-cific to the SCO profile (Creveaux et al., 1998). We alsocharacterized a PCR product whose open reading framelinked the two partial SCO-spondin and RF-GlyI se-quences, and we extended the SCO-spondin sequence upto the CTCK domain. Taken together, the data reportedhere demonstrate that RF-GlyI and SCO-spondin aremerely two designations for the same glycoprotein. Eventhough we emphasize that both RT-PCR products hy-bridized mainly to the same 14 kb transcript, very faintsignals could have escaped the analysis of transcripts gen-erated by a complex regulation of the SCO-spondin gene.We have already shown that probes located in the middleof the SCO-spondin coding sequence revealed minor,shorter transcripts (Creveaux et al., 1998). Thus, depend-ing on a specific mosaic organization, each isoform ofSCO-spondin may have a different function. A strikingfeature of the carboxy-terminal region of SCO-spondin isits modular organization without a particular arrangementof the various domains.

As with a number of molecules expressed in thedeveloping nervous system, such as F-spondins 1 and 2and mindins 1 and 2 (Klar et al., 1992; Higashijima et al.,1997) secreted by the floor plate or transmembrane sema-phorins F and G (Adams et al. 1996), repeated domainswith homology to the TSP type 1 repeats found in throm-bospondins 1 and 2 (for review see Adams et al., 1995) aredetected in this part of SCO-spondin. Several lines ofevidence implicate the TSP type 1 repeats of throm-bospondins 1 and 2 in cell adhesion and neurite outgrowthof several types of neurons (Neugebauer et al., 1991;O’Shea et al., 1991; Osterhout et al., 1992; DeFreitas etal., 1995). In addition, within this consensus domain thereare two well-defined functional sequences, CSXXCG andWSXWS, that each play a fundamental role in ligand–receptor interactions in other biological systems (Miyazakiet al., 1991; Tusynski et al., 1992; Adams et al., 1995).Likewise, peptides derived from an SCO-spondin TSP

Fig. 4 (figure appears on following page). Analysis of the 3 kb PCRproduct. A: Nucleotide and deduced amino acid sequence. SCO-spondin sequence (AC number AJ 132106) is underscored with acontinuous line and that of RF-GlyI (AC number AF 078930) with adashed line. B: Alignment of the TSP type 1 repeats in the 3 kb PCRproduct compared to a TSP type 1 repeat of thrombospondin 1. C:Alignment of the D-like domains of SCO-spondin compared to aD-domain of prepro-von Willebrand factor. Sequences were alignedwith Clustal W 1.6 (Thompson et al., 1994). The final output of thealignment was generated with BOXSHADE (K. Hoffman and M. Barron:http://ulrec3.unil.ch/software/BOX_form.html). Dashes indicate gaps inthe sequence. A black background indicates amino acid identity, andgray shading indicates amino acid similarity.

Fig. 3. Northern blot hybridization of embryonic SCO poly-A1 usingboth PCR products specific for RF-GlyI (A) and SCO-spondin (B).The same major 14 kb transcript was detected with these two probes.Arrowhead shows the start point of electrophoresis migration.

SCO-Spondin, A Brain-Secreted Glycoprotein 503

Figure 4 (figure legend appears on preceding page).

type 1 repeat and corresponding to the WSGWSSCSR-SCG sequence have been shown to increase adhesivity andneurite outgrowth of cortical neurons in primary cellcultures (Monnerie et al., 1998). Regarding the presenceof a great number of TSP type 1 repeats in the SCO-

spondin sequence including a WSXWSXCSXXCG se-quence, this molecule may be highly potent in promotingneurite outgrowth. The SCO-spondin gene is expressedearly in the SCO primordium located at the boundary ofthe dorsal diencephalum and mesencephalum beneath theposterior commissure (Meiniel et al., 1996). Thus, in away similar to that of the floor plate in the spinal cord, theSCO in the dorsal midbrain could steer commissural ax-ons, and SCO-spondin could participate to the posteriorcommissure formation.

The carboxy-terminal part of SCO-spondin also dis-plays homologies with mucins and the vWf. These cysteine-rich regions consist of repetitive elements with pronouncedsimilarities to the D-domain present in the N-terminal regionof human prepro-vWf (ppvWf; Sadler, 1998). In addition totheir presence in ppvWf, D-domains are also found in severalfunctionally diverse proteins, including some of the secretedmucins, MUC2 (Gum et al., 1992; 1994), MUC5 (Desseynet al., 1997), porcine submaxillary mucin (Eckhardt et al.,

Fig. 6. Schematic representation of the modular structure of thecarboxy-terminal region of the SCO-spondin. A: PCR product of 3 kbcharacterized in this study. B: Modification and extension of the 39 endof SCO-spondin compared to the sequence published by Nualart et al.(1998).

Fig. 5. Nucleotide and deduced amino acid sequence of the extensionof the terminal end of SCO-spondin. Arrow indicates the additionalbase (G) identified in the SCO-spondin sequence, which led to achange in the reading frame compared to the sequence published byNualart et al. (1998). The thrombospondin (TSP) type 1 repeat is

boxed. The vWf D-like domain is underscored with a continous line.The vWf C-like domain is shaded. The CTCK domain is underscoredwith a dashed line. An asterisk indicates the stop codon. The polyad-enylation signal is marked by a double underscore.

SCO-Spondin, A Brain-Secreted Glycoprotein 505

1997), insect humoral lectin hemocytin (Kotani et al., 1995),mouse inner ear matrix protein a-tectorin (Legan et al.,1997), and sperm membrane protein zonadhesin (Gao andGarbers, 1998). D-domains in human ppvWf, as in theproteins listed above, mediate the formation of disulfide-linked oligomers required for their biological function (Sa-dler, 1991; Ho et al., 1995). This suggests that similar do-mains present in SCO-spondin may also serve to forminterchain disulfide bonds as with several types of mucins(Dekker et al., 1991; Sheehan et al., 1991).

Compared to the sequence published by Nualart etal. (1998), extension of the carboxy-terminal region ofSCO-spondin led to the identification of additional do-mains, including a vWf C-domain with functional simi-larity to the D-domains and a CTCK domain. ThisCTCK structural domain is also found at the 39 end ofvarious secreted proteins, including vWf (Sadler, 1998);several mucins, i.e., MUC2 (Gum et al., 1992), MUC5(Dekker et al., 1991); FIM-B.1 (Hoffmann and Hauser,1993); and some growth factors, such as nerve growthfactor, transforming growth factor-b, platelet-derivedgrowth factor, and human chorionic gonadotrophin (Mc-Donald and Hendrickson, 1993; Isaacs, 1995; Sun and Da-vies, 1995). In all these molecules, the CTCK motif is in-volved in the dimerization of polypeptidic chains throughintermolecular disulfide bridges. Thus, SCO-spondin mole-cules very likely form dimers by their carboxy-terminal re-gion, a primary step before multimer assembly mediatedby the C- and D-domains as described above.

Thus, the structural feature of SCO-spondin arguefor its propensity to polymerize in the central canal of thespinal cord, where it forms a thread-like structure calledReissner’s fiber. Nevertheless, mechanisms for the assem-bly into multimers in vWf and SCO-spondin may besimilar but not strictly identical.

Modular or mosaic proteins, assembled from a vari-ety of repeating protein modules, are very frequent notonly in the extracellular matrix (Engel et al., 1994) but alsoin the immune, blood clotting, and other biological sys-tems (Bork, 1991; Engel, 1991; Doolittle, 1995). Thesemodules are mostly involved in both the structure of theproteins and the functional aspects leading to specificinteractions with other proteins, such as specific mem-brane receptors (Giry-Loringuez et al., 1994). In the CNS,the SCO-spondin gene encodes several isoforms with avariable multidomain organization, which together withthe primary structure suggests a wide variety of functions.However, the nature of the receptors interacting with thevarious motifs and the signals triggered by their activationremain to be elucidated before the exact significance of theSCO-spondin molecules in the brain can be determined.

ACKNOWLEDGMENTWe thank R. Ryan for help with English.

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