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Proc. NatI. Acad. Sci. USA Vol. 91, pp. 3334-3338, April 1994 Developmental Biology Drosophila syndecan: Conservation of a cell-surface heparan sulfate proteoglycan (orcptor/glycamnglcas/Wgrowth factors/evolution) JURG SPRING*t, STEPHENIE E. PAINE-SAUNDERSt, RICHARD 0. HYNES*, AND MERTON BERNFIELD*§ *Joint Program in Neonatology, Harvard Medical School, 300 Longwood Avenue, Enders 9, Boston, MA 02115; and tHoward Hughes Medical Institute and Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 Communicated by Marilyn Gist Farquhar, December 20, 1993 ABSTRACT In mmals, ceil-surface heparan sulfate is (12-14) and proposed to be involved in pioneer axon guidance required for the action of basic fibroblast growth factor, in cockroaches (15), but there is no evidence for cell-surface fibronectin, antithrombln m, as well as other effectors. The heparan sulfate proteoglycans in invertebrates. We describe syndecans, a gene family offour transmembrane proteoglycans here a transmembrane proteoglycan in Drosophila.1 This that participates in these interactions, are the major source of proteoglycan is related structurally to members of the ver- this heparan sulfate. Based on the conserved transmembrane tebrate syndecan family. As in mammals, this proteoglycan and cytoplasmic domains ofthe ma syndecans, a single bears heparan sulfate chains, is expressed at the cell surface, syndecan-like gene was detected and localized in theDrosophila and is shed as an intact extracellular domain. However genome. As in mammals, Drosophila syndecan is a heparan Drosophila, unlike mammals, appears to contain only a single sulfate proteoglycan expressed at the cell surface that can be syndecan, apparently derived from a common ancestor with shed from cultured cells. The single Drosophila syndecan is the vertebrate syndecan family, which is expressed in a wide expressed in embryonic tissues that correspond with those variety of tissues. tissues in mammals that express distinct members of the syndecan family predominantly. Conservation of this class of molecules suggests that Drosophila, like mammals, uses cell- MATERIALS AND METHODS surface heparan sulfate as a receptor or coreceptor for extra- Molecular Cloning. For PCR, two oligonucleotides (o79, cellular effector molecules. o96), corresponding to the most conserved regions ofthe stop transfer domain, and one reverse primer (o97), matching the Analyses of the fruit fly Drosophila melanogaster have nucleotide sequence encoding the C-terminal four amino revealed many developmentally important genes with coun- acids Glu-Phe-Tyr-Ala and the stop codon, were synthesized terparts in humans-e.g., genes for homeobox proteins, as a combination of the respective sequences of murine growth factors, or tyrosine kinases (1). Moreover, recent data syndecan 1(16) and human syndecan 2 (17). These contained on' polysialic acid in Drosophila suggest phylogenetic con- 15-18 specific nucleotides and, at the 5' end, restriction servation ofpotentially important glycans (2). It has become enzyme cleavage sites: (o79) CGTCGACGSATGARRAA- apparent that heparan sulfate, a glycosaminoglycan that is GAAG, (o96) CGTCGACATGARRAAGAAGGAYGA, and ubiquitous on adherent cells in mammals, is involved in the (o97) CTCTAGAYYASGCRTARAAYTC, using the code Y functions of several classes of effector molecules. For ex- = CorT, S = CorG, andR = A orG. ForPCR, 1 pgof ample, to induce proliferation, migration and differentiation, genomic DNA was heated at 950C for 5 min in 100 A& of PCR fibroblast growth factor (FGF) family members must bind to mixture (10 mM Tris-HCl, pH 8.3/50 mM KCl/1.5 mM heparan sulfate to activate their tyrosine kinase receptors MgCl2/200 AM dNTPs/1 jkM primers) and cooled to the (3-5). The binding specificity of the heparan sulfate may temperature for 30 sec. Then 2.5 units of Taq switch developmentally to accommodate different FGFs (6). annealing This coreceptor function of cell-surface heparan sulfate (7) polymerase and a drop ofmineral oil were added, followed by also involves the binding of a variety of extracellular matrix a 1-min extension at 720C. After 20 cycles of 1 min at 950C, components like fibronectin, cell adhesion molecules like 30 sec at 350C, and 1 min at 720C with primers o79 and o97, N-CAM, or protease inhibitors like antithrombin III. In 90 Ad ofthe reaction products was separated on a 3% NuSieve mammals, the syndecans are a family of four distinct gene 3:1 agarose gel. The region corresponding to the expected products that contain a cluster ofheparan sulfate attachment size of syndecan-related products of 90-120 nt was cut out sites near the N terminus in the extracellular domain and from the gel and transferred to a Spin-X centrifuge filter unit highly conserved transmembrane and cytoplasmic domains and centrifuged at 14,000 x g for 30 min to elute the DNA. (for review, see ref. 7). These proteoglycans are develop- Ten microliters of the eluate was reamplified as above with mentally regulated and are thought to play an important role the primers o96 and o97 for 10 cycles at an annealing in signal transduction because they link the cytoskeleton via temperature of 37TC followed by an additional 30 cycles at the cytoplasmic domains to extracellular ligands bound to the 550C. The reaction products were separated on a 5% NuSieve heparan sulfate chains (7). 3:1 agarose gel; visible bands were cut, purified, sequenced, Whether Drosophila contains proteins, like the FGFs and and if they were similar to syndecan sequences, subcloned fibronectin, that bind to heparan sulfate and function in a into the plasmid pBluescript SK-. An aliquot ofan 8- to 12-hr comparable manner is not known. These ligands have been Drosophila embryo cDNA library in the plasmid pNB40 was elusive in Drosophila, although homologues of their recep- screened by PCR using an SP6 primer with two specific tors-e.g., integrins and FGF receptors, are well studied (8-11). Heparan sulfate has been documented in insects Abbreviation: FGF, fibroblast growth factor. tPresent address: Institute ofZoology, University ofBasel, CH-4051 Basel, Switzerland. The publication costs ofthis article were defrayed in part by page charge §To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" 1The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U03282). 3334 Downloaded by guest on September 29, 2021 i-Ov.velopmental Biology: Spring et al. Proc. Natl. Acad. Sci. USA 91 (1994) 3335 primers for the candidate Drosophila syndecan sequence, Immunoanalysis. Conditioned medium was brought to 2 M (o106) GCGGTTATTCGCATTTT and (o108) GGCGATC- urea/150 mM NaCl/50 mM sodium acetate, pH 4.5/0.1% TCTTTGGCTC. After 40 cycles with primers SP6 and o106 Triton X-100 and rocked overnight with DEAE-Sephacel. with 1 min at 950C, 30 sec at 450C, and 2 min at 720C, 1 ul of The beads were washed with 2 M urea/150 mM NaCl/50 mM the 100-IlI reaction was reamplified with primers SP6 and sodium acetate, pH 4.5/0.1% Triton X-100 and the same o108 in a new reaction under the same conditions. Both buffer without urea. Elution was achieved by raising the salt reactions were run on a 3% NuSieve 3:1 agarose gel revealing concentration to 1 M. The samples were precipitated by the appearance of a prominent band in the second amplifi- adding 3 vol ofethanol with 1.3% potassium acetate at -200C cation. This 654-nt PCR fragment, corresponding to a part of overnight, centrifuged at 14,000 x g for 30 min, dried, the extracellular, the transmembrane, and a part of the suspended in 100 mM Tris-HCl, pH 7.2/5 mM EDTA/1 mM cytoplasmic domain, was purified, subcloned into the plas-' phenylmethylsulfonyl fluoride, and incubated with hepariti- mid pBluescript SK-, sequenced, and used as a probe nase at 10 milliunits/ml or chondroitinase ABC at 250 miii- throughout this work-e.g., RNA blot, screening for full- units/ml as indicated for 1 hr at 370C or for 10 min with an length cDNA clones from a Agtll library, and chromosomal equal volume of freshly prepared nitrous acid at pH 1.5, localization. For the latter, the fragment was used as a according to the method of Shively and Conrad (20). template in a random-primed labeling reaction incorporating biotin-16-dUTP, according to manufacturer's instructions RESULTS AND DISCUSSION (Boehringer Mannheim). Molecular Cloning of a Drosophia Syndecan. To determine Sequences were analyzed with the Genetics Computer whether the syndecans are evolutionarily conserved, we used Group package (18), MacVector 4.0, or at the National PCR and degenerate oligonucleotide primers derived from Center for Biotechnology Information using the BLAST net- the sequences encoding the conserved cytoplasmic domains work service. of the murine syndecan 1 (16) and human syndecan 2 genes Northern Analysis. The syndecan probe was labeled with (17). Amplification products from a first set of primers were digoxigenin by PCR and hybridized overnight to 5 pg of separated by agarose gel electrophoresis, the gel region with poly(A)+ RNA from 7.5- to 15-hr Drosophila embryos, sepa- the size expected for a syndecan-like sequence was cut out, rated on a denaturing formaldehyde gel, and blotted on a nylon and the eluted materials were reamplifled by using a new membrane. Hybridization was done at 42°C in 50% (vol/vol) internal primer in combination with one of the original formamide/5 x standard saline/citrate (SSC) (0.75 M NaCl/75 primers. In identical experiments, genomic DNA from Dro- mM sodium citrate, pH 7.0)/0.1% N-lauroylsarcosine/0.2% sophila yielded a single product with a syndecan-like se- SDS/yeast tRNA at 50 Ag/ml/2% Boehringer Mannheim quence, and genomic DNA from the mouse yielded four such blocking reagent, prepared and autoclaved as a 10o solution products, corresponding in sequence to syndecan 1, 2, 3, and in 0.1 M maleic acid buffer, pH 7.5.

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