©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Repetitive Ser-Gly Sequences Enhance Heparan Sulfate Assembly in Proteoglycans (*)

(Received for publication, January 31, 1995; and in revised form, September 1, 1995)

Lijuan Zhang (§) Guido David (1) Jeffrey D. Esko (¶)

From the Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294 Center For Human Genetics, University of Leuven, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We showed previously that the synthesis of heparan sulfate on betaglycan occurs at a Ser-Gly dipeptide flanked by a cluster of acidic residues and an adjacent tryptophan (Zhang, L., and Esko, J. D.(1994) J. Biol. Chem. 269, 19295-19299). A survey of the protein data base revealed that most heparan sulfate proteoglycans contain repetitive (Ser-Gly) segments (n geq 2) and a nearby cluster of acidic residues. To study the role of these amino acid sequences in controlling heparan sulfate synthesis, we have examined the assembly of glycosaminoglycans on Chinese hamster ovary (CHO) cell syndecan-1. The glycosylation sites were mapped by making chimeric proteoglycans containing segments of CHO syndecan-1 cDNA fused to Protein A. Two sites near the transmembrane domain (-EGSGEQ- and -ETSGEN-) were used solely for chondroitin sulfate synthesis, whereas three sites near the N terminus (-DGSGDDSDNFSGSGTG-) supported both heparan sulfate and chondroitin sulfate synthesis. The strongest sites for heparan sulfate synthesis consisted of the repeat unit, -SGSG-. An unusual coupling phenomenon occurred across the adjacent SG dipeptides, leading to a greater proportion of heparan sulfate than predicted by the behavior of each site acting independently. The clusters of acidic residues adjacent to the heparan sulfate sites play important roles as well. These sequence motifs suggest a set of rules for predicting whether heparan sulfate assembles at glycosylation sites in proteoglycan core proteins.


INTRODUCTION

Chondroitin sulfate and heparan sulfate proteoglycans contain glycosaminoglycan (GAG) (^1)chains attached to specific serine residues of core proteins. Studies of hybrid proteoglycans (^2)such as betaglycan, ryudocan, and syndecan-1 showed that two types of GAG attachment sites exist(1, 2, 3, 4) . One type carries heparan sulfate or chondroitin sulfate chains, whereas the other type bears only chondroitin sulfate. In the proteoglycan, betaglycan, a specific amino acid sequence drives heparan sulfate synthesis(2) . This site consists of a Ser-Gly dipeptide, a nearby cluster of acidic residues, and an adjacent tryptophan that augments the proportion of heparan sulfate made(2) . These structural elements may enhance the interaction of glycosylated core protein intermediates with a key alpha-GlcNAc transferase that initiates the formation of heparan sulfate(5, 6) .

Almost all cloned heparan sulfate proteoglycans contain a cluster of acidic residues near one or more putative heparan sulfate attachment sites, but only a few contain nearby tryptophan. To study whether other amino acid sequences surrounding GAG attachment sites might enhance heparan sulfate assembly, we have examined the assembly of GAGs on syndecan-1, a hybrid proteoglycan containing both heparan sulfate and chondroitin sulfate(7) . Kokenyesi and Bernfield (4) showed recently that the N-terminal half of mouse syndecan-1 contains heparan sulfate and chondroitin sulfate chains, whereas the C-terminal half contains only chondroitin sulfate. Five conserved Ser-Gly dipeptides may act as the sites for GAG addition, but the identity of the sites that prime heparan sulfate was not determined. Our study reveals that most of the heparan sulfate occurs at an attachment site containing (Ser-Gly)(2) through an unusual coupling mechanism. This sequence motif occurs frequently in heparan sulfate proteoglycans and always includes a cluster of acidic amino acids flanking the site. We also show that the biosynthetic capacity of the cell to make heparan sulfate and the amount of core protein expression affect the proportion of heparan sulfate assembled.


EXPERIMENTAL PROCEDURES

Cell Culture

Chinese hamster ovary cells (CHO-K1) were obtained from the American Type Culture Collection (CCL-61, Rockville, MD). Cells were grown in Ham's F12 medium supplemented with 7.5% (v/v) fetal bovine serum (Hyclone, Salt Lake City, UT), penicillin G (100 units/ml), and streptomycin sulfate (100 µg/ml) under an atmosphere of 5% CO(2), 95% air and 100% relative humidity. Cells were passaged with trypsin every 3-4 days and revived periodically from frozen stocks. Sulfate-deficient F12 medium was prepared with chloride salts instead of sulfate(8) , penicillin G (100 units/ml), and 10% (v/v) fetal bovine serum that had been exhaustively dialyzed against phosphate-buffered saline (PBS)(9) .

Radiolabeling Studies

Na(2)SO(4) (25-40 Ci/mg; 1 Ci = 37 GBq) was purchased from Amersham Corp. Syndecan-1 was labeled biosynthetically by incubating cells in sulfate-deficient medium containing SO(4) (100 µCi/ml) for 12 h. The medium was discarded, the radiolabeled monolayer was rinsed five times with cold PBS, and the proteoglycans were extracted with Triton X-100 buffer (1% (w/v) Triton X-100, 150 mM NaCl, 10 mM Na(2)HPO(4), 2 mM KH(2)PO(4), pH 7.4) containing protease inhibitors (5 mMN-ethylmaleimide, 5 mM benzamidine-HCl, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 1 µg/ml of pepstatin A). The extract was clarified by centrifugation (10,000 times g, 60 min), and the pellet was re-extracted with 5 ml of Triton X-100 buffer for 1 h at room temperature. The supernatants were combined, and solid urea was added to achieve a final concentration of 6 M. Bovine tracheal chondroitin sulfate A (1 mg, Calbiochem) was added as carrier, and the samples were applied to a 0.5-ml column of DEAE-Sephacel (Pharmacia Biotech Inc.) prepared in a disposable pipette tip and equilibrated in urea buffer (6 M urea, 0.2 M NaCl, 0.5% (w/v) Triton X-100, 25 mM Tris-HCl, pH 7.0, and protease inhibitors). The columns were washed with urea buffer (15 ml), and radiolabeled proteoglycans were eluted with 2.5 ml of urea buffer containing 1 M NaCl. The proteoglycans were desalted over a PD-10 column (Pharmacia) equilibrated in 10% (v/v) ethanol in water containing protease inhibitors. After lyophilizing the samples, they were dissolved in PBS containing protease inhibitors (10 ml).

Syndecan-1 Purification and Characterization

The proteoglycan extract was passed over an affinity column (0.5 ml) of anti-syndecan-1 2E9 monoclonal antibody (^3)coupled to Sepharose CL-4B(10) . The column was washed with 8 ml of PBS, followed by 2 ml of PBS supplemented with 0.5 M NaCl. Syndecan-1 was eluted from the resin with 5 ml of a solution containing 4 M guanidine hydrochloride, 50 mM sodium acetate (pH 5.8), and protease inhibitors. Salt and guanidine were removed from purified syndecan-1 by chromatography over a PD-10 column before further analysis. More than 95% of purified syndecan-1 rebound to the affinity column, and binding was independent of GAG composition(10, 11) .

A portion of affinity-purified syndecan was resuspended in 0.5 M NaOH containing 1 M sodium borohydride and incubated at 4 °C for 24 h to beta-eliminate the chains. The base was neutralized by adding 10-µl aliquots of 10 M acetic acid until bubble formation ceased. Radiolabeled syndecan-1 and the released GAG chains were analyzed by gel filtration HPLC (TSK G4000SW, 30 cm times 7.5 mm inner diameter, Pharmacia). Samples were eluted with 0.5 M NaCl in 0.1 M KH(2)PO(4) (pH 6.0) containing 0.2% Zwittergent 3-12 at a flow rate of 0.5 ml/min, and radioactivity in the effluent was determined by in-line liquid scintillation spectrometry using Ultima Gold XR scintillant (Packard Instrument Company, Downers Grove, IL). GAGs were digested with 20 milliunits of chondroitinase ABC (Seikagaku) in 50 mM Tris-HCl and 50 mM sodium acetate buffer (pH 8.0) containing protease inhibitors. Complete digestion of chondroitin sulfate by chondroitinase ABC was assured by monitoring the extent of conversion of the carrier to disaccharides (1 mg = 11.4 absorbance units at 232 nm). Nitrous acid-catalyzed deaminative cleavage of heparan sulfate was performed according to the low pH method of Shively and Conrad(12) .

cDNA Cloning of CHO Syndecan-1^4

Sequences coding for syndecan-1 were amplified from a CHO-K1/cDNA quick-clone library (Clonetech, Palo Alto, CA). The reaction mixture contained 2 units of pfu polymerase (Stratagene, La Jolla, CA), 1 ng of cDNA, and 100 pmol of sense and antisense primers. The primers (Oligo etc., Inc., Wilsonville, OR) were identical to the murine syndecan-1 cDNA sequence from nucleotides 240-264 and 1151-1175(13) . After 30 thermal cycles (1 min of denaturation at 94 °C, 2 min of annealing at 55 °C, and 3 min of extension at 72 °C), the amplification products were analyzed by gel electrophoresis in 1% agarose gels and detected by ethidium bromide staining. The 900-base pair amplification product was excised from the gel and treated with the Gene Clean Kit (BIO 101, La Jolla, CA). The PCR product was phosphorylated, ligated into the SmaI restriction site of plasmid pBluescript SK (Stratagene), and introduced into competent E. coli DH5alpha cells. Positive clones from two separate PCR reactions were chosen and sequenced by the dideoxy chain termination method (14) using supercoiled plasmid, Sequenase version 2.0 kit (U.S. Biochemical Corp.), T3 and T7 primers, dGTP, and dITP.

PCR Fragments

pBluescript SK containing 927 base pairs of CHO syndecan-1 cDNA was used as a template for PCR of syndecan-1 fragments. In the oligonucleotides listed below, Nxxx refers to the 5`-end primer, and Cxxx refers to the 3`-end primer in the following oligonucleotides: N26, 5`-GGCGAATTCCGCTGTCAATGTGCCTCCTGAGG-3`; N32A, 5`-GGCGAATTCCGAGGATCAGGATGGCTCTGG-3`; N32B, 5`-GGCGAATTCCCAGAATCAG; AATGGCTCTGGGGATG-3`; N34, 5`-CAGGATGGCGCTGGGGATG-3`; N39, 5`-GATGACTCTAACAACTTCTCTGGCTCAGGC-3`; N42A, 5`-GACAACTTCGCTGGCGCAGGCACAGG-3`; N42B, 5`-GACAACTTCACTGGCTCAGGCACAGG-3`; N42C, 5`-GACAACTTCTCTGGCACAGGCACAGG-3`; N49, 5`-GGCGAATTCCACAGGTGCTTTGCCAGACATC-3`; N187, 5`-GGCGAATTCCATCAAAGAGGTTGCCGAGGATGG-3`; N207, 5`-GGCGAATTCCGAGCAGGATTTCACCTTTGAGAC-3`; C53, 5`-GGCGAATTCATGGCAAAGCACCTGTGCCTGAG-3`; C64, 5`-GGCGAATTCAAGTGGAAGAAGTCTGCCGTG-3`; C204, 5`-GGCGAATTCAGCCCTCCCCTGTAGGAAGTTG-3`; C214, 5`-GGCGAATTCATGTCTCAAAGGTGAAATCCTG-3`; C240, 5`-GGCGAATTCAACCTGTGGCTCCTTCATCCAC-3`. The numbered oligonucleotides are based on the cDNA sequence coding for CHO syndecan-1.^4 The boldface nucleotides stand for mutations in the underlined codons. The megaprimer PCR method was used for site-directed mutagenesis(15) .

The numbers before the colon in the following list of constructs refer to a segment of amino acids from CHO syndecan-1 (mutations in parentheses), and the information after the colon refers to the set of oligonucleotide primers and templates used to amplify the desired sequence. 26/64:N26/C64; 26/64(S37A):N26/(N34/C64); 26/64(S45T):N26/C(N42B/C64); 26/64(S47T):N26/C(N42C/C64); 26/64(S45A,S47A):N26/C(N42A/C64); 26/64(S37A,S45T):N26/C(N42B/C64), and the plasmid containing 26/64(S37A) was used as a template; 26/64(S37A,S47T):N26/C(N42C/C64), and the plasmid containing 26/64(S37A) was used as a template; 187/214:N187/C214; 49/204:N49/C204; 207/240:N207/C240; 32/53:N32A/C53; 32/53(EDQD/QNQN):N32B/C53; 32/53(DDSD/DDSN): N32A/C(N39/C53); 32/53(EDQD/QNQN, DDSD/DDSN):N32B/C(N39/C53).

The plasmid, pC17, containing the full-length fibroglycan cDNA sequence (M81687, a gift from Dr. John Gallagher, Manchester, United Kingdom) was used as a template for PCR of the fibroglycan fragment (DMYLDSSSIEEASGLYPIDDDDYSSASGSGAYEDKGSPDLTTSQ).

The wild-type betaglycan construct, SPGDSSGWPDGYEDLE, and the mutant, SPGDSSGAPDGYEDLE, were made using primers described previously(2) . Overlapping PCR primers were designed to generate the following betaglycan fragments (M77809).

Overlapping PCR primers were designed for the following peptide sequences.

PCR fragments were extracted with phenol/chloroform, precipitated with ethanol, digested with EcoRI, purified by agarose gel electrophoresis, and transferred to DEAE paper (Schleicher & Schuell). After elution, the fragments were ligated into a derivative of the eukaryotic expression vector pPROTA (16) that had been treated with EcoRI and calf intestinal alkaline phosphatase. All constructs were sequenced to confirm their identity.

Transfection

For transient transfection experiments, each well of a 12-well plate was seeded with 2 times 10^5 cells in F12 medium containing 10% (v/v) NuSerum (Collaborative Research, Inc., Bedford, MA). After one day, the medium was removed, and 0.5 ml of F12 medium containing 0.25 mg/ml of DEAE-Dextran (Sigma D-9885), 50 mM Tris-HCl (pH 7.4), 50 µg/ml of chloroquine, and 10 µg/ml of plasmid DNA was added to each well. After 2 h at 37 °C, the solution was aspirated, and 0.5 ml of 10% (v/v) Me(2)SO in PBS was added. After 2 min, the solution was aspirated and the cells were washed with 2 ml of F12 medium without serum. Cells were then labeled at 37 °C for 2 days with 50 µCi/ml of SO(4) in sulfate-deficient growth medium or with 50 µCi/ml of TRANS-LABEL, a mixture of [S]methionine and [S]cysteine (ICN, Irvine, CA).

Stable transfectants were created by transfecting a monolayer of wild-type CHO cells (50% confluence) with 16 µg of pPROTA containing CHO syndecan-1 cDNA coding for amino acid residues 26-240. The vector pMAMNEO was included (2 µg) along with 20 µg of Transfectam reagent using conditions recommended by the manufacturer (Promega Corp., Madison, WI). Two days later, the cells were harvested with trypsin, and a serial dilution (1:3, v/v) series was prepared in a 96-well plate. Strains resistant to G418 (400 µg/ml, corrected; Life Sciences Technologies, Inc.) were selected. The medium was changed every 4 days for 2 weeks, and individual colonies were picked, expanded, and evaluated for expression of chimeras as described above.

Fusion Protein Purification

Spent culture medium (1 ml) was mixed in an Eppendorf tube with 10 µl of a solution containing 1 M Tris-HCl (pH 7.5), 5% (w/v) Triton X-100, and 0.2% (w/v) sodium azide. Samples were centrifuged at 10,000 times g for 5 min, and the supernatant was decanted into a fresh tube. IgG-Agarose beads (20 µl, Sigma) were added, and the sample was mixed end-over-end overnight at 4 °C. Samples were centrifuged at 10,000 times g for 2 min. After aspirating the supernatant, the pellets were washed 3 times with 1.4 ml of buffer containing 50 mM Tris-HCl (pH 8), 0.15 M NaCl, and 0.02% sodium azide. Samples were dissolved in 20 mM Tris buffer (pH 7.0) and treated at 37 °C for 4 h with 10 milliunits of chondroitinase ABC, 2 milliunits of heparin lyase III (EC 4.2.2.8), or both enzymes. The reactions were stopped by adding SDS-PAGE sample buffer and heating them for 7 min in a boiling water bath. Samples were loaded on a 5-16% linear gradient SDS-PAGE gel (200 times 160 times 1.5 mm) and electrophoresed overnight at constant voltage (80 V). The gel was dried onto a piece of filter paper and visualized by either autoradiography or by imaging (Molecular Dynamics PhosphoImager, Sunnyvale, CA). The distribution of counts was quantitated using ImageQuant software (Molecular Dynamics).

Another set of samples was treated for 24 h at 4 °C with 100 µl of 0.5 M NaOH containing 1 M NaBH(4) to beta-eliminate the GAG chains. [S]GAGs were isolated by anion exchange chromatography as described(17) . Samples labeled with TRANS-LABEL were dissolved in 1 ml of 25 mM Tris-HCl buffer (pH 7.0) containing 6 M urea, 0.5% Triton X-100, and 0.25 M NaCl. An aliquot was counted by liquid scintillation spectrometry.


RESULTS

CHO Syndecan-1

Syndecan-1 represents 15-20% of CHO cell S-proteoglycans based on binding to an affinity column composed of the 2E9 monoclonal antibody against human syndecan-1 described by Lories et al.(10) . All of the affinity-purified material behaved like proteoglycan since the S-counts eluted in the V(o) of a gel filtration column and shifted to included fractions after beta-elimination (Fig. 1). Treating purified syndecan-1 with low pH nitrous acid, which depolymerizes heparan sulfate chains by cleaving at N-sulfated glucosamine residues(12) , converted 80-85% of the counts to small S-oligosaccharides and free SO(4). A residual core protein containing heparan sulfate stubs and chondroitin sulfate emerged at a K of 0.27. Treating a sample with chondroitinase ABC, which depolymerizes chondroitin sulfate chains to S-disaccharides, caused 15-20% of the S-counts to elute in the V(t) of the column (Fig. 1A). Thus, CHO syndecan-1 consisted of 80-85% heparan sulfate and 15-20% chondroitin sulfate. The same composition was obtained when the released GAG chains were analyzed by enzymatic digestion (Fig. 1B).


Figure 1: Gel filtration chromatography of CHO syndecan-1. Nearly confluent monolayers of cells were incubated for 12 h in sulfate-deficient medium containing 100 µCi/ml of SO(4). Syndecan-1 was purified from Triton X-100-extracted proteoglycans by affinity chromatography (see ``Experimental Procedures''). One portion was treated with alkali to beta-eliminate the GAG chains. Samples were analyzed by gel filtration HPLC before and after treatment with nitrous acid or chondroitinase ABC (see ``Experimental Procedures''). A, syndecan-1 proteoglycan. B, syndecan-1 GAG chains. bullet, intact syndecan-1 or GAG chains; up triangle, nitrous acid-treated material; , chondroitinase ABC-treated material.



There are five possible GAG attachment sites in mouse, human, and rat syndecan-1 based on the presence of Ser-Gly dipeptides(7) . The same five sites were present in CHO syndecan-1, which was cloned by PCR using the mouse cDNA sequence to design appropriate primers (Fig. 2A; (13) ). The CHO syndecan-1 amino acid sequence showed 94, 87, 86, and 77% homology to the Syrian golden hamster, rat, mouse, and human sequences, respectively (Fig. 2B). More importantly, the amino acid sequences near the putative GAG attachment sites are highly conserved among all the different species(7) .


Figure 2: Sequence of CHO syndecan-1 and comparison with sequences from other species. A, nucleotide sequence (upper line) and predicted amino acid sequence (lower line) of CHO syndecan-1 cDNA. CHO syndecan-1 was amplified from the CHO-K1 quick-clone cDNA library by PCR. The sequence of the PCR primers, indicated in lowercase letters, was derived from mouse syndecan-1(13) . The underlined sequences define the fragments for generating the chimeras shown in the tables (GenBank accession number L38991). B, comparison of predicted protein sequence of syndecan-1 from CHO, Syrian golden hamster (SGH, M29967)(36) , mouse (Mur, X15487)(13) , rat (Rat, M81785)(37) , and human (Hum, X60306)(10) . -, identical amino acids; *, a gap to improve alignment; boldface letters, potential GAG attachment sites.



Kokenyesi and Bernfield (4) reported that both heparan sulfate and chondroitin sulfate chains were present on the N-terminal half of murine syndecan-1, but only chondroitin sulfate was found on the C-terminal half. Of the five potential GAG attachment sites, three reside in the N-terminal half and two in the C-terminal half. We showed previously that the synthesis of heparan sulfate on betaglycan requires a cluster of acidic residues located near a specific Ser-Gly attachment site(2) . Two clusters of acidic amino acids border the three Ser-Gly sites on the N-terminal part of the syndecan-1 (-EDQDGSGDDSDNFSGSGTG-, Fig. 2B), consistent with the idea that one or more of these sites might support heparan sulfate assembly.

To examine how GAGs assemble at the individual sites, we prepared chimeras composed of CHO syndecan-1 segments fused to the IgG binding domain of Staphylococcal Protein A with a signal peptide from the secreted metalloprotease, transin, attached to the N terminus(16) . The chimeras were introduced into wild-type CHO cells by transient transfection, and the attachment of GAG chains was assessed by SO(4) incorporation into secreted chimeras (``Experimental Procedures''). The activity of each Ser-Gly site was measured independently by mutating other potential sites in the constructs (Table 1). All three Ser-Gly dipeptides toward the N terminus (-EDQDGSGDDSDNFSGSGTG-) were capable of priming heparan sulfate chains, but their relative ability varied from 9% at Ser to 23% at Ser. In contrast, the two sites near the transmembrane domain (-PTGEGSGEQDFTFETSGENTA-) primed only chondroitin sulfate. A sixth Ser-Gly dipeptide exists in both CHO and Syrian golden hamster syndecan-1 (Ser), but not in mouse, rat, and human syndecan-1. Expression of a 156-amino acid chimera containing residues 49-204 did not result in incorporation of SO(4) into GAGs (Table 1), suggesting that Ser does not act as a glycosylation site.



The extent of substitution of the chimeras with heparan sulfate was less than expected from the study of endogenous syndecan-1, which contained 80-85% heparan sulfate (Fig. 1). The difference was not due to variation in the extent of sulfation of heparan sulfate and chondroitin sulfate chains, since comparable results were found when the cells were labeled with [6-^3H]GlcN and chain length did not vary significantly (Fig. 3). The difference also was not due to poor expression of the chimera since only 20% of material labeled with TRANS-LABEL was converted to high molecular weight proteoglycan.


Figure 3: Gel filtration chromatography of [S]syndecan-1 chimeras. Syndecan-1 chimeras containing the sequences -EDQDGAGDDSDNFSGTGTG- and -EDQDGAGDDSDNFSGSGTG- were affinity-purified from SO(4)-labeled cells (see ``Experimental Procedures''). A portion of material was beta-eliminated to liberate the [S]GAGs from the chimeras, and an aliquot was digested with chondroitinase ABC or nitrous acid (see ``Experimental Procedures''). The samples were then analyzed by gel filtration HPLC (see ``Experimental Procedures''). A, intact chimeras. B, beta-eliminated GAG chains. C, heparan sulfate and chondroitin sulfate chains. circle, chimeras with a single acceptor site (-EDQDGAGDDSDNFSGTGTG-64); bullet, chimeras with two acceptor sites (-EDQDGAGDDSDNFSGSGTG-); up triangle, beta-eliminated chains from the single site chimera; , beta-eliminated chains from the two-site chimera; box, heparan sulfate chains from the two-site chimera; , chondroitin sulfate chains from the two-site chimera.



We also tested the chimeras in CHO ldlD cells, which cannot obtain UDP-GalNAc from UDP-GlcNAc due to a deficiency in the 4`-epimerase that interconverts the nucleotide sugars(18) . This strain makes less chondroitin sulfate when deprived of exogenous GalNAc(19) . When the chimeras were introduced into ldlD cells, the amount of heparan sulfate was enhanced (up to 79%), but the relative amount made at each N-terminal site remained about the same (Table 1, values in parentheses). Interestingly, Ser (-TGEGSGEQDF-), which primed only chondroitin sulfate in wild-type cells, generated about 28% heparan sulfate in ldlD cells. The other chondroitin sulfate site at Ser (-TFETSGENTA-) remained ineffective.

Coupling across Adjacent Ser-Gly Attachment Sites

A key factor controlling the extent of heparan sulfate substitution emerged from studies of chimeras containing more than one GAG attachment site. A 39-amino acid chimera covering residues 26-64 and containing the two adjacent sites at Ser and Ser yielded about 48% heparan sulfate (Table 1). An identical construct containing all three N-terminal sites gave 60% heparan sulfate. Enhanced synthesis also occurred in constructs containing either Ser and Ser or Ser and Ser (Table 1). These findings suggested that some type of coupling occurs across nearby attachment sites that increases the proportion of heparan sulfate.

To test whether coupling was dependent on flanking amino acid sequences, a repetitive SGSG sequence was introduced into betaglycan. In betaglycan, the site that supports heparan sulfate synthesis contains Ser-Gly-Trp flanked by a cluster of acidic residues (Table 2). We showed previously that converting the Trp residue to Ala reduced the level of heparan sulfate from 54 to 21% (2) . When a second Ser-Gly dipeptide was substituted for the Ala residue, the normal level of heparan sulfate synthesis was restored (51%, Table 2). Thus, the coupling across adjacent Ser-Gly sites appeared to be independent of surrounding sequence. Separating the Ser-Gly repeats by one or more residues reduced the proportion of heparan sulfate, suggesting that the coupling depended on proximity (Table 2). Inserting a Trp residue next to the repeat Ser-Gly segment gave a slight enhancement of heparan sulfate (58 versus 51%).



A stable transfectant expressing a chimera with all five sites (residues 26-240) was analyzed (Table 3). Unlike the behavior of the transient transfectants, the stably transfected cell line converted all of the chimera to high molecular weight proteoglycan (data not shown) and produced about 3-fold more total S-proteoglycan than nontransfected control cells (Table 3). The proportion of heparan sulfate was reduced in the chimera (55%) compared with the proteoglycans found in nontransfected parental cells (72%). Interestingly, the endogenous CHO cell proteoglycans also contained less heparan sulfate in the stable transfectant (54-62%). These findings suggested that the reduced level of heparan sulfate may have been due in part to enhanced core protein expression. Similar effects have been observed when full-length syndecan and betaglycan constructs were expressed in cells(1, 4) .



To gain insight into the mechanism that results in a higher proportion of heparan sulfate at adjacent attachment sites, we analyzed the GAG substitution pattern of syndecan-1 chimeras containing one site (-EDQDGAGDDSDNFSGTGTG-) and two sites (-EDQDGAGDDSDNFSGSGTG-). As expected, the two-site chimera migrated with a greater hydrodynamic volume during gel filtration than the chimera containing one site (Fig. 3A). beta-elimination shifted the [S]GAG to a more included position, but the mixture of GAG chains on both chimeras had essentially the same elution position (Fig. 3B). Analysis of the heparan sulfate and chondroitin sulfate chains on the two-site chimeras showed that they were comparable in size as well (Fig. 3C).

The two-site chimera presumably consisted of glycoforms containing one or two GAG chains, with various combinations of heparan sulfate and chondroitin sulfate chains. Analysis of S-labeled material by SDS-PAGE revealed smeared bands characteristic of proteoglycans (Fig. 4, lane 3). The material of faster mobility migrated in the same position as a chimera containing only one attachment site (compare lane 3 with lanes 1 and 2), suggesting that it represented material with only one GAG chain. The smear above this region presumably contained S-chimeras with two chains, although some overlap occurred with molecules containing one chain. Using the divisions shown in Fig. 4, we found that 50% of the total S-counts were in chimeras containing two chains. This material represented about one-third of the chimeras, given that the size of the chondroitin sulfate and heparan sulfate chains did not vary significantly (Fig. 3C). The degree of sulfation of heparan sulfate and chondroitin sulfate differs somewhat (0.8 sulfate groups/disaccharide versus 1 sulfate/disaccharide)(20) , but this effect only moderately underestimates the extent of substitution by heparan sulfate.


Figure 4: Electrophoresis of [S]syndecan-1 chimeras. Chimeras were purified from SO(4)-labeled cells by IgG affinity chromatography and dissolved in SDS-PAGE protein sample buffer. The samples were analyzed on SDS-polyacrylamide (5-16%) gradient gels, and the dried gel was imaged for data collection (see ``Experimental Procedures''). The positions of chimeras containing one and two GAG chains are indicated to the right of the gel, and the protein molecular weight standards are indicated to the left of the gel. Each lane contained the following insert derived from syndecan-1: lane 1, -EDQDGAGDDSDNFS45GTGTG-; lane 2, -EDQDGAGDDSDNFTGSGTG-; lane 3, -EDQDGAGDDSDNFSGSGTG-; lane 4, -EDQDGAGDDSDNFSGSGTG- after treatment with chondroitinase ABC; lane 5, -EDQDGAGDDSDNFSGSGTG- after treatment with heparin lyase III; lane 6, -EDQDGAGDDSDNFSGSGTG- after treatment with both chondroitinase ABC and heparin lyase III. Equal portions of the samples were analyzed to show the reduction in signal due to enzymatic digestion.



Digesting a sample with chondroitinase ABC and heparin lyase III prior to electrophoresis removed all of the upper band material (lane 6). Chondroitinase ABC (lane 4) removed all of the proteoglycans from the upper smear that contained chondroitin sulfate chains, leaving behind only those molecules containing two heparan sulfate chains. This resistant material represented about 42% of the counts in the upper smear or about 14% (one-third of 42%) of the total S-proteoglycans. Treating samples with heparin lyase III (lane 5) removed all of the glycoforms containing heparan sulfate, and only those molecules containing two chondroitin sulfate chains remained. The resistant material represented about 32% of the counts in the upper smear or about 11% (one-third of 32%) of the total S-proteoglycans. By subtraction, hybrid chimeras containing one chondroitin sulfate and one heparan sulfate chain represented 26% of the upper smear counts (100 - (42 + 32)), or 9% of the total proteoglycans. These findings are summarized in Table 4.



The extent of substitution by heparan sulfate in the two-chain glycoforms (14% of total chimeric proteoglycan) was greater than one would predict based on the independent behavior of each site. Constructs containing only Ser or Ser produced 15 and 23% heparan sulfate proteoglycans, respectively (Table 1). If the sites acted independently in the chimera containing both Ser and Ser, then the proportion of chimeras containing two heparan sulfate chains should have been 1.1% based on the probability of producing heparan sulfate at each site (0.15 times 0.23) multiplied by the proportion of chimeras containing two chains (one-third). Therefore, glycoforms with two heparan sulfate chains accumulated more than 10-fold over the predicted value (14 versus 1.1%). The proportion of the other glycoforms was less than predicted (11 versus 22% for proteoglycans containing two chondroitin sulfate chains (one-third of 0.85 times 0.77] and 9 versus 10% for hybrids containing one heparan sulfate and one chondroitin sulfate chain).

Previous studies of betaglycan showed that a cluster of acidic residues downstream from the site that supported heparan sulfate synthesis dramatically affected GAG composition(2) . The sites supporting heparan sulfate synthesis in syndecan-1 also have nearby clusters of acidic residues (Fig. 2). To examine if these clusters played a similar role in syndecan-1, a chimera containing residues 32-53 and all three N-terminal sites was prepared. Mutating the Asp and Glu residues in the clusters to Asn and Gln, respectively, reduced the proportion of heparan sulfate from 38% in the control construct to 8 and 13%, depending on the cluster (Table 5). Mutating both clusters diminished heparan sulfate synthesis to background (4%). Thus, the clusters of acidic residues are important elements in both syndecan-1 and betaglycan.




DISCUSSION

This report describes the use of protein chimeras to probe the assembly of heparan sulfate chains on proteoglycans. The results suggest a set of rules for predicting whether heparan sulfate will assemble at glycosylation sites. These rules include features of the core protein, the cellular capacity to produce individual GAGs, and the relative abundance of core proteins. Together, they support a model for GAG chain assembly in which specific core protein determinants interact with a key biosynthetic enzyme involved in heparan sulfate biosynthesis.

Clusters of Acidic Residues Are Necessary but Not Sufficient

Studies of betaglycan suggested that one type of heparan sulfate attachment site consists of a Ser-Gly dipeptide located near a cluster of acidic residues(2) . Mutation of the acidic residues diminished heparan sulfate synthesis and led to greater substitution of the site with chondroitin sulfate. Studies of syndecan-1 showed that sites supporting heparan sulfate synthesis also contain nearby clusters of acidic residues (-EDQDGSGDDSDNFSGSGTG-). Mutation of the Asp and Glu residues to Asn and Gln in either cluster reduced heparan sulfate synthesis, and altering both clusters caused full inhibition (Table 5).

Inspection of various cloned proteoglycans reveals that a cluster of acidic residues always flanks sites thought to contain heparan sulfate chains, although the position and exact composition of the cluster varies (Table 6). These clusters occur less frequently in chondroitin sulfate proteoglycans. beta-Glycan, decorin, invariant chain, and HI-30 contain clusters of acidic residues, but the natural proteoglycans contain only chondroitin sulfate chains at the indicated sites(1, 21, 22, 23) . Decorin and the betaglycan chimeras behaved similarly (Table 6). These findings suggest that other elements work along with the cluster of acidic residues to drive heparan sulfate synthesis. Thus, clusters of acidic residues appear to be necessary but not sufficient determinants for heparan sulfate assembly.



Hydrophobic Amino Acids Can Act as Enhancer Elements

In betaglycan, the site that supports heparan sulfate synthesis contains a Trp residue adjacent to the Ser-Gly unit(2) . Changing this residue to Ala reduced heparan sulfate synthesis. More importantly, inserting Trp next to a chondroitin sulfate site with a nearby cluster of acidic residues enhanced heparan sulfate formation. Although Trp occurs relatively rarely in proteoglycans (Table 6), aromatic and aliphatic residues are quite common, enhancing the overall hydrophobicity of the immediate region by the GAG attachment site(4) . The hydrophobicity of chondroitin sulfate sites tends to be less pronounced (Table 6).

Earlier studies of beta-D-xylosides in CHO cells showed that priming of chondroitin sulfate most likely occurs by default(24, 25) . Simple xylosides containing alkyl chains as an aglycone prime chondroitin sulfate but not heparan sulfate. Compounds containing fused aromatic rings, which may simulate the structure of the indole side chain of tryptophan, drive heparan sulfate as well as chondroitin sulfate synthesis(25) . Thus, aromatic beta-D-xylosides may mimic the hydrophobic patch found near heparan sulfate attachment sites. Synthesis of heparan sulfate by aromatic beta-D-xylosides requires a relatively high concentration of primer (>30 µM) compared with endogenous intermediates, possibly because they lack negative charges imparted by the acidic residues in natural core proteins.

Adjacent Ser-Gly Attachment Sites Are Coupled

Most heparan sulfate proteoglycans contain (Ser-Gly)(n) segments, where n geq 2 (Table 6). Experiments with peptides derived from a number of proteoglycans that contain repeating Ser-Gly units primed heparan sulfate, albeit to different extents: fibroglycan, 60% (-DMYLDSSSIEEASGLYPIDDDDYSSASGSGAYEDKGSPDLTTSQ, syndecan-3, 56% (-DDELDDIYSGSGSGYFEQESGLE), epican, 24% (-EDERDRHLSFSGSGIDDDEDFIS), glypican, 20% -DFQDASDDGSGSGSGDGCLDDLCG). This variation may reflect differences in primary sequence immediately adjacent to the site or secondary and tertiary structure in the segments. This possibility may explain why natural glypican contains only heparan sulfate(26) , whereas the glypican chimera primed heparan sulfate relatively poorly compared with other constructs (Table 6).

The enhanced synthesis of heparan sulfate across adjacent Ser-Gly sites deserves further study. The upstream site in syndecan-1 (Ser) couples to the downstream sites at Ser and Ser (Table 1). Shworak et al.(3) showed that stable transfectants expressing ryudocan contain more heparan sulfate when multiple attachment sites were present. These findings may indicate that coupling can occur at a distance, possibly by juxtaposing the sites through secondary and tertiary structure. Interestingly, chimeras containing the sites in syndecan-3 (-SDLEVPTSSGPSGDFEIQEEEETT-) and in N-syndecan (-TTTQDEPEVPVSGGPSGDFELQEE-) do not prime much heparan sulfate (7 and 8%, respectively). Both sequences contain a proline residue between the Ser-Gly units, which may act as an inhibitor. The low degree of coupling between closely spaced Ser-Gly sites in betaglycan also may have been due to an intervening proline residue(2) , which may have altered the conformation of the peptide (Table 3). If correct, this idea is reminiscent of the inhibitory effect of Pro on the attachment of Glc(3)Man(9)GlcNAc(2) oligosaccharides from dolichyl-P intermediates to Asn-Xaa-Ser/Thr sites in glycoproteins(27) . Perhaps negative regulatory sequences/amino acids play a role in proteoglycan assembly as well.

As shown in Table 6, repetitive Ser-Gly dipeptides with a flanking cluster of acidic residues represent a common motif in a variety of heparan sulfate proteoglycans. Thrombomodulin also contains adjacent Ser-Gly dipeptides, but lacks a cluster of acidic residues and therefore does not prime heparan sulfate. Searching the Wisconsin gene bank for sequences consisting of (Ser-Gly)(n) and a nearby cluster of acidic amino acids ((D/E)(n)) within 6 residues yielded 15 out of 16 heparan sulfate proteoglycans cloned to date. The search also yielded proteins from bacteria, viruses, parasites, and eukaryotic subcellular organelles. Cell surface and secreted proteins, including agrin, a tyrosine kinase receptor, prostatic spermine-binding protein, and sporozoite surface antigen have a high chance of bearing heparan sulfate chains based on their location on the surface or outside the cell. Among them, agrin was shown recently to bear heparan sulfate chains(28) . Interestingly, a chimera prepared from the tyrosine kinase receptor sequence (M35196) primed heparan sulfate, suggesting that the native protein might contain a heparan sulfate chain.

Biosynthetic Capacity Affects GAG Composition

A few heparan sulfate proteoglycans lack the repetitive Ser-Gly sequences of syndecan-1 or the aromatic residue found in betaglycan (e.g. proline-rich proteoglycan), suggesting that other enhancing factors may exist. One possibility is that the proportion of chains depends on the relative capacity of cells to produce GAG chains. In ldlD cells, which have reduced ability to make chondroitin sulfate, the relative proportion of heparan sulfate increases at the expense of the chondroitin sulfate chains (Table 1). Some sites that do not make much heparan sulfate will even become active when expressed in ldlD cells (Table 1). Conversely, the proportion of chondroitin sulfate increases dramatically in pgsD mutants, defective in heparan sulfate synthesis(6, 29) .

Proteoglycans vary in composition in different tissues and cells. For example, serglycin contains chondroitin sulfate chains when expressed in various myeloid cells (30) and a mixture of heparin and chondroitin sulfate chains when expressed in connective tissue mast cells. (^5)Syndecan-1 also varies in composition in different cells (31) and in response to growth factors (32) . No systematic study of the biosynthetic enzymes has been undertaken in these tissues, but it is reasonable to assume that changes in enzyme expression could alter GAG composition. Thus, the actual complement of chains borne by a proteoglycan depends on biosynthetic capacity as well as permissive elements embedded in the core protein sequence.

The Amount of Core Protein Can Affect GAG Composition

The amount of core protein substrates passing through the biosynthetic compartments may also affect GAG composition. Stable transfectants expressing a syndecan chimera overproduce GAG and cause a decline in the proportion of heparan sulfate (Table 2). The effect was not limited to the chimera, since endogenous proteoglycans also contained less heparan sulfate chains. Stable transfectants expressing syndecan-1 and betaglycan show similar effects(1, 2, 4) . Recent studies suggest that core protein sequences outside the GAG attachment sites may regulate the fine structure of the chains as well(33) .

Although the proportion of heparan sulfate declines, the actual amount of material increases after transfection. The data presented in Table 2show that wild-type cells produced about 3.2 times 10^6 cpm of [S]heparan sulfate (4.4 times 10^6 cpm times 72%). The stable transfectant produced 8.1 times 10^6 cpm of [S]heparan sulfate (1.5 times 10^7 times 54%). This 2-3-fold increase in heparan sulfate synthesis is offset by a much larger increase in chondroitin sulfate synthesis, causing the relative composition to change. The compositional difference mimics the effect of beta-D-xylosides, which depress the proportion of heparan sulfate because the primers preferentially stimulate chondroitin sulfate assembly(25) .

A Model for Heparan Sulfate Biosynthesis

The above information suggests a model for controlling GAG composition, in which a key enzyme in the biosynthetic pathway senses permissive elements of the core and controls if heparan sulfate assembles. The identity of this enzyme emerged from earlier enzymatic studies showing that a unique alpha-GlcNAc transferase (alpha-GlcNAc TI) acts on the tetrasaccharide intermediate, -GlcAbeta1-3Galbeta1-3Galbeta1-4Xyl-protein(5, 6) . This unique transferase may bind to structural elements in the core protein and select specific linkage tetrasaccharides for heparan sulfate assembly. In betaglycan and related proteoglycans, the recognition element consists of the cluster of acidic residues and the adjacent Trp, which together may determine the affinity of the substrate for the transferase. Analogous interactions between distal glycosyltransferases and nascent glycoproteins occur during the addition of GalNAc to GlcNAc on the termini of N-linked oligosaccharides of glycohormones (34) and mannose 6-phosphate to the termini of chains found on lysosomal glycoproteins(35) . In syndecan-1, the mechanism involves a cluster of acidic residues and perhaps a hydrophobic pocket defined by aromatic and aliphatic residues. In addition, a coupling phenomenon occurs across nearby Ser-Gly attachment sites. The juxtaposition of two or more sites may raise the probability that alpha-GlcNAc TI will act on adjacent linkage fragments. Thus, initiation of the heparan sulfate chains may involve both sequence recognition and a quasi-processive mechanism with regard to the carbohydrate acceptors.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Bldg. 68-480, Cambridge, MA 02139.

To whom correspondence and reprint requests should be addressed. Tel.: 205-934-6034; Fax: 205-975-2547; jesko@bmg.bhs.uab.edu.

(^1)
The abbreviations used are: GAG, glycosaminoglycan; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.

(^2)
A hybrid proteoglycan contains more than one type of GAG chain, such as heparan sulfate and chondroitin sulfate.

(^3)
Recent studies suggest that 2E9 also recognizes syndecan-3.

(^5)
Lidholt, K., Eriksson, I., and Kjellén, L. (1995) Biochem. J.311, 233-238.

(^4)
The GenBank accession number for CHO syndecan-1 is L38991[GenBank].


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