Different Usage of the Glycosaminoglycan Attachment Sites of Biglycan*

Hans KresseDagger §, Daniela G. SeidlerDagger , Margit MüllerDagger , Egon BreuerDagger , Heinz HausserDagger , Peter J. Roughley||, and Elke SchönherrDagger

From the Dagger  Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, D-48149 Münster, Germany and the || Shriners Hospital for Children, McGill University, Montreal, Quebec H3G 1A6, Canada

Received for publication, October 12, 2000, and in revised form, January 5, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Biglycan is a member of the small leucine-rich proteoglycan family. Its core protein comprises two chondroitin/dermatan sulfate attachment sites on serine 42 and serine 47, respectively, which are the fifth and tenth amino acid residues, respectively, after removal of the prepro peptide. Because the regulation of glycosaminoglycan chain assembly is not fully understood and because of the in vivo existence of monoglycanated biglycan, mutant core proteins were stably expressed in human 293 and Chinese hamster ovary cells in which i) either one or both serine residues were converted into alanine or threonine residues, ii) the number of acidic amino acids N-terminal of the respective serine residues was altered, and iii) a hexapeptide was inserted between the mutated site 1 and the unaltered site 2. Labeling experiments with [35S]sulfate and [35S]methionine indicated that serine 42 was almost fully used as the glycosaminoglycan attachment site regardless of whether site 2 was available or not for chain assembly. In contrast, substitution of site 2 was greatly influenced by the presence or absence of serine 42, although additional mutations demonstrated a direct influence of the amino acid sequence between the two sites. When site 2 was not substituted with a glycosaminoglycan chain, there was also no assembly of the linkage region. These results indicate that xylosyltransferase is the rate-limiting enzyme in glycosaminoglycan chain assembly and implicate a cooperative effect on the xylosyl transfer to site 2 by xylosylation of site 1, which probably becomes manifest before the removal of the propeptide. It is shown additionally that biglycan expressed in 293 cells may still contain the propeptide sequence and may carry heparan sulfate chains as well as sulfated N-linked oligosaccharides.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The biosynthesis of proteoglycans is a complex process in which, in the case of chondroitin and heparan sulfate proteoglycans, the assembly of the glycosaminoglycan side chains begins with the transfer of xylose from UDP-xylose to the hydroxyl group of selected serine residues of the core protein (1). A recognition consensus sequence for the attachment of xylose residues may consist of two acidic amino acids closely followed by the tetrapeptide Ser-Gly-Xaa-Gly, where Xaa is any amino acid (2-4). However, a sequence comparison of several proteoglycan core proteins indicated that the second glycine residue was present in only 19% of the cases, and there was also only an ~50% abundance of acidic residues among the three amino acids preceding the glycosaminoglycan attachment site (5). Furthermore, a Gly-Ser-Ala sequence instead of the Xaa-Ser-Gly sequence is found at the chondroitin sulfate attachment site of chicken type IX collagen (6), and threonine substitutions of serine residues can serve as acceptors for chain assembly, albeit at low efficiency (7). The situation is additionally complicated by the fact that established attachment sites are not necessarily used regularly, giving rise to so-called part-time proteoglycans. This has been considered as a result of the possibility that the attachment site is not always recognized by xylosyltransferase and that there may be competition for substrate between xylosyltransferase and glycoprotein N-acetylgalactosaminyltransferase (8). Furthermore, in the glycosaminoglycan-free form of the part-time proteoglycan thrombomodulin, the respective serine residue is substituted with the linkage region tetrasaccharide GlcAbeta 1-3Galbeta 1-3Galbeta 1-4Xyl (9), raising the possibility that it is the transfer of the fifth monosaccharide (either GalNAc or GlcNAc) that represents the most critical step in glycosaminoglycan chain biosynthesis.

Considering the complexity of glycosaminoglycan chain assembly, we have focused on the biosynthesis of biglycan. Biglycan belongs to the family of small leucine-rich proteoglycans (for recent reviews see Refs. 10-12) and carries most often two chondroitin/dermatan sulfate chains that are linked to serines 42 and 47 of the full-length human core protein sequence (13). The sequence around these serine residues (marked by an asterisk) is DEEAS*GADTS*GVLD. However, usage of the two sites is normally not complete (14), and up to 30% of the core protein may be substituted with a single chain only (15). Biglycan contains a prepro sequence of 38 amino acids. The propeptide whose function is not fully known is not necessarily removed prior to or after secretion (16). Additional proteolytic processing may take place after removal of the propeptide, accounting for nonglycanated forms of the molecule (17, 18). The presence of only two glycosaminoglycan attachment sites that are located in close proximity made biglycan an ideal candidate to study glycosaminoglycan chain assembly in a still simple, yet somewhat more complex system than in the case of the monoglycanated small proteoglycan decorin, where glycosaminoglycan biosynthesis has been studied in great detail (2, 7). It will be shown that the attachment of the two chains is regulated differently.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of Recombinant Biglycan in Human 293, Chinese Hamster Ovary, and COS-7 Cells-- Total RNA was isolated from human MG-63 osteosarcoma cells, which are known to produce high quantities of biglycan. It was subjected to reverse transcription with AMV reverse transcriptase and an oligo(dT)15 primer. Then the complete biglycan coding sequence was amplified by the Expand High Fidelity polymerase chain reaction system (Roche Diagnostics; polymerase chain reaction conditions: denaturation for 1 min at 94 °C, annealing for 1 min at 58 °C, and elongation for 2 min at 72 °C) using the forward primer 5'-GTTGGAATTCGAGTAGCTGCTTTCGGTCC-3' and the reverse and complement primer 5'-ATAAGAATGCGGCCGCCAAGGCTGGAAATG-3'. The primers introduce an EcoRI and a NotI restriction site, respectively, near the 5' and 3' ends of the amplified sequence. Upon EcoRI/NotI digestion, the purified amplification product was ligated into the EcoRI/NotI-digested pcDNA3.1 (+) vector (Invitrogen). The plasmid was isolated after transformation of competent Escherichia coli TOP 10F'.

Biglycan mutations were introduced by using the QuikChange site-directed mutagenesis kit (Stratagene) and HPSF quality oligonucleotides (MWG Biotec) as primers. The method uses forward and reverse primers that carry the base exchange in the center of their sequence and anneal to identical positions of the plasmid. High fidelity Pfu Turbo DNA polymerase was used for amplification. The polymerase chain reaction conditions were as follows: denaturation for 30 s at 94 °C, annealing for 1 min at 55 °C, and elongation for 14 min at 68 °C. After 30 polymerase chain reaction cycles, the methylated template was degraded by DpnI, and XL1-Blue supercompetent cells were used for transformation without prior ligation. The forward primers used to introduce mutations of the wild-type sequence were as follows: BGN S42A, 5'-GATGAGGAAGCTGCGGGCGCTGACAC-3'; BGN S42T, 5'-CGATGAGGAAGCTACGGGCGCTGACAC-3'; BGN S47A, 5'-CGCTGACACCGCAGGCGTCCTGGAC-3'. The latter primer was also used to mutate BGN S42A for the generation of the double mutant BGN S42A/ S47A. The primer pair 5-AAGGTGCCCAAGGGAGATTTCAGCGGGCTCC-3' and 5-GGAGCCCGCTGAAATCTCCCTTGGGCACCTT-3' served for the construction of the triple mutant BGN S42A/S47A/ V178D. With the use of the BGN S42A construct as template, BGN S42A/A44D was made by using 5'-GAAGCTGCGGGCGATGACACCTCAGG-3' as forward primer. The BGN S47A construct served as template to generate BGN E39Q/S47A with the forward primer 5-'CATGATGAACGATCAGGAAGCTTCGGGCG-3'. Sequencing of all constructs verified the introduction of the desired point mutations and the absence of mutations in the other parts of the cDNA sequence. Finally, a hexapeptide, IGPQVP, which is similar to the hexapeptide following the Ser-Gly attachment site in decorin (IGPEVP), was placed between Gly43 and Ala44 of the S42A biglycan mutant. In the first step 5'- and 3'-portions of the desired sequence were generated by using the primers corresponding to the vector sequences mentioned above, the reverse and complement primer 5'-GGGCACCTGGGGGCCGATGCCCGCAGCTTCCTCATC-3' (5' portion) and the forward primer 5'-ATCGGCCCCCAGGTGCCCGCTGACACCTCAGGCGTCC-3' (3' portion), respectively. The amplified products were purified by agarose gel electrophoresis and subjected to a second polymerase chain reaction by using the two primers designed from the multicloning site. The cDNA thus obtained was ligated into the pcDNA3.1 (+) vector after EcoRI/XbaI digestion. The mutants used are summarized in Fig. 1.


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Fig. 1.   Alignment of the glycosaminoglycan chain attachment consensus sequence with the sequence of mature wild-type and mutant biglycan. Mutated amino acids are indicated by bold single letters in italics. a designates an acidic amino acid, and x designates any amino acid. GAG, glycosaminoglycan; KO, knockout.

Cultured human 293 kidney cells were transfected with the unmodified pcDNA3.1 (+) vector or with this vector containing one of the above mentioned constructs by using LipofectAMINE 2000 (Life Technologies, Inc.) according to the instructions of the manufacturer. The cells were selected for neomycine resistance by adding 750 µg/ml G418 (Life Technologies, Inc.). The expression of human decorin cDNA in 293 cells has been described before (19). Wild-type BGN, BGN S42A, and BGN S47A were also used to transfect Chinese hamster ovary cells and COS-7 cells by an analogous procedure. COS cells were used for experiments 2 days after transfection.

Metabolic Labeling and Proteoglycan Isolation-- Unlabeled reference biglycan was purified from the culture media of 293 cells stably transfected with wild-type human biglycan cDNA. Purification included ammonium sulfate precipitation and anion exchange chromatography as described (20), except that during the latter step NaCl was replaced by KCl. Biglycan-containing fractions that were eluted from the DEAE column with 0.6 M KCl were made 2 M with (NH4)2SO4 and directly applied to a phenyl-Sepharose column (Sigma; 1 ml of gel/50 ml of conditioned medium) equilibrated with 10 mM K2SO4, pH 6.8, containing 2 M (NH4)2SO4. The column was washed with 3 volumes of starting buffer and then with 3 volumes each of this buffer in which the (NH4)2SO4 concentration had been reduced to 1 M, 0.5 M, and 0.2 M, respectively. Analytically pure biglycan (purity >95%) was desorbed with water.

Metabolic labeling of subconfluent 293, Chinese hamster ovary, and COS cells with [35S]sulfate (incubation period 24 h) and [35S]methionine (incubation period 8 h) was performed as described earlier (19). When [35S]methionine labeling was performed in the presence of p-nitrophenyl-beta -D-xyloside (Sigma), the xyloside was already present during the 1-h preincubation period in methionine-free medium. For labeling with [1-3H]galactose (ICN Pharmaceuticals; specific radioactivity 18 Ci/mmol), Waymouth MAB 87/3 medium containing 4% fetal calf serum and only 1.4 mM glucose was used. Incubation was for 24 h in the presence of 14 ml of culture medium with 500 µCi of 3H radioactivity per 75-cm2 culture flask. At the end of the incubations, medium was mixed with proteinase inhibitors and made 70% saturated with (NH4)2SO4. After centrifugation, the pellet from one 25-cm2 culture flask was dissolved in 300 µl of 0.1 M Tris/HCl buffer, pH 7.4, containing 1 M NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, and proteinase inhibitors. The solution was first allowed to react with protein A-Sepharose (Sigma; 1.5 mg of dry gel per 25-cm2 flask), which had been preincubated with preimmune rabbit antisera as described (15). Biglycan was subsequently immunoprecipitated by applying 6 mg of protein A-Sepharose per 25-cm2 flask, which had been coated with a monospecific rabbit antiserum against a KLH-coupled biglycan-derived peptide raised analogously as described (13).

Enzyme Treatment-- Protein A-Sepharose-bound immune complexes were subjected to digestions with chondroitinases ABC and ACII (20) and/or heparinases I and III (21) as described. Treatment with peptide:N-glycosidase F was for 20 h at 37 °C in 60 µl of sodium phosphate buffer, pH 7.5, containing 0.3 mM phenylmethylsulfonyl fluoride, 3.3 mM EDTA, 0.4% (v/v) Nonidet P-40, 0.06% (w/v) SDS, 0.3% (v/v) 2-mercaptoethanol, and 500 units of enzyme (New England Biolabs). After enzyme treatment, the protein A-Sepharose-bound material was washed twice with 10 mM Tris/HCl, pH 6.8, prior to SDS-PAGE.1 Use of [35S]methionine-labeled proteoglycan established the complete removal of the glycosaminoglycan chains and the N-linked oligosaccharides by this methodology.

Other Methods-- SDS-PAGE followed either by fluorography or by Western blotting was performed as described previously (20, 22). Fluorograms were evaluated with the ImageQuant 5.0 software program (Amersham Pharmacia Biotech). Western blots were probed with antisera against biglycan (see above), biglycan propeptide (16), and the HNK-1 carbohydrate epitope (a kind gift of Dr. M. Schachner, University of Hamburg, Germany). Automated protein sequencing was performed on a Procise 492-01 gas phase sequencer (PerkinElmer Life Sciences). A search for tyrosine O-sulfation was attempted after hydrolysis of core proteins under nitrogen in 1 M NaOH for 24 h at 110 °C (23).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Properties of Recombinant Biglycan Synthesized by 293 Cells-- Within 24 h stably transfected 293 cells synthesized about 0.5 pg of biglycan core protein per cell. Surprisingly, however, its glycosaminoglycan chains were not completely sensitive toward degradation by chondroitin ABC lyase. Between 5 and 15% of total [35S]sulfate incorporated into biglycan remained in a monoglycanated form. The same observations were made with Chinese hamster ovary cells. The label could be completely removed by treatment with heparinases I and III (Fig. 2), indicating that a subset of biglycan, synthesized by these cells, is a hybrid proteoglycan. However, glycosaminoglycan-free core protein was not observed when biglycan was treated with heparinases only. Experiments with [35S]methionine-labeled biglycan corroborated these results (data not shown). Thus, only when two glycosaminoglycan chains were present could one of the chains be composed of heparan sulfate.


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Fig. 2.   Heparinase sensitivity of recombinant biglycan. [35S]Sulfate-labeled biglycan was isolated from 293 cells transfected with wild-type biglycan cDNA and subjected to treatments with chondroitin ABC lyase (ABC) and/or heparinases I and III (HSase) prior to SDS-PAGE in a 10% separation gel followed by fluorography. The migration distance of reference proteins is indicated in the right margin.

It was also investigated whether or not the secreted proteoglycan still carries the propeptide sequence. With the use of propeptide-specific antibodies, the presence of such a sequence could be verified in Western blots of biglycan purified from 293 cells (Fig. 3). However, amino acid sequencing revealed that at most 15% of the total quantity of secreted core protein contained the propeptide sequence, which comprised amino acids Phe19-Asn37.


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Fig. 3.   Identification of biglycan propeptide. Biglycan was purified from the conditioned medium of 293 cells transfected with wild-type biglycan cDNA. After digestion with chondroitin ABC lyase, it was subjected to SDS-PAGE, Western blotting, and immunochemical reaction with a propeptide-specific antibody (lane 1) and an antibody directed against the mature core protein (lane 2). The prosequence obtained by gas phase sequencing is shown in the right panel.

Different Use of Glycosaminoglycan Attachment Sites-- For studying the structural prerequisites for glycosaminoglycan chain attachment to serines 42 and 47, respectively, of biglycan core protein, a series of mutations was created that is summarized in Fig. 1. In a first series of experiments the serine residues were replaced by alanine and used for studying biglycan biosynthesis in human 293 cells. Complementary experiments were performed with Chinese hamster ovary and COS cells. The consequences for glycosaminoglycan chain attachment turned out to be different depending on which one of the two serine residues was mutated. In the S47A mutant biglycan, the core protein became substituted with a single glycosaminoglycan chain, which was to be expected (shown for 293 cells in Fig. 4). In contrast, in the S42A mutant about 80% of the core protein was secreted into the culture medium without being substituted with a glycosaminoglycan chain. This percentage did not differ between the three cell types under study. The glycosaminoglycan chain-bearing species exhibited a somewhat slower and a less heterogeneous migration pattern during SDS-PAGE than did the monoglycanated proteoglycan resulting from the S47A mutation. Such a migration behavior is best explained by assuming that the glycosaminoglycan chain of the S42A mutant is somewhat larger and less heterogeneous in size than that of the other mutant. As an alternative to alanine, serine-42 was also replaced by threonine. In this mutant, too, the majority of serine 47 residues remained unused (Fig. 5). In the double mutant S42A/S47A, an alternative use of other potential glycosaminoglycan attachment sites could not be observed. As will be discussed below, the glycosaminoglycan-free, but still N-glycanated, core proteins were nevertheless substituted with [35S]sulfate residues.


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Fig. 4.   Glycosaminoglycan substitution of wild-type biglycan and biglycan mutants S42A, S47A, and S42A/S47A. [35S]Sulfate- and [35S]methionine-labeled biglycan was isolated after biosynthetic labeling and subjected to SDS-PAGE and fluorography. CV, control vector; WT, wild type.


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Fig. 5.   Glycosaminoglycan substitution of biglycan mutants S42T, S42A/A44D, and E39Q/S47A as determined by [35S]sulfate incorporation. Secreted biglycan was obtained by immunoprecipitation from the mutants indicated and from 293 cells transfected with wild-type (WT) biglycan or insert-free control vector (CV) prior to SDS-PAGE and fluorography.

The proposed consensus sequence for glycosaminoglycan chain attachment is fitted better for use of serine 42 than of serine 47 (Fig. 1). Further constructs were, therefore, generated to optimize the use of the latter site by introducing an additional acidic residue and to make the first site less efficient by removal of an acid residue. Serine 42 was always fully substituted by a glycosaminoglycan chain regardless of whether or not a preceding acidic residue had been exchanged for a neutral one. Use of serine 47, however, could remarkably be stimulated, although complete substitution was never approached (Figs. 5 and 6). The results of three or more separate incubations are summarized in Table I.


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Fig. 6.   Glycosaminoglycan substitution of biglycan mutants S42T, S42A/A44D, and E39Q/S47A as determined by [35S]methionine incorporation. The experiment was performed analogously to that described in the legend to Fig. 5. ABC denotes digestion with chondroitin ABC lyase. WT, wild type.

                              
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Table I
Composition of glycosaminoglycan-containing biglycan secreted by 293 cells

From the data described so far it appeared that use of serine 47 was markedly dependent on a substitution of serine 42. To study this point further, a hexapeptide was inserted in the S42A mutant in front of the serine 47 residue. This novel mutant protein, too, was secreted normally. However, [35S]sulfate incorporation into a chondroitin ABC lyase-sensitive glycosaminoglycan chain was just above the limit of detection (Fig. 7), whereas the secretion of the glycosaminoglycan-free core protein remained unaltered. Similarly, attempts to create a new glycosaminoglycan attachment site at serine 180 in the S42A/S47A double mutant by inserting aspartate in position 178 were unsuccessful (result not shown). This negative result should be seen in the context that of all unused Ser-Gly sites, serine 180 should have the greatest distance from N-glycosylation sites and, in analogy to the proposed horseshoe structure of decorin (24), should be located at the unrestricted convex site of the molecule.


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Fig. 7.   Further decline of glycosaminoglycan substitution by hexapeptide insertion into biglycan mutant S42A. Cultures of 293 cells expressing wild-type (WT) biglycan, biglycan S42A, and S42A with a hexapeptide insert were incubated in parallel with [35S]sulfate prior to SDS-PAGE of secreted biglycan followed by fluorography. Note that wild-type biglycan was overexposed. ABC denotes digestion with chondroitin ABC lyase.

Absence of Linkage Region Saccharides on Serine 47 in the S42A Mutant-- The majority of serine 47 residues were not substituted with a full-length glycosaminoglycan chain in the S42A mutant. The substitution of this residue with a linkage region tetrasaccharide, however, would have escaped detection. To study this possibility, 293 cells harboring the S42A mutation were, therefore, incubated with a high quantity of [1-3H]galactose for 24 h. The medium was then passed over a DEAE-Trisacryl M column, and unbound biglycan core protein was recovered by immunoprecipitation. The immune complex was exhaustively digested with endoglycosidase F to remove all asparagine-bound oligosaccharides. No 3H radioactivity remained associated with the immune complex. As a control for a proteoglycan with a single glycosaminoglycan chain, 293 cells transfected stably with human decorin cDNA were used and labeled under identical conditions. In this case, the whole medium was subjected to decorin immunoprecipitation followed by digestion of the immune complex with chondroitin ABC and ACII lyases and then with endoglycosidase F. The immune complex still contained 1920 cpm of 3H radioactivity. It was therefore concluded that serine 47 was either substituted with a full-length glycosaminoglycan chain or was devoid of a galactose-containing linkage region oligosaccharide. This conclusion was further supported by the observation of a slightly different migration behavior during SDS-PAGE of the unsubstituted S42A core protein and the S42A core protein obtained by chondroitin ABC lyase treatment.

In further experiments, competition for glycosaminoglycan chain assembly by exogenously added p-nitrophenyl-beta -D-xyloside was studied. Neither in the S42A nor in the S47A mutant was the use of the remaining glycosaminoglycan chain attachment site influenced by increasing doses of the competitor. However, shorter glycosaminoglycan chains were assembled at high xyloside concentrations (Fig. 8). These data suggest that the activity of galactosyltransferases is not the rate-limiting step during the formation of the linkage region. Hence, it seems likely that serine 47 was also not substituted with xylose in the glycosaminoglycan-free species of the S42A mutant.


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Fig. 8.   Influence of p-nitrophenyl-beta -D-xyloside on biglycan biosynthesis. Cells stably transfected with wild-type (WT) biglycan cDNA or one of the mutant constructs were preincubated with the xyloside concentrations given in the figure (µM) for 1 h before they were incubated further for 6 h in the presence of [35S]methionine and the same concentrations of xyloside. Medium was then subjected to immunoprecipitation, SDS-PAGE, and fluorography.

Core Protein-associated [35S]Sulfate-- As seen in Fig. 2 and subsequent figures, the core protein of biglycan could be labeled with [35S]sulfate. Combined digestion with chondroitin ABC lyase and chondroitin ACII lyase for complete removal of the glycosaminoglycan chains with the exception of the linkage region tetrasaccharide did not result in complete desulfation. Because small proteoglycans may carry tyrosine O-sulfate residues (25, 26), biglycan core proteins were subjected to alkaline hydrolysis. Thereafter, all radioactivity could be removed as insoluble barium salt, thereby excluding the presence of alkali-stable tyrosine O-sulfate residues. However, the label could completely be removed by treatment with endoglycosidase F, indicating that sulfate ester may be present on one or both of the two asparagine-linked oligosaccharides of biglycan (Fig. 9). N-linked oligosaccharides may carry a terminal 3-sulfated GlcA residue as part of the HNK-1 carbohydrate epitope (27). Westerns blots with antibodies against this epitope were negative for biglycan core protein although proteins of higher molecular weight in the medium of transfected 293 cells gave a positive immune reaction (result not shown). Whether or not asparagine-linked oligosaccharides of biglycan core protein contain sulfated N-acetyllactosamine units (28) was not investigated further.


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Fig. 9.   Sensitivity of core protein sulfation to endoglycosidase F treatment. Cells transfected with the double mutant S42A/S47A were labeled with either [35S]methionine or [35S]sulfate before biglycan was obtained by immunoprecipitation and subjected to digestion with endoglycosidase F (Endo F) prior to SDS-PAGE and fluorography. Note that in this experiment some biglycan had undergone limited proteolysis of the core protein.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The main result of this study is the observation that of the two glycosaminoglycan attachment sites of biglycan the more C-terminally located one is used nearly completely only when the first attachment site is available. This result should be considered in light of the high wild-type biglycan production of transfected 293 and CHO cells. Hence, it appears unlikely that the data obtained with mutant core proteins simply reflect the limited capacity for glycosaminoglycan chain assembly of these cells. Furthermore, [35S]sulfate labeling studies with p-nitrophenyl-beta -D-xyloside as primer molecule also supported this conclusion. Monoglycanated biglycan has also been extracted from normal tissue. However, it has not yet been investigated systematically which of the two glycosaminoglycan attachment sites were used in these cases. In bovine articular cartilage both sites were almost completely substituted (14) whereas, as anticipated from the present study, the second site was less frequently used in biglycan from human bone (29). Analogous data were obtained with chicken decorin, which resembles mammalian biglycan because it may also carry two chondroitin/dermatan sulfate chains linked to serine 4 and serine 16, respectively. In the monoglycanated proteoglycan form, serine 4 was used exclusively (30). Information on the usage of the multiple sites of other chondroitin/dermatan sulfate proteoglycans is not available.

In a study on the glycosaminoglycan attachment sites of perlecan it has been shown that the serine residues 65, 71, and 76 could be used individually when the two other sites were mutated to threonine (31). As in our model, the remaining serine residue did not serve as acceptor site in all molecules, but a sequence-dependent change in the efficiency of usage did not become evident. A direct comparison of the data with respect to site effectiveness, however, is difficult because a deletion of 110 amino acids was present in the perlecan construct eight amino acids C-terminal of serine 76, the last glycanated serine residue. The existence of core protein-specific pathways may also be envisaged (32).

Direct biosynthetic labeling of the linkage region together with the results of competition experiments with p-nitrophenyl-beta -D-xyloside strongly suggested that in the S42A mutant, serine 47 was either linked with a full-length glycosaminoglycan chain or not substituted at all with carbohydrate. This finding indicated that UDP-D-xylose:protein beta -D-xylosyltransferase (EC 2.4.2.26) is the rate-limiting step in glycosaminoglycan chain assembly and not, as in the case of thrombomodulin, a beta -N-acetylgalactosaminyltransferase (9). Also in other aspects of glycosaminoglycan chain initiation, thrombomodulin appears to exhibit special features, because the attachment consensus sequence is less well preserved, and, most strikingly, different serine residues can be used in different cells (33).

Xylose addition appears to begin in the endoplasmic reticulum and to continue in intermediate compartments, perhaps including the cis-Golgi, where galactose is added prior to the assembly of the repetitive disaccharide structures (34-36). For an explanation of the apparent cooperativity between the usage of the two glycosaminoglycan attachment sites that are located close to the N terminus of the mature core protein, it is tempting to assume that the propeptide contributes to optimal recognition of the first serine residue by the xylosyltransferase. The second serine residue then becomes more easily recognized, although an independent usage of it may occur and may become facilitated by an appropriate introduction of an additional acidic amino acid close to its N-terminal site. The compartment where the propeptide sequence of biglycan and decorin is normally removed has not yet been directly defined. The recent observation that bone morphogenetic protein-1 (procollagen C-proteinase) processes probiglycan (37) strongly suggests that this cleavage occurs after secretion or immediately prior to it. Thus, a regulatory influence of the propeptide on chain initiation is compatible with the topography of probiglycan processing. The N terminus of the propeptide was found to be generated by proteolysis at an unusual cleavage site between Pro18 and Phe19 and not as often after a small amino acid (38). The predominant form of biglycan from the secretions of rat and bovine smooth muscle cells was generated by hydrolysis between Ala16 and Leu17. However, an alternative rat propeptide sequence also began with Phe19, indicating that there might be either cell- and species-specific differences in the processing of the prepro peptide of biglycan or further degradation of the generated propeptide by aminopeptidases (39). The functional consequences of these differences are not yet known.

The present study yielded several additional results. First, the glycosaminoglycan-free biglycan core protein is transport-competent and becomes secreted as the fully N-glycosylated species. Its extracellular stability, however, may be decreased, considering the finding that the conformational stability of biglycan depends to a greater extent than in the case of decorin on the presence of chondroitin/dermatan sulfate chains (40). Second, as expressed in 293 and CHO cells, biglycan may be substituted with a single heparan sulfate chain, thereby representing a hybrid proteoglycan. Recombinant biglycan synthesized by HT-1080 cells also appears as a hybrid species because its glycosaminoglycan chains cannot completely be removed by chondroitin ABC lyase (41). The factors that govern the substitution with either two galactosaminoglycans or a single galactosaminoglycan and an additional glucosaminoglycan chain and their possible cell type specificity have not yet been defined. Some experimental variability was also noted in the present investigation. Third, sulfation of asparagine-bound oligosaccharides of a chondroitin/dermatan sulfate proteoglycan was shown for the first time. This sulfate substitution may have escaped detection in previous studies because of its low relative amount. The functional significance and the detailed structure of the sulfated oligosaccharides remain to be determined.

    ACKNOWLEDGEMENT

The expert technical help of Barbara Liel is gratefully acknowledged.

    FOOTNOTES

* This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 496, Project A6).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Inst. of Physiological Chemistry and Pathobiochemistry, Waldeyerstr. 15, D-48149 Münster, Germany. Tel.: 49-251-8355581; Fax: 49-251-8355596; E-mail: kresse@uni-muenster.de.

Present address: Dept. of Orthopedics, University of Ulm, 89069 Ulm, Germany.

Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M009321200

    ABBREVIATIONS

The abbreviation used is: SDS-PAGE, SDS-polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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