Different Usage of the Glycosaminoglycan-Attachment Sites of

Different Usage of the Glycosaminoglycan-Attachment Sites of Biglycan∗

Hans Kresse1, Daniela G. Seidler1, Margit Müller1, Egon Breuer1, Heinz Hausser1,2,

Peter J. Roughley3, and Elke Schönherr1

1Institute of Physiological Chemistry and Pathobiochemistry, University of Münster,

Germany, and 3Shriners Hospital for Children, McGill University, Montreal, Canada

Running title: Glycosaminoglycan Substitution of Biglycan

Correspondence: Dr. Hans Kresse, Institute of Physiological Chemistry and Pathobiochemistry, Waldeyerstr. 15, D-48149 Münster, Germany Phone: +49-251-8355581 Fax: +49-251-8355596 E-mail: [email protected]

∗ This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 496, Project A6) 2 Present address: Department of Orthopedics, University of Ulm, Germany

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on January 5, 2001 as Manuscript M009321200 by guest on A

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Synopsis

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 residue after removal of the prepro

peptide. Since 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) an 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

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

is probably becoming 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.

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Introduction

The biosynthesis of proteoglycans is a complex process where 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 about 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 GlcAβ1-3Galβ1-3Galβ1-4Xyl (9), raising the possibility that it is the transfer

of the fifth monosaccharide (either GalNAc or GlcNAc) which represents the most critical

step in glycosaminoglycan chain biosynthesis.

Considering the complexity of glycosaminoglycan chain assembly we have focussed

on the biosynthesis of biglycan. Biglycan belongs to the family of small leucine-rich

proteoglycans (see 10-12 for recent reviews) and carries most often two chondroitin/dermatan


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sulfate chains which 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 pro-peptide 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 pro-peptide accounting for non-glycanated

forms of the molecule (17,18). The presence of only two glycosaminoglycan attachment sites

which 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 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.


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EXPERIMENTAL PROCEDURES

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 PCR system (Roche Diagnostics; PCR

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'-ATAAGAATGCGGCCGCCAAGGCT

GGAAATG-3'. The primers introduce an EcoRI- and a NotI-restriction site, respectively, near

the 5' and 3' end 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 E. coli TOP 10F'.

Biglycan mutations were introduced by using the QuikChange Site Directed

Mutagenesis Kit (Stratagene) and HPSF quality oligonucleotides (MWG Biotech) as primers.

The method uses forward and reverse primers which 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 PCR conditions were denaturation for 30 sec at

94 °C, annealing for 1 min at 55 °C, and elongation for 14 min at 68 °C: After 30 PCR 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'-CGATGAGGAAGC

TACGGGCGCTGACAC-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'


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and 5-GGAGCCCGCTGAAATCTCCCTTGGGCACCTT-3' served for the construction of

the triple mutant BGN S42A/S47A/V178D. Using 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 Gly-43 and Ala-44 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.

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 (GibcoBRL) according to the instructions of the manufacturer. The

cells were selected for neomycine resistance by adding 750 µg/ml G418 (Life Technologies).

The expression of human decorin cDNA in 293 cells had 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 two

days after transfection.


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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 which 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 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 where 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-β-D-xyloside (Sigma), the xyloside was present already during the

1 h preincubation periode 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 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 immune precipitated by applying 6 mg of protein A-Sepharose per 25 cm2 flask


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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. 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 (PE

Biosystems). 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).


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RESULTS

Properties of recombinant biglycan synthesized by 293 cells - Stably transfected 293

cells synthesized within 24 h about 0.5 pg of biglycan core protein per cell. Surprisingly,

however, its glycosaminoglycan chains were not completely sensitive towards 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 completely be 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, one of the chains could be composed of heparan

sulfate.

It was also investigated whether or not the secreted proteoglycan is still carrying the

propeptide sequence. By using 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 the most 15 % of the total quantity of secreted core

protein contained the propeptide sequence which comprised amino acids Phe19 - Asn37.

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 which 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, respectively. 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 core protein became substituted with a single


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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 the monoglycanated proteoglycan resulting from the S47A mutation. Such a migration

behaviour is best explained by assuming that the glycosaminoglycan chain of the S42A

mutant is somewhat larger and less heterogeneous in size than in 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.

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 optimise 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 against 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.

From the data described so far it appeared that use of serine-47 was markedly

dependend 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


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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 horse-

shoe structure of decorin (24) - should be located at the unrestricted convex site of the

molecule.

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

harbouring 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 immune precipitation. 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 immune precipitation followed by digestion of the

immune complex with chondroitin ABC and ACII lyases and then with endoglycosidase F.

The immune complex still contained 1920 c.p.m. 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 behaviour during SDS-

PAGE of the unsubstituted S42A core protein and the S42A core protein obtained by

chondroitin ABC lyase treatment.


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In further experiments competition for glycosaminoglycan chain assembly by exogeneously

added p-nitrophenyl-β-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.

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. Since 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 media 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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DISCUSSION

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-β-xyloside as primer molecule also supported this conclusion.

Monoglycanated biglycan has also been extracted from normal tissue. However, it had not yet

been investigated systematically which of the two glycosaminoglycan-attachment sites had

been 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 in so far as it may also carry two

chondroitin/dermatan sulfate chains being linked to serine-4 and serine-16, respectively. In

the monoglycanated proteoglycan form serine-4 was used exclusively (30). Informations on

the usage of the multiple sites of other chondroitin/dermatan sulfate proteoglycans are not

available.

In a study on the glycosaminoglycan attachment sites of perlecan it had 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).


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Direct biosynthetic labeling of the linkage region together with the results of

competition experiments with p-nitrophenyl-β-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-xylose: core protein

xylosyltransferase (EC 2.4.2.26) is the rate-limiting step in glycosaminoglycan chain

assembly and not - as in case of thrombomodulin - a β-N-acetylgalactosaminyltransferase (9).

Also in other aspects of glycosaminoglycan chain initiation thrombomodulin appears to

exhibit special features, since 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

which 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


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between Ala16 and Leu17. However, an alternative rat propeptide sequence began also 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, biglycan as expressed in

293 and CHO cells 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 since its glycosaminoglycan chains cannot completely be removed

by chondroitin ABC lyase (41). The factors which govern the substitution with either two

galactosaminoglycan 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 Ms. Barbara Liel is greatfully

acknowledged.


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Figure Legends

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 and x any amino acid.

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 on the right margin.

FIG. 3. Identification of biglycan propeptide. Biglycan was purified from the

conditioned media of 293 cells transfected with wild-type biglycan cDNA. After digestion

with chondroitin ABC lyase it was subjected to SDS-PAGE and 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.

FIG. 4. Glycosaminoglycan substitution of wild-type biglycan and biglycan

mutants S42A, S47A, and S42A/S47A. [35S]Sulfate- and [35SS]methionine-labeled biglycan

was isolated after biosynthetic labeling and subjected to SDS-PAGE and fluorography.

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


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with wild-type biglycan (WT) or insert-free control vector (CV) prior to SDS-PAGE and

fluorography.

FIG. 6. Glycosaminoglycan substitution of biglycan mutants S42T, S42A/A44D,

and E39Q/S47A as determined by [35S]methionine incorporation. The experiment was

performed analogously as described in Fig. 5. ABC denotes digestion with chondroitin ABC

lyase.

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 over-exposed.

FIG. 8. Influence of p-nitrophenyl-β -D-xyloside on biglycan biosynthesis. Cells

stably transfected with wild-type biglycan cDNA (WT) 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. Media were then subjected to immunoprecipitation, SDS-PAGE and

fluorography.

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 prior to SDS-PAGE and fluorography. Note that in this experiment

some biglycan had undergone limited proteolysis of the core protein.


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TABLE I

Composition of glycosaminoglycan - containing biglycan

secreted by 293 cells

wild type

S42A

S42T

S47A

S42A

A44D

E39Q

S47A

% of total

2 GAG chains 1 GAG chain Free core protein

84 - 94

9 - 14

0 - 6

0

15 - 25

75 - 85

0

25 - 35

65 - 75

0

98 - 100

0 - 2

0

50 - 56

44 - 50

0

96 - 100

0 - 4


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FIG. 1.

Consensus sequence

Mature BGN

Consensus sequence

aaxSGxG

||||||

38DEEASGADTSGVL50

|||||

aaxSGxG

GAG KO site 1

GAG KO site 2

GAG KO sites 1 and 2

Site 1, decreased efficiency

Site 2, increased efficiency

Site extension

Novel site V178D in GAG

KO sites 1 and 2

38DEEAAGADTSGVL50

38DEEATGADTSGVL50

38DEEASGADTAGVL50

38DEEAAGADTAGVL50

38DQEASGADTAGVL50

38DEEAAGDDTSGVL50

38DEEAAGIGPEVPADTSGVL56

177GDFSGLRNMN187


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FIG. 2.


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FIG. 3.


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FIG. 4.


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FIG. 5.


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FIG. 6.


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FIG. 7.


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FIG. 8.


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FIG. 9.


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Roughley and Elke SchönherrHans Kresse, Daniela G. Seidler, Margit Müller, Egon Breuer, Heinz Hausser, Peter J.

Different Usage of the Glycosaminoglycan-Attachment Sites of Biglycan

published online January 5, 2001J. Biol. Chem.

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