Nov 15, 1993 - From the Cancer Research Campaign and University of Manchester Department of Medical Oncology, Christie Hospital,. Wilmslow Road ...
THEJOURNAL OF BIOUXICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 269,No. 15,Issue of April 15,p p . 11208-11215, 1994 Printed in U.S.A.
Liver Heparan Sulfate Structure A NOVEL MOLECULAR DESIGN* (Received for publication, November 15, 1993,and in revised form, January 18, 1994)
Malcolm LyonS, Jon A. Deakin, andJohn T.Gallagher From the Cancer Research Campaign and University of Manchester Department of Medical Oncology, Christie Hospital, Wilmslow Road, Manchester M20 9BX, United Kingdom
complexity and diversity of structure (3,4),which appear tobe The structure of rat liver heparan sulfate (HS) has been investigated using a combination(a) ofchain scis- cell- and possibly differentiation-specific, together with their sion with specific reagents, ( b ) disaccharide composi- propensity for interaction with a wide range of extracellular tional analysis, and( c ) end-referenced sequence analy- proteins, including growth factors, matrix proteins, enzymes, sis of the proximal, protein-linked region of the chain. and enzyme inhibitors (5, 6 ) . This study reveals that the liver synthesizes a highly The structuralcomplexity of HS (and thechemically related sulfated HS species (1.34 sulfateddisaccharide), particuheparin)arises from an extensive series of post-polymeric larly high in N-sulfation (6Wo) and 2-0-sulfate content modifications of a nonsulfated precursor (heparan) with the (36%).Approximately half of the latter is found in trisul- structure (-4GlcUAPl4GlcNAcal-),. These modifications are fateddisaccharides, Le. IdceA(2-OS03)cul-4G1cNS0,(6- carried out by specific enzymes that act in a stepwise and OSO,). End-referencing methodology established the exinterdependent manner tosynthesize the mature, sulfated poistence of an extended, unmodified heparan (GlcUAP1- lysaccharide (7). Consequently, the number of potential disac4GlcNAc) sequence, 8-11 disaccharides in length, charide combinations are constrained, although the relative attached to the linkage tetrasaccharide, similar to that abundance of each can vary considerably in different HS spefound in a number of other HS species. Directly followcies. The transformation of heparan to heparan sulfatebegins ingthis is a mixedHexUA14GlcNR(6-OS03)(where GlcNR represents a-D-glucosamine with an unspecified with the concomitant de-N-acetylationlN-sulfationof GlcNAc N-substituent)-containingsequence of variable length, residues, followed by conversion of P-D-glucuronate on the reducingside of GlcNSO, residuesto a-L-iduronate. Subseculminating in the appearance of the first IdceA(P-OS0,) residue -20 disaccharides from the linkage region, Le. quently, 0-sulfate groups are added at various positionswithin “40%along the length of the chain. The distal 6Wo of the the constituentdisaccharides of the nascent HS chain (7). The it is generally (-2 sulfated modifications are subquantitative in nature, and polysaccharide is highly sulfated disaccharide) and mainly comprises three heparin-like the case that whereas thedegree of N-sulfation of HS species falls withina relatively restricted rangeof 4O-50%, the level of domains,highlyenrichedinIdceA(2-OS0,)residues. Overall, liverHS qualifies as an extreme member of the0-sulfation is more variable, falling in the range of 0.2-0.75 HS family, with a considerable proportion of heparin0-sulfatesldisaccharide (8).Heparin is distinguished from HS like structure asymmetrically concentrated to the distal by the much higher levels of N- and 0-sulfation, and the popart of the chain. lysaccharide contains an average of 2.4 sulfates/disaccharide unit (4). Detailed structural analyses have been undertaken of two Heparan sulfates (HS)’are complex polysaccharides synthe- HS species from cultured cells, namely human adult skin fisized by nearly all mammalian cells. They occur as proteogly- broblast HS (9-12) and human umbilical vein endothelial HS cans that can be expressed at the cell surface in the form of (13, 14). These in-depth studies have confirmed and extended transmembrane proteins(e.g. the syndecan family and the al- observations made for many other HS species (8, 15-18) that ternatively spliced variants of CD44) or glycosylphosphatidyl- the polymer modifications (i.e. iduronate formation and both Nevenly distributed throughout inositol-linked proteins (e.g. glypican), or alternatively, they and O-sulfate additions) are not can be directly secreted and incorporated into the extracellular the chain, but tendt o be colocalized such that highly modified matrix (e.g. perlecan) (for reviews, see Refs. 1 and 2). The (i.e. high in both iduronate and sulfate) block sequences are heparan sulfates are of considerable interest because of their generally separated by predominantly unmodified, nonsulfated domains, with some mixed sequences of N-acetylated and N* This work was supported by the Cancer Research Campaign. The sulfated disaccharides occurringat theboundaries. In addition, costs of publication of this article were defrayed in part by the payment they have revealed the occurrence of an extended unmodified of page charges. This article must therefore be hereby marked “aduer- heparan sequence directly adjacent to the protein linkage retisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate gion. In thecase of skin fibroblast HS,this sequence was shown this fact. in $ To whom correspondence should be addressed. “el.: 061-446-3203; to be 8-11 disaccharides in length (9,12), whereas endotheFax: 061-446-3109. lial HS, it was estimated to be 10-17 disaccharides (14). This The abbreviations used are: HS, heparan sulfate(s); HSPGs, hepa- sequence alsoappears to exist in a number of other HS species ran sulfate proteoglycans; CHAPS, 3-[(3-cholamidopropyl)dimethylam- (19-22). These various features of chain organization may be moniol-1-propanesulfonicacid; HPLC, high pressure liquid chromatogp-D- common to the heparan sulfates and may distinguish them raphy; PAGE, polyacrylamide gel electrophoresis; GlcUA, glucuronate; IdceA, a-L-iduronate; IdceA(2-OS03), a-L-iduronate from heparin. 2-sulfate; HexUA, unspecified hexuronate; AHexUA, A4t5-unsaturated Knowledge of the variations in composition and organization hexuronate; AHexUA(2-OS03), A4,5-unsaturated hexuronate 2-sulfate; of HS from different cells and tissues is becoming increasingly GlcNAc, a-D-N-acetylglucosamine;GlcNAc(G-OSO,),a-D-N-acetylgluto elucidate the relationships important as attempts are made cosamine 6-sulfate; GlcNSO,, a-D-N-sulfoglucosamine; GlcNS03(6between HS structure and function, e.g. the sequence-specific OSO,), a-D-N-sulfoglucosamine 6-sulfate.
11208
Structure SulfateHeparan Liver
11209
binding and activationof proteins suchas antithrombin I11 (23) matic or chemical scission were analyzed by gel filtration chromatog0.2 M and basic fibroblast growth factor (24-27). In this context, the raphy on a Bio-Gel P-10column (1 x 135 cm)elutedwith NH,HCO, a t a flow rate of 4 ml/h. Fractions were counted, and spill HS synthesized by the liver is potentially interesting because it correction was performed so that the distribution of oligosaccharides has been known sinceits earlydescription (28)that itpossesses and the percentage of glycosidic bond cleavage could be calculatedfrom a relatively high level of overall sulfation for a HS. However, its the 3H profile using the formulaof Malmstrom et al. (32). structure has not been analyzed using the more recent techPreparation and Analysis ofNitrousAcid- a n d Heparinase I-resistant niques that would allow a detailed comparison with the well Protein Linkage Fragrnents-3W5S-Radiolabeled HSPG was degraded characterized, butonly moderately sulfated, fibroblast and en- with low pH nitrous acid (31).An aliquot wasremoved and checked for dothelial HS species. This paper describes such a n analysis of maximumdegradation by chromatography on Bio-Gel P-10 (as described earlier). The nitrous acid digest was neutralized with ammothe HS chainsderived from pure, liver cell-associated HSPGs, nium carbonate and then dialyzed against 80 m~ NaC1,20 m~ sodium the purification and characterization of which have been de- phosphate, 0.1% CHAPS, pH 7.0 (coupling buffer).Specific recovery of scribed previously (29, 30). The results indicate that although protein-linked oligosaccharides was achieved by mixing the dialysate liver HS clearly conforms to the general pattern of a HS spe- with a suspension of washed activated AtE-Gel 15 on a n end-over-end cies, it containsa remarkable proportion of heparin-like struc- mixer for 3 h at 4 "C. The gel suspension was then packed into a small ture, andwe suggest thatit represents one extreme end of the column and sequentially washed with (i) couplingbuffer; (ii) distilled water; (iii) 4 M guanidinium chloride, 1% 'hiton X-100, 50 mM sodium known HS spectrum. phosphate, pH 7.0; and finally, (iv) distilled water. Bound oligosaccharides were released from the Aff-Gel-coupled core protein by incubation EXPERIMENTAL PROCEDURES with 50 m~ NaOH, 1 M sodium borohydride a t 45 "C for 24 h. The supernatant, recovered by centrifugation, was neutralized by careful Materials-~-[G-~H]G1ucosamine hydrochloride (20-45 Ci/mmol) and N%,%04 (carrier-free; 1200-1400 Ci/mmol) were obtained from DuPont addition of 2 M acetic acid, dialyzed against 0.2 M NH,HCO, in Speclyophilized. The NEN.HeparinaseI(Flavobacteriumheparinurn;heparinlyase, EC trapor low M,cut-off (> 1000) dialysis tubing, and then 4.2.2.7) was from Seikagaku Kogyo Co. (Tokyo, Japan). Heparinase I1 molecular size of the recovered nitrous acid-resistant fragments was analyzed by gel filtration chromatography on a Sepharose CL-GB col( R heparinurn; no EC number assigned) and heparinase III/ heparitinase ( E heparinurn; heparin-sulfate lyase, EC 4.2.2.8) were umn (1.5 x 67.5 cm) eluted with 0.2 M NH4HC03at a flow rate of 12 obtained from Grampian Enzymes (Aberdeen, UnitedKingdom). A m - myh. Fractions were counted for radioactivity. In addition, the fragby electrophoresis on a 2 6 3 0 % (w/v) total acrylamide, Gel 15 was purchased from Bio-Rad Laboratories (Heme1 Hempstead, ments were sized 2-5% (w/v) cross-linker gradient polyacrylamide gel with a 5% (w/v) United Kingdom). acrylamide stacking gel essentially as described by Turnbull and GalPreparation of Liver Heparan Sulfate Proteoglycans and Heparan Sulfate Chains-Liver proteoglycans were metabolically radiolabeled inlagher (33). After gradient PAGE, the resolved oligosaccharides were electrotransferred t o a positively charged BiotraceRP nylon membrane vivo by intraperitoneal injectionof adult rats with 0.5 ml each of 0.15 M (Gelman Instrument Co.). The membrane was air-dried, sprayed with NaCl, 20 m~ sodium phosphate, pH 7.4, containing1mCi of Na,3'S04 and 250 pCi of ~-[G-~H]glucosamine hydrochloride. m e r 2 h, the rats EN3HANCE (Amersham International), air-dried again, and then exposed t o preflashed KodakX-Omat AR film at -70 "C. were killed, and their livers were excised. Three radiolabeled livers Heparinase I-resistant linkage fragments were prepared a similar in were normally pooled with six unlabeled ones. Cell-associated HSPGs were extracted and purified as described by Lyon and Gallagher (29). way tothe nitrous acid-resistant fragments.3H/%-Radiolabeled HSPG Heparan sulfate chains were prepared by exhaustive proteolytic diges- was exhaustively digested with three additions of 20 mIU/ml hepariof 18h in 0.1 M sodium acetate, 0.1 tion of HSPGs with Pronase(1 mg/ml) in 0.5 M NaCI, 50 mM Tris-HC1, nase Iover a total incubation period 0.1% CHAPS, pH 8.0, for 24 hat 37 "C. HS was thenrecovered by gel m~ calcium acetate,0.1% CHAPS, 0.1 mg/ml bovine serum albumin, pH filtration chromatographyon a SepharoseCL-GB column (1.5 x 67.5 cm) 7.0, at 37 "C. After checking the digest for maximum degradation by eluted with 0.2 M NH,HCO, at a flow rate of 12 mVh. Fractions were chromatography of an aliquot on Bio-Gel P-10, the core protein was coupled to Am-Gel 15, and the protein-bound oligosaccharides were monitored by liquid scintillation counting, and those corresponding to the HS chains werepooled, lyophilized twice, and then concentrated to recovered a s described above. The size profile of the resistant oligosaccharides was analyzed by gel filtration on a Sepharose CL-GB column dryness on a centrifugal evaporator. Disaccharide Compositional Analy~is-~HPS-Labeled heparan sul- (1.5 x 96 em). The nitrousacid susceptibility of the heparinase I-resisby rechromatography on fate chains were exhaustively digested with 20 mIU/ml each of hepari- tant linkage oligosaccharides was assessed nases I, 11, and I11 in 0.1 M sodium acetate, 0.1 m~ calcium acetate, 1 Sepharose CL-GB and Bio-Gel P-10 columns (conditions as described above) after treatment withlow pH nitrous acid. mg/ml bovine serum albumin, pH 7.0, at 37 "C for 18 h. The same The gradient polyacrylamide gel used for analysis of the nitrous quantities of enzymes were then again added, and the mixture was acid-resistant oligosaccharides was calibrated with a ladder of hyaluincubated for an extra 4 h. The digest was analyzed by gel filtration ronate oligosaccharides. These were prepared by partial digestion of chromatography on a Bio-Gel P-2 column(1 x 111c m ) eluted with 0.2 M [3Hlhyaluronate (isolated from the culture mediumof skin fibroblasts NH,HCO, a t a flow rate of 4 ml/h. Fractions were collected and analyzed for radioactivity. Those fractions corresponding t o disaccharides metabolically radiolabeled with [3Hlglucosamine) with 100 pg/ml bowere pooled and repeatedly lyophilized to remove the NH,HCO,. The vine testicular hyaluronidase in 0.1M sodium acetate, 0.15 M NaCl, pH 5.0, at 37 "C. Portions were periodically removed from the digest and sample was then redissolved in distilled acidified water to pH 3.5by the addition of HCI. The constituent disaccharides were resolved by strong heated at 100 "C for 10 min to terminate enzyme activity. The digests were then recombined and centrifuged to remove denatured protein, anion-exchange chromatography on a 5-pm Spherisorb SAX column a Bio-Gel P-30 gel filtra(Technicol, Stockport, United Kingdom) linked to a Dionex HPLC sys- and the resulting supernatant was applied to tem. After sample injection, the column was washed with5 ml of acidi- tion column (1 x 135 cm) eluted with 0.2M NH,HCO, at a flow rate of 0.8 mVh. Fractions were analyzed by liquid scintillation counting. Olified water, pH 3.5, followed by elution witha 40-ml gradient of (M.75 gosaccharide peaks of known size were individually collected and lyM NaCI, pH 3.5, at a flow rate of 1 mumin. Radioactivity elution was monitored using an on-line Radiomatic Flo-Onemeta Series A-200 de- ophilized. tector (Canberra Packard) employing a0.5-ml flow-through liquid cell and a scintillantsample ratio of 3:l. Where possible, the eluant was RESULTS simultaneously monitoredfor UV absorbance a t 232 nm. Disaccharides Intact Chains-Gel filtration chromatography on Sepharose were identifiedby comparison withthe elution positions of eight known CL-GB of dual radiolabeled HS chains released from intact disaccharide standards (see legend to Fig, 3). Spill correction was perHSPG by alkaline borohydride elimination revealed a single formed on the radioactivity data so that the disaccharide composition could be calculatedby integration of the ,H-labeled disaccharide peaks. major homogeneous peak eluting with a K,, of 0.49, together Specific Degradation ofHeparan Sulfate Chains-Nitrous acid digeswith a small amountof lowmolecular weight 3Hlabel (probably tion was performed by the low pH method of Shively and Conrad (31). N- and/or 0-linked oligosaccharides) and free 35S in theV,(Fig. Exhaustive enzymatic digestion with heparinase or II11 was performed 1). The relative M , of the HS chain is -22,000 by comparison with three additions of enzyme (20 mIU/ml each) in 0.1 M sodium acetate, 0.1 m~ calcium acetate, 1mg/ml bovine serum albumin, pH7.0, with the published calibration of Wasteson (34), and it comover a n 18-h period a t 37 "C to ensure that the maximum breakdown pares well with a K,, of 0.44 ( M , 25,000) obtained previously was achieved. The size profiles of the degradation products from enzy- from analysis of the HS released directly from rat liver plasma
11210
Liver Heparan Sulfate Structure
membranes (35).Release of the HS chainsby proteolysis using a broader peak eluting witha similar K,, of Pronase resulted in 0.44 (data not shown). To maximize access of enzymes to the full length of the HS chain, butto avoid any loss of potentially alkali-labile sulfate groups, all subsequent enzymatic degradations were performed where possible on Pronase-released HS chains. Specific Scission of H S - G e l filtration profiles of the oligosaccharides produced by specific chain scission of liver HS (Fig. 2) provided clear evidence for a sulfate-rich polymer with a domain structure as reported for other HS species, but with distinct characteristics. Nitrous acid caused extensive breakdown of liver HS (Fig. 2A ), indicating a high N-sulfate content (58.5% N-sulfation), with the majority of the N-sulfatesbeing in continuous disaccharide sequences (Table I). There was a correspondingly low frequency of resistant N-acetylated regions, although among these was a proportion that was sufficiently large in size to be excluded from the Bio-Gel P-10 column (Fig. 2 A ) . The nitrousacid profile was noticeably different from those of other HS species, which yield a high proportionof tetrasaccharides and a prominent and reproducible pattern of resistant oligosaccharides in the6-20 saccharide size range (8). Liver HS showed considerable sensitivity to heparinase I (cleavage of GlcNS0,(~6-OS03)-IdceA(2-OS0,)), with -30% (Table I) of the linkages cleaved to yield di- and tetrasaccharides as the major products (Fig. 2 B ) . This reflects the close proximity of the heparinase I-susceptible sites in the polymer chain. The relative abundance and proximity of these sites are compatible with thepresence of significant block structures. In other HS species analyzed to date, the IdceA(2-OSOJ residue
essential for heparinase I action is present inlower concentration (6.510% of total HexUA) and probably exists inrelatively small clusters (10, 11, 13). Heparinase I11 specifically attacks hexosaminidic linkages adjacent to GlcUA residues andis therefore useful to estimate GlcUA content and distribution along the HS chain. This enzyme cleaved 45% of the disaccharides in liver HS (Fig. 2C and Table I), which is substantially less than the 63 and 78% obtained with skin fibroblast (10) and endothelial (13)HS, respectively. The susceptible sites were highly contiguous(75%; Table I), and the resistant oligosaccharides, containing an internal repeat of IdceA-GlcNSO, (with possible 0-sulfation), comprised a well-defined population from four to nine disaccharides in length, with the majority in the 7-9-disaccharide size range (Fig. 2C). In view of the extent of degradation of liver HS by heparinase I, it is likely that the majority of the iduronates in these sequences are sulfated at (2-2. Disaccharide Composition-Disaccharides were prepared from liver HS by combined heparinase digestion and separation La60 LOO0
760
F
I
I
I
0000
I
e
(A) 4
EO00
600
a000
860 0 EO0
0
4000
7a Y
EO0
8000
EO0
1600
m 400
0 EO0 n
n
II
8
Y
400
1000
aooo
i
400
5
.
0
t
110
EO0
m
1
eo0
500
zoo
a60
’
0
3 I
0
a0
40
60
EO
70
hmction nnmbmr
FIG.1. Gel filtrationchromatography of intact liverHS chains. ,W6S-Labeled HS chains were released from liver HSPG by treatment with alkaline borohydride and chromatographed on a Sepharose CL6B column.
FIG.2. Specific degradationsof liver HS and analysisof resulting oligosaccharides by gel filtration chromatography. 3W35SLabeled HS was degraded by exhaustive treatment with low pH nitrous acid (A), heparinase I ( B ) ,and heparinase I11 ( 0 as described under “Experimental Procedures.” The digests were analyzed by chromatography on a Bio-Gel P-10 column. Oligosaccharide peaks are labeled according to their number of monosaccharide units.
TABLEI Susceptibility of liver HS to cleavage with various specificreagents The 3H profiles in Fig. 2 were used to calculate the levels of glycosidic bond cleavageand the relative distributions of the susceptible linkages. Specific cleavage reagent
Linkage specificity’
Disposition of cleaved linkagesb
Total linkages cleaved Contiguous
Alternate
%
%
58.5 HNO, Heparinase I GlcNR‘-GlcUA Heparinase 21.0 I11
10.5 GlcNS0,-HexUA GlcNSO,-IdceA(2-OSO,)54.2 3.8 75.2
22.5 30.1 45.3
Spaced
67.0 27.8
18.0
These disaccharide structures can also contain 0-sulfate at appropriate C-2 and C-6 positions. Only 0-sulfates absolutely essential to the reagent specificity are detailed. * Contiguous linkages give rise to disaccharide products. Alternate susceptible linkages possess single intervening resistant linkages and therefore give rise to tetrasaccharide products. Spaced susceptible linkages have two or more intervening resistant linkages, thereby giving hexasaccharide and larger oligosaccharide products. e a-D-Glucosamine with an unspecified N-substituent.
11211
Liver Heparan Sulfate Structure
60000 on Bio-Gel P-2, followed by strong anion-exchange HPLC anal0.76 ysis. The disaccharide yield was consistently 9697% (data not shown) despite exhaustive digestion and, in one instance, the additional useof heparitinase IV (similar specificity to heparinase I, but more efficient at cleaving small oligosaccharides; 0.60 40000 generous gift of Dr. K. Yoshida, Seikagaku Kogyo Co., Tokyo, Japan). The residual resistant material appeared be to primarily highly sulfated tetrasaccharides, which may therefore contain 3-0-sulfate groups. Yamada et al. (36) havesuggested, on the basisof studies with heparin, that tetrasaccharides derived 0.86 from the antithrombin 111-binding site that bear 3-0-sulfation eoooo on the terminal GlcN residue are specifically resistant to this heparinase combination. c. c* *** Analysis of the disaccharide composition confirms the dis# v tinctive structureof liver HS. Nearly 61% of the disaccharides 0 +"-F O :u were N-sulfated, and36% contained a n IdceA(2-OS03) residue K (Fig. 3 and Table 11).These findings are ingood agreement with 0.76 j 90000 the nitrous acid and heparinase I scission data, respectively (Fig.2 and Table I). By comparisonwith other HS species analyzed, the content of trisulfated disaccharides (IdceA(2OSO,)-GlcNSO3(6-OSO3))was very high (18.9%; see Table 11). El 2 0 0 0 0 a 0.60 The overall polymer sulfation of 1.34 sulfates/disaccharide (N0 sulfate: O-sulfate ratio of 0.83) was also significantly higher m than that of HS from other sources (Table 11). However, the level of sulfation is still well below that of heparin, inwhich the 1oooa polymer sulfation is -2.4 residueddisaccharide and trisulfated o.a6 disaccharides are the major structural unit. N-Acetylated disaccharides (GlcNAc-GlcUA) in liver HS ** *.* were mainly nonsulfated (Table 11), and their total content (39%)was slightly below the 45% level of scission by heparinase 0 a 16 80 e6 SO 36 46 40 111, which cleaves these disaccharides as well as any N-sulfated disaccharides that contain the GlcUA residue. The additional Time (min) 6% of GlcUA residues must therefore be linked to GlcNSO,. It FIG.3. Strong anion-exchange HPLC analysis of liver HS follows that in the vast majority of N-sulfated units, theGlcUA disaccharides. Disaccharides were prepared by exhaustive digestion residue has been epimerized to IdceA during polymer biosyn- with a combination of heparinases, purified by Bio-GelP-2 gelfiltration thesis, therebyproviding a high substrate concentration for the chromatography, and then resolved on a 5-pm Spherisorb SAX HPLC column eluted with a gradient of NaCl as described under "Experimeniduronate 2-0-sulfotransferase. tal Procedures." The eluant was monitoredusing an on-line radioactivChain Structure Adjacent to Protein Linkage Region-In ity detector, and the (A) and 35S( B ) profiles are shown.The numcommon with most other glycosaminoglycans, HS chains are bered peaks correspondto the elution positions of eight known covalently attached to the core protein via the GlcUA-(Gal),-Xyl disaccharide standards as follows. Peak 1, AHexUA-GlcNAc; peak 2, linkage tetrasaccharide at the reducing end of the chain. Be- AHexUA-GlcNSO,; peak 3, AHexUA-GlcNAc(6-OS03); peak 4, AHexUA(2-OSO,)-GlcNAc;peak 5,AHexUA-GlcNS03(6-OS0,);peak 6, cause of this, it is possible to utilize the protein anchor to AHexUA(2-OS0,)-GlcNS03; peak 7, AHexUA(2-OS03)-GlcNAc(6specifically retain oligosaccharides derived from the reducing OSO,); peak 8,AHexUA(2-OS03)-GlcNS03(6-OS03). In addition, three end, after the chain hasbeen fragmented. These specific frag- unknown minor peaks were found and are indicated by asterisks. The ments can thus be separated from the other oligosaccharides earliest eluting one has anelution position and 35S:3Hratio suggestive (derived from the more distal partsof the chain), and their sizes of a disulfated disaccharide. However, it did not exhibit the characteristic absorbance at 232 nm of a AHexUA residue (data not shown). One and compositions ascertained. This is the basis of the endpossibility is that it represents a specific nonreducing terminal "capreferencing technique that has been previously utilized, in two ping" disaccharide that is released with its uronate residue unmodified. slightly different ways, to probe the proximal regions of both Interestingly, its abundance (2.1%) does correlate with an occurrence of the skin fibroblast (9, 12) and umbilical vein endothelial (14) approximately one such structure per HS chain (-50 disaccharides). second unidentified peak occurs as a late shoulder on the major to determine theloca- The HS species. We have used this approach trisulfated disaccharide (8).This may correspond toanother trisulfated tion of the N-sulfate and iduronate 2-0-sulfate residues nearest species, which would have to be 3-0-sulfated. The third and very late to the core protein. eluting minor peak is highly sulfated and possesses a U V absorbance at Metabolically ,W5S-labeled HSPG was treated with low pH 232nm (data not shown). It may represent another highly sulfated nitrous acid, and oligosaccharides that remained on the core disaccharide of unknown structure. protein were recovered by covalent coupling of the protein to MI-Gel 15. Oliogsaccharides were then released from the pro- hyaluronate oligosaccharides as molecular weight standards. tein using alkalineborohydride. Analysis by gel filtration chro- Separation by gradient PAGE revealed a cluster of bands cormatography on Sepharose CL-GB revealed a singlepeak of responding to oligosaccharides of 10-13 disaccharides in length 3H-labeled material (Fig. 4A)with a K,, of 0.73, corresponding (Fig. 4B ). Thus, the structureof liver HS from the core protein to a M , of -5500 (34). The molecular weight of this peak is to the most proximal N-sulfate group can be represented by the almost identical to that of the largest resistantfraction recov- following general formula: HexUA-GlcNS0,-HexUA-GlcNAcered from a low pH nitrous acid digest of the whole HS chain (GlcUA-GlcNAc),~,,-GlcUA-(Gal),-Xyl. The extended size and (i.e. V, peak from Bio-Gel P-10; Fig. 2 A ) . These nitrous acid- relatively narrow size distribution of this internal sequence resistant linkage fragments (nitrous acid fragment) will con- compared remarkably well withthose of the corresponding tain repeatsequences of N-acetylated disaccharides, and these proximal N-acetylated sequences in skin fibroblast HS (9,12). can be accurately sized by using gradientPAGE calibrated with Heparinase I digestion was also employed to locate the apT
I
Y
I
1
Structure SulfateHeparan Liver
11212
TABLEI1 Disaccharide composition of liver HS The areas under the peaks in Fig. 3A were integrated to obtain the disaccharide composition. The data are presented as the average of analyses of two separate enzymedigests, with the standard deviations given in parentheses. For comparison,the disaccharide compositionsof three other HS species (one tissue HS and two cell culture HS) are also listed. These are bovine kidney HS(sample from Sigma), human adult skin fibroblast HS (J.E. Turnbull and J. T. Gallagher, unpublisheddata), and human umbilical vein endothelial cell HS (derived from the data in Ref. 13). Standard eak No. (in or&, of elution)
Total disaccharides Disaccharide structure
Rat liver“
Bovine kidney
Human skin thelia1 fibroblast
Human endo-
cells
I
1 2 3 4
*
5 6 7 8
** ***
AHexUA-GlcNAc AHexUA-GlcNAc(6-OS0,) AHexUA(2-OS03)-GlcNAc AHexUA-GlcNSO, unknown AHexUA-GlcNS03(6-OS03)
AHexUA(2-OS03)-GlcNAc(6-OS03) AHexUA(2-OS0,)-GlcNS03
AHexUA(2-OS03)-GlcNS03(6-OS0,) Unknown Unknown NSOdlOO disaccharides OSO$lOO disaccharides Total SO$lOO disaccharides
31.4(0.4) 6.2 1.2 18.0(0.7) 2.UO.l) 5.0(0.1) 0.5(0.1) 14.4(0.2) 18.9(0.6) 1.7(0.3) 0.6(0.1) 60.7 72.9 133.6
49.4 18.9 0.9 12.0
46.0 5.4 1.1 27.7
56 9
7.2
2.4
2
3.7 7.9
15.4 2.0
5 2
30.8 46.5 77.3
47.5 28.3 75.8
37 20 57
28
a In the calculation of N- and 0-sulfate contents, the three unknown “disaccharide” structures are assumed to be di-, tri-, and trisulfated, respectively, with one of the sulfates being an N-sulfate in each case (see legend to Fig. 3). As each of these structures is of minor abundance,any misattribution will not have a significant effect on the overall composition.
proximate position of the first susceptible IdceA(2-OS0,) residue relative to the protein core. As formation of IdceA(2-OS0,) requires an adjacent GlcNSO,, then a heparinase I-resistant linkage fragment (heparinase I fragment) should beat least as large as the nitrous acid fragment, if not larger. Gel filtration chromatography of the heparinase I fragment on Sepharose CL-GB (Fig. 5 A ) gave a broader, more asymmetric peak than was found with the nitrous acid fragment. The major peak eluted with a K,, of 0.71, slightly larger thanthat of the nitrous acid fragment, although the average size is skewed to a higher molecular weightby a discernible shoulder at a K,, of 0.63 and a further minor shoulder at a K,, of 0.57. All the heparinase I fragments had appreciable label associated with them. This profile was similar to that obtained following Sepharose CL-GB chromatography (data not shown) of the largest heparinase I-resistant sequences isolated from the whole HS chain (ie. material in theV, peak from Bio-Gel P-10; Fig.2 B ) . These findings indicate that theheparinase Ifragments correspond to the longest heparinase I-resistant sequences in liver HS. Unlike the nonsulfated nitrous acid fragments, these heparinase I fragments are larger, sulfated, and less homogeneous in structure and therefore cannot be banded and accurately sized by gradient PAGE (33). However, all these fragments, by definition, must contain the nitrous acid fragment. Therefore, the major part of the heparinase I fragment was recovered from the Sepharose CL-GB column (Fig. 5 A ) , treated with lowpH nitrous acid, and thenrechromatographed on the same column. Two partially resolved peaks were obtained (Fig. 5B).The content, larger M , peak, which contained 45% of the total but no 35S,corresponds t o the nitrous acid fragment (Kav= 0.83). The lower M , peak, which eluted near to and within the V, of the column, contained 55% of the total content (and all of the 35Scontent) and representssmaller degradation products derived from the region between the most proximal GlcNSO, and IdceA(2-OSOJ residues. Thus, this region contained additional GlcNSO, residues and all of the sulfate. From the distribution of label between the two peaks, it can be calculated that, on a number-average basis, the IdceA(2-OS0,) residue is -20 disaccharides from the protein core, twice the distance of the most proximal GlcNSO, residue and equivalent to twofifths of the length of the HS chain, although clearly there is
polydispersity in thepositioning of the first IdceA(2-OS0,) residue. The remaining IdceA(2-OS0,) residues are therefore concentrated in the remaining three-fifths of the polysaccharide, relatively remote from the core protein. Gel filtration of a nitrous acid digest of the heparinase I fragments on a Bio-Gel P-10 column allowed more detailed analysis of the N-sulfation pattern (Fig. 5C). Although disaccharides were released by nitrous acid (derived from contiguthe major peak ous sequences of HexUA-GlcNSO3(+6-OSO3)), corresponded to tetrasaccharides. There were also significant hexasaccharide and octasaccharide fragments, but ,H-labeled material eluting in the higher molecular weight range will be the nitrous acid fragments from the reducing end of the chain. Thus, the region of the HS chain in between the first GlcNSO, and IdceA(2-OS0,) positions willbe rather varied in structure, but comprising mainly N-sulfated and N-acetylated disaccharides in alternate sequences together with some 6-0-sulfate groups also present within these mixed sequences (tetrasaccharide peak contains some 35Slabel; Fig. 5 0 . These structural features are quite different from those of both the proximal N-acetylated domain and the distal highly sulfated areas of the polymer chain. DISCUSSION
Detailed analysis of the composition and organization of liver heparan sulfate has revealed a distinctive structure that combines aspects of both heparan sulfate and heparin. The Nsulfation level is -60%, which compareswith earlier estimates of 50% (37) and 50-60% (38). This represents a rather high N-sulfate content for a HS species and further extends the upper limit of the range (40-50%) previously suggested as encompassing the degree of variation encountered in HS species (8). It is clear from a number of recent studies that this range may also need to be less restrictive at the lower end as Nsulfation levels of 37 and 29% have recently been encountered in endothelial (13) and arterial (mainly smooth muscle) (39) HS, respectively. In addition to high N-sulfation, liver HS also possesses a remarkably high 0-sulfation level (-0.73 residues/ disaccharide), also placing it at thetop of the range (0.2-0.75 residueddisaccharide) indicated by Gallagher and Walker (8).
Liver Heparan Sulfate Structure 160
400
1
I
I
I
1
so0
100
-
60
-
;
A
I
I
I
I
160
I
n
X
200
I
v
m
-
100
;
v.
J.
U
3
I
I
(A)
I
(A)
El
11213
U
0
I
200
-
100
-
I
m
I
a
n
(B)
0
100
0 40
0 20
so
6 07 0
40
80
80
Fraotion numbar
eo0
,
I
1
I
60
eo
70
eo
loo
00
110 120
Fraotlon numbar I
I
I
I
I
,
BOO
Fraction number
282624
22
-
FIG. 5. Analysis of heparinase I-resistant proximal linkage fragments. 3W35S-Labeledliver HSPG was degraded with heparinase I, and the protein-linked resistant oligosaccharides were retrieved by coupling to Affi-Gel 15. The oligosaccharides were released by treatment of the matrix with alkaline borohydride. This material was then analyzed by gel filtration chromatography on a Sepharose CLGB column (A). The major fraction of the resistantoligosaccharides,indicated by the horizontal bar in A, was recovered and treated with lowpH nitrous acid. One half of the digest was rechromatographed on the Sepharose CLGBcolumn ( B ) , while the other half was chromatographed on a Bio-Gel P-10column ( 0 .
The combination of high N- and 0-sulfation translates to a total sulfate content of1.34 sulfateddisaccharide. This is at least 50% higher than that of most of the HS species cited by Gallagher and Walker (8) as well as skin fibroblast (Table 111, umbilicalvein endothelial (Table 111, and Chinese hamster ovary cell(40) HS species and over twiceas high as thatof some such as kidney (Table 11) and arterial (39) HS, but less than that of heparin (-2.4 sulfateddisaccharide). It is also relatively unusual, although not unique (e.g. kidney HS), that liver HS displays an excess of 0-sulfates over N-sulfates, a property also shared by the heparins. Taken together, these characteristics clearly place liver HS at the most sulfated, extreme end of the FIG.4. Analysis of nitrous acid-resistant proximal linkage HS spectrum, although the overall quantitative distinctions fragments.A, 3w5S-Labeledliver HSPG was degraded with low pH between it and heparin still clearly remain. nitrous acid, and the remaining resistant protein-linked oligosaccharThe structure of liver HS can be predicted with reasonable ides were recovered by coupling to Mi-Gel 15. The oligosaccharides accuracy from data on the size of the IdceA-rich sulfated dowere then released from the matrix by treatment with alkaline borohydride and analyzed by gel filtration chromatography on a Sepharose mains (defined by heparinase I11 scission), sequencing of the CLGB column. Onlythe 3H profile is shown as thematerial was essen- proximalregion, and disaccharide composition.Combining tially devoid of 35S0, label. B, nitrous acid-resistant oligosaccharides data from nitrous acid and heparinase I sequencing, the proxiprepared as described forA were analyzed by gradient polyacrylamide gel electrophoresis as described under “Experimental Procedures.” The mal portion of the HS chain can be describedas a region of low electrophoretic banding pattern was electrotransferred to a cationic sulfation enriched in N-acetylated disaccharides and conformnylon membrane and visualized by fluorography. Oligosaccharidesizes ing to the following general sequence: GlcNSO3-IdceA(2-OSO,)were determined by comparison with the migration positions of a series of [3Hlhyaluronateoligosaccharides of known size (molecular size indicated as number of monosaccharide units).
GlcNSO,-[HexUA-GlcNAcSO,],_,,-HexUA-GlcNSO3-HexUAGlcNAc-[GlcUA-GlcNAcl,~,o-GlcUA-[Gal],-Xyl. Considering HS chain contains -50 disaccharides (M, thattheentire
Liver Heparan Sulfate Structure
11214 Core Protein
1st
I
I
1st
GlcNSO, IdoA(2-OSO,)
f
t
* “ E
Ser-Xyl-(Gal)
I
8-1 1
- 20
75%
GlcNSO,
60% IdoA(2-OSO, )
-
- 50 disaccs.
4
w
GIcNAc - GlcA GlcNSO, ( 2 6 - 0 S 0 , ) - IdoA/GlcA GlcNS03(*6-OSO3) - IdoA(2-OSO3)
FIG.6. Diagrammatic modelof structure of liver €ISchain. The model is based ona combination of the disaccharide composition (TableII), the patternsof chain scission with different specific reagents (Fig. 2 and Table I), andthe detailed analyses of the proximal regionof the chain (Figs. 4 and 5 ) , accommodated within an estimated chain length of -50 disaccharides (Fig. 1).The model proposes a highly asymmetric structure with the sulfate density concentrated toward the distal portion of the chain. The proximal region comprises a nonsulfated sequence, 8-11 disaccharides in length, immediately adjacent to the linkage tetrasaccharide, followed by a similarly sized domain of mixed GlcUA (GlcA)lIdceA (Id&) and GlcNAdGlcNSO, content, relatively low in sulfation and devoid of IdceA(2-OS0,) residues. The remaining 60% of the chain comprises a small number of highly sulfated blocks, 7-9 disaccharides in length, heavily enriched in GlcNSO, and IdceA(2-OSO,) residues separated by short intervening sequences containing a few GlcUA residues. The model represents a simplified average structure, and there may be significant interchainvariation.
22,000-25,000), thisinternal sequence will comprise about two-fifths of the length of the polysaccharide and will contain well over half of the N-acetyl groups. The remainder of the chain will be predominantly N-sulfated, with the N-sulfates mainly arranged ina series of structural domains composed of, on average, 7-9-disaccharide-long repeats of IdceA-GlcNSO, units. This arrangement of the N-sulfates was identified by their resistance to heparinaseI11 scission (Fig. 2C). These domains will be heavily substituted with 0-sulfates, mainly at C-2 of iduronate (approximately two out of every three substituted) and, to a lesser extent, at C-6 of GlcNSO,. Three such domains could be accommodated in a single HS chain, with the domains separated by short sequences of2-3 disaccharides containing at least 1 GlcUA residue, providing for heparinase I11 sensitivity. On the basis of these conclusions, we have constructed a model of liver HS (Fig. 6) that describes a highly asymmetric structure in which the proximal sequence is typical of other HS species, whereas the distal portion contains the distinct featuresof close spacing of extended NIO-sulfated domains (>2 sulfateddisaccharide) that are clearly heparin-like in composition. It is interesting that this highly distinctive HS is not synthesized on a unique liver-specific core protein that could in some way have influenced the structure of the attached HS chains. Theliver core proteins from which this HS was derived have been shown previously to belong to the syndecan family (29,30). These same core proteins are abundantly expressed in many other cells, including fibroblasts (41) and endothelial cells (42), where they receive HS chains of radically different structure from liver HS. A surprising feature of liver HS is that even though the N-acetyl content is relatively low (40%), it still contains an extended N-acetylated sequence adjacent to the protein linkage. In anearly investigation of liver membrane HSPG, it was noted that treatment of HSPG with platelet heparitinase (an endoglucuronidase) generates a smaller “core protein” than did nitrous acid treatment (43). This enzyme may have been capable of cleaving within this heparan sequence. The length of the N-acetylated sequence is remarkably similar to that pre-
sent in skin fibroblast HS, even though the HS chains are considerably different in composition and sulfate content as well as in length (M,22,000-25,000 uersus 45,000, respectively). In heparin,by contrast, N-sulfation occurs to within1-3 disaccharides of the proteinlinkagesequence (44-46). The presence of a n extended proximal N-acetylated region in many structurally diverse HS species would therefore appear tobe a further diagnostic feature of HS, in addition to specific N - and 0-sulfation levels. The molecular organizationof the distalregion of the chain is a striking characteristic of liver HS. This may help to explain the restricted activity of endoglycosidases on this polysaccharide. There have been a number of reports describing HS-degrading endoglycosidases in theliver, which may be associated with a lysosomal compartment (4749) andpossibly the plasma membrane (35). These are probably endoglucuronidases (48, 49), and they appear to cleave only at a very limited number of sites within the chain, releasing a small number of relatively large (M,6000-7000) protein-free oligosaccharides from liver HS (35, 49). These enzymes may be restricted to acting on the limited areas of mixed sequence containing GlcUA residues that separate the highly sulfated domains, thus excising the latter intact. All of the protein binding activities and specificities of HS require moderate to high levels of sulfation (23, 24, 261, and it therefore seems likely that the distal regions will determine the protein recognition properties of liver HS. The fundamental question is why does this polysaccharide have such a distinct (and possibly unique) structure from other HS species. The high sulfation and preponderance of IdceA(2-OS0,) residues suggest the potential for a strong interaction with the fibroblast growth factors,but whether this aisphysiological mechanism in the liver is unclear. What is intriguing,however, is that the most potent mitogen for hepatocytes is the so-called hepatocyte growthfactor or scatter factor. This cytokine binds strongly to heparin (50) and also HS (511, and the sulfation pattern of HS in the liver may be especially compatible with binding to hepatocyte growth factor and regulating its biological activity.
Liver Heparan Sulfate Structure Acknowledgments-We thank Dr. Jeremy Turnbull for Droviding unpublished data o n the disaccharide composition of skin fibroblast HS and Marjorie Evans for secretarial assistance.
REFERENCES 1. Gallagher, J. T.(1989) Curc Opin. Cell Biol. 1, 1201-1218 2. Fransson, L.-A. (1989) in Heparin (Lane, D. A,, and Lindahl, U., eds) pp. 115-134, Edward Arnold Ltd., London 3. Gallagher, J. T.,Lyon, M., and Steward, W.P. (1986)Biochem. J. 236,313-325 4. Gallagher, J. T.,and Lyon, M. (1989)in Heparin (Lane, D. A,, and Lindahl, U., eds) pp. 135-158, Edward Arnold LM., London 5. Jackson, R. L., Busch, S.J., andCardin,A. D. (1991)Physiol. Reu. 71,481-539 6. KjellBn, L., and Lindahl, U. (1991)Annu. Reu. Biochem. 60, 443475 7. Lindahl, U. (1989) in Heparin (Lane, D. A,,and Lindahl, U.,eds) pp. 159-189, Edward Arnold Ltd., London 8. Gallagher, J. T.,and Walker, A. (1985)Biochem. J. 230, 665-674 9. Lyon, M., Steward, W.P., Hampson, I. N., and Gallagher, J. T. (1987)Biochem. J. 242.493498 10. Turnbull, J. E., and Gallagher, J. T. (1990) Biochem. J. 266, 715-724 11. Turnbull, J. E., and Gallagher, J. T.(1991a) Biochem. J. 273, 553-559 12. k b u l l , J. E., and Gallagher, J. T.(1991b) Biochem. J. 277, 297-303 13. Lindblom, A,, and Fransson, L.-A. (1990) G'lycoconj, J . 7, 545-562 14. Lindblom, A,, Bengtsson-Olivecrona,G., and Fransson, L.-A. (1991) Biochem. J. 279,821329 15. Linker, A., and Hovingh, P. (1975) Biochim. Biophys. Acta 386,324-333 16. Cifonelli, J.A,, and King, J. (1977) Biochemistry 16,2137-2141 17. Fransson, L.-A., Sjoberg, I., and Havsmark, B. (1980) Eur. J. Biochem. 106, 59-69 18. Winterbourne, D. J., and Mora, P. T.(1981) J. Biol. Chem. 266,43104320 19. Knecht, J., Cifonelli. J. A., and Dorfman, A. (1967) J. Biol. Chem. 242, 46524661 20. Cifonelli, J. A. (1968) Carbohyd,: Res. 8, 233-242 21. Parthasarathy, N., and Spiro, R. G. (1984) J. Biol. Chem. 259, 12749-12755 22. Edge, A. S . B., and Spiro, R. G. (1990) J. Biol. Chem. 266, 15874-15881 23. Lindahl, U., Thunberg, L., Biickstidm, G., Riesenfeld, J., Nordling, K., and Bjork, I. (1984) J. B i d . Chem. 269, 1236s12376 24. Turnbull, J. E., Fernig, D. G., Ke, Y., Wilkinson, M. C., and Gallagher, J. T. (1992) J. Biol. Chem. 267, 10337-10341 25. Walker, A., k b u l l , J. E., and Gallagher, J. T. (1994) J. Biol. Chem. 269, 931-935
11215
26. Maccarana, M., Casu, B., and Lindahl, U.(1993) J. Biol. Chem. 268,2389s 23905 27. Guimond, S., Maccarana, M., Olwin, B. B., Lindahl, U., and Rapraeger, A. C. (1993) J. Biol. Chem. 268,23906-23914 28. Oldberg, A., Hiiiik, M., Obrink, B., Pertoft, H., and Rubin, K. (1977) Biochem. J. 164,75431 29. Lyon, M., and Gallagher, J. T.(1991)Biochem. J. 273,415-422 30. Pierce, A., Lyon, M.,Hampson, I. N., Cowling,G. J., and Gallagher, J. T.(1992) J. Biol. Chem. 267, 38943900 31. Shively, J. E.,and Conrad, H. E. (1976) Biochemistry 15,3943-3950 32. Malmstrom,A., Carlstedt, I.,Aberg, L., and Fransson, L.-A. (1975)Biochem. J. 161.477489 33. k b h l , J. E.,and Gallagher, J. T. (1988) Biochem. J. 261,597-608 34. Wasteson, A. (1971) J. Chromntogc 69,87-97 35. Gallagher, J. T.,Walker, A,, Lyon,M.,and Evans, W. H. (1988)Biochem. J. 260, 719-726 36. Yamada, S., Yoshida, K., Sugiura, M., Sugahara, K., Khoo, K.-H., Moms, H. R., and Dell, A. (1993) J. Biol. Chem. 268,4780-4787 37. Akasaki, M., Kawasaki, T.,and Yamashima, I. (1975)FEBS Lett. 69, 100-104 38. Riesenfeld,J.,Hook, M., and Lindahl, U. (1982)J.Biol. Chem. 267,7050-7055 39. Schmidt, A,, Yoshida, K., and Buddecke, E.(1992)J. Biol. Chem. 267,1924219247 40. Bame, K. J., Reddy, R. V., and Esko, J. D. (1991) J. Biol. Chem. 266, 1246112468 41. Lories, V., Cassiman, J . J . , Van den Berghe, H., and David, G . (1992) J. Biol. Chem. 267, 1116-1122 42. Mertens, G., Cassiman, J.J.Van , den Berghe, H., and David, G. (1992)J. Biol. Chem. 267, 20435-20443 43. Oldberg, A,, KjellBn, L., and Hook, M. (1979) J. Biol. Chem. 264, 8505-8510 44. Lindahl, U. (1966) Biochim. Biophys. Acta 130,368-382 45. Cifonelli, J. A., and King, J. (1972) Carbohydc Res. 21, 173-186 46. Rosenfeld, L., and Danishefsky, I. (1988) J. Biol. Chem. 263,262-266 47. Hiiiik, M., Wasteson, A,, and Oldberg, A. (1975)Biochem. Biophys. Res. Commun. 67,1422-1428 48.Oldberg, A,, Heldin, C.-H.,Wasteson, A,, Busch, C., and Hiiiik, M. (1980) Biockemistry 19,57555762 49. KjellBn, L., Pertoft, H., Oldberg, A,, and Hiiiik, M. (1985) J. Biol. Chem. 260, 8416-8422 50. Nakamura, T., Nawa, K., and Ichihara, A. (1984)Biochem. Biophys. Res. Commun. 122, 145CL1459 51. Lyon, M., Deakin, J. A,, Mizuno, K.,Nakamura, T., and Gallagher, J. T.(1994) J. Biol. Chem. 269, 11216-11223
