iV-Acetylated Domains in Heparan Sulfates Revealed by a M onoclonal Antibody against the Escherichia coli K 5 Capsular Polysaccharide DISTRIBUTION OF THE COGNATE EPITOPE IN NORMAL HUMAN KIDNEY AND TRANSPLANT KIDNEY WITH CHRONIC VASCULAR REJECTION

The Escherichia coli K5 capsular polysaccharide has the same (GlcUA-->GlcNAc)n structure as the nonsulfated heparan sulfate/heparin precursor polysaccharide. A monoclonal antibody (mAb 865) against the K5 polysaccharide has been described (Peters, H., Jürs, M., Jann, B., Jann, K., Timmis, K. N., and Bitter-Sauermann, D. (1985) Infect. Immun. 50, 459-466). In this report, we demonstrate the binding of anti-K5 mAb 865 to N-acetylated sequences in heparan sulfates and heparan sulfate proteoglycans but not to heparin. This is shown by direct binding and fluid phase inhibition of mAb 865 in an enzyme-linked immunosorbent assay. In this system we found that the binding of the mAb decreased with increasing sulfate content of the polysaccharide. By testing chemically modified K5 and heparin polysaccharides, we found that each of the modifications that occur during heparan sulfate (HS) synthesis (N-sulfation, C-5 epimerization, and O-sulfation) prevents recognition by mAb 865. Samples of heparan sulfate from human aorta (HS-II) were selectively degraded so as to allow the separate isolation of N-sulfated and N-acetylated block structures. N-Sulfated oligosaccharides (obtained after N-deacetylation by hydrazinolysis followed by nitrous acid deamination at pH 3.9) were not recognized by mAb 865, in contrast to N-acetylated oligosaccharides (obtained after nitrous acid deamination at pH 1.5), although the reactivity was lower than for intact HS-II. Analysis of the latter's pH 1.5 deamination products by gel filtration indicated that a minimal size of 18 saccharide units was necessary for antibody binding. These results lead us to propose bivalent antibody-heparan sulfate interaction, in which both F(ab) domains of the mAb interact with their epitopes, both of which are present in a single large (>/=18 saccharide units) N-acetylated domain and additionally with single epitopes present in two N-acetylated sequences (each <18 saccharide units) bridged by a short N-sulfated domain. Immunohistochemistry with mAb 865 on cryostat sections of normal human kidney tissue, revealed its binding to most but not all renal basement membranes. However, all renal basement membranes contain heparan sulfate, as shown by a mAb against heparitinase-digested heparan sulfate stubs (mAb 3G10). This finding indicates that not all heparan sulfate chains present in basement membranes express the mAb 865 epitopes. Besides the normal distribution, mAb 865 staining was found in fibrotic and sclerotic lesions in vessels, interstitium, and mesangium in transplant kidneys with chronic vascular rejection. Occasionally, a decrease of staining was observed within tubulo-interstitium and glomeruli. These findings show that N-acetylated sequences in heparan sulfates can be demonstrated by anti-K5 mAb 865 in normal and diseased kidneys.

units) bridged by a short iV-sulfated domain. Immune* histochem istry with mAb 865 on cryostat sections of normal human kidney tissue, revealed its binding to most but not all renal basem ent membranes. However, all renal basement membranes contain heparan sulfate, as shown by a mAb against heparitinase-digested hepa ran sulfate stubs (mAb 3G10). This finding indicates that not all heparan sulfate chains present in basem ent mem branes express the mAb 865 epitopes. Besides the nor mal distribution, mAb 865 staining was found in fibrotic and sclerotic lesions in vessels, interstitium , and mesangium in transplant kidneys with chronic vascular rejec tion. Occasionally, a decrease of staining was observed within tubulo-interstitium and glomeruli. These find ings show that iV-acetylated sequences in heparan sul fates can be demonstrated by anti-K5 mAb 865 in normal and diseased kidneys.
The biosynthesis of heparan sulfate (HS)1 involves the forma tion of a nonsulfated (GlcUA/31,4->GlcNAcal,4)" precursor po lysaccharide AT-acetylheparosan, which subsequently undergoes a series of polymer modification reactions. These reactions start with N-deacetylation/N-sulfation of GlcNAc residues, which is followed by C-5 epimerization of GlcUA to Idee A units, and fi nally by O-sulfation at various positions (1). The GlcUA C-5 epimerization and O-sulfation reactions occur in the close vicinity of iV-sulfate groups, pointing to a key role for the glucosaminyl AMeacetylase/A^sulfotransferase enzyme in determining the overall extent of modification of the HS chain. Structural analy sis of HS from various sources has revealed that these modifica tions tend to colocalize in block sequences, separated by rela tively unmodified domains (2)(3)(4)(5)(6). The extent of biosynthetic modification, affecting the number, length, and substitution pat terns of the modified domains as well as their position along the HS chain, may differ among cell types (7), alter during prolifer ation (8), and change as a result of cell transformation (9,10).
Many biological activities of heparan sulfate proteoglycans (HSPGs) are due to interactions between the HS polysaccha ride side chains and a variety of proteins such as extracellular matrix molecules, enzymes, enzyme inhibitors, growth factors, and other cytokines (1,(11)(12)(13), These interactions can be either specific, dependent on defined sulfation patterns within given sequences of sugar residues, as described for antithrombin (14), basic fibroblast growth factor (15,16), hepatocyte growth factor (17), and interferon-7 (18); or they can be mainly based on relatively nonspecific electrostatic interactions and involve pro teins such as lipoprotein lipase (19), platelet factor 4 (20) and mast cell protease I (2 1 ) (see Ref. 22 for a general discussion).
Structural analysis of HS is complicated by the fact that even highly purified and uniform preparations consist of mixtures of polysaccharide chains that have reached different levels of modification. Monoclonal antibodies (mAbs) that specifically recognize well-defined epitopes in HS could be major tools in such analysis. Already in 1985 a mAb (designated as mAb 865) against the Escherichia coli K5 polysaccharide had been de scribed (23). This bacterial polysaccharide has the same (GlcUA/31,4->GlcNAcal,4)/z structure as the nonsulfated HS/ heparin precursor polysaccharide (24), suggesting that anti-K5 mAb 865 might recognize the iV-acetylated (K5-like) domains in HS. In the present report, we show that this is indeed the case. Immunohistological application of the mAb on normal human renal tissue and on transplant kidneys with chronic vascular rejection revealed these iV-acetylated HS sequences in extracellular matrix and basement membranes. Since in most protein-HS interactions highly sulfated, IdceA-rich domains are involved, the possible biological significance of these rela tively unmodified iV-acetylated domains in HS is discussed.

MATERIALS AND METHODS
Glycosaminoglycans and Heparan Sulfate Proteoglycans ~ HS (prep aration HS-II) was isolated from human aorta, essentially according to Iverius (25). HS from pig intestine, the E, coli K5 capsular polysaccha ride, with the same (GlcUA->GlcNAc),t structure as the nonsulfated HS/heparin precursor polysaccharide (24) and chemically O-sulfated K5 polysaccharide were kindly provided by Dr. G. van Dedem (Organon Corp., Oss, The Netherlands). iV-Sulfated K5 was given by Dr. B. Casu (Instituto di Chimica e Biochimica G. Ronzoni, Milan, Italy). Intact heparin (stage 14) from pig intestinal mucosa was obtained from Inolex Pharmaceutical Division (Park Forest South, IL) and purified by re peated precipitation with cetylpyridinium chloride from 1.2 M NaCl (26). Heparan sulfate proteoglycans were isolated from the mouse Engelbreth-Holm-Swarm sarcoma (Becton Dickinson Labware, Bedford, MA) or from rat glomerular basement membranes, essentially accord ing to van den Heuvel et al. (27). Briefly, glomeruli were isolated from rat kidney cortex by a sieving method. Glomerular basement mem branes were isolated from glomeruli by the detergent method, extracted twice in 4 M guanidine and dialyzed against 7 M urea, and HSPG was isolated by ion exchange chromatography. HSPG containing fractions were pooled, concentrated, and extensively dialyzed against phosphatebuffered saline (PBS).
Chemical Modifications o f Polysaccharides-N -Acetylated oligosac charides were derived from HS-II by low pH nitrous acid deamination, Reaction at pH 1.5 (10 min) was performed as described (28) and resulted in selective attack of iV-sulfated GlcN units, with cleavage of the corresponding glucosaminidic linkages (29). The deamination prod ucts were reduced with NaBH4. Deamination products, obtained after degradation of 3 m g of HS-II by nitrous acid, pH 1.5 followed by reduction with NaBH4 were analyzed by gel chromatography on a column (1 X 140 cm) of Bio-Gel P-10 fine (Bio-Rad Laboratories, Her cules, CA) in 0.5 M ammonium hydrogen carbonate, eluted at a rate of 2.4 ml/h. Effluent fractions of 0.6 ml were collected and analyzed for hexuronic acid by the carbazole reaction (30), lyophilized twice to get rid of the ammonium hydrogen carbonate, dissolved in PBS, and tested in the inhibition ELISA for mAb 865 binding (see below). To obtain Nsulfated oligosaccharides, HS-II was deacetylated by hydrazinolysis followed by deamination at pH 3.9 (31). For JV-deacetylation by hydrazinolysis, samples (1 mg) of HS-II were dissolved in 1 ml of hydrazine hydrate (Fluka; H20 content, 36%) containing 1% (w/v) hydrazine sul fate and heated in sealed glass tubes at 96 °C. After 4 h, the samples were repeatedly evaporated to dryness and desalted on PD-10 columns (Pharmacia, Uppsala, Sweden) according to the instructions of the manufacturer. After this procedure, deamination at pH 3.9 (28) cleaved the polysaccharide chain at A^-unsubstituted GlcN residues (29). The deamination products were reduced with NaBH4. Completely (Nand 0-) desulfated heparin was prepared according to Jacobsson et al. (32), and iV-acetylated by treatm ent with acetic anhydride as described by Danishefsky et al. (33). E L IS A -Binding properties of mAb 865 to polysaccharides were tested in ELISA. Polysaccharides (50 pig/ml) were coated overnight at room temperature in PBS to the wells of polystyrene flat-bottom microtiter plates (NUNC Maxisorp, Life Technologies, Inc., Breda, The N eth erlands), 100 /xlAvell. Alternatively, HSPG (1 ¿xg/ml) was coated under the same conditions. Wells were washed 6 times with PBS containing 0.05% Tween 20 (PBS-T) and incubated for 2 h at room temperature with PBS containing 1% gelatin, 120 ¿d/well, to avoid nonspecific anti body binding. After washing again with PBS-T, a dilution range of mAb 865 in PBS-T, 100 juJ/well, was incubated for 1 h at room temperature in the ELISA plates. Detection of the mAb and substrate reaction was identical to the inhibition ELISA and has been described before (34). The mAb 865 inhibition ELISA is based on the inhibition of mAb 865 binding to coated K5 or HS-II by liquid phase polysaccharides, HS-IIderived oligosaccharides, or intact HSPGs, Percentage of inhibition was calculated as (1 -(A450 with inhibit or/A450 without inhibitor)) x 100%. IC50 (/xg of inhibitor/ml) is defined as the concentration of inhib itor that gives 50% inhibition in the ELISA system.
Immunohistology-Normal human kidney specimens in = 8 ) were obtained during surgery or were from cadaveric donor kidneys not suitable for transplantation. Specimens of renal transplant tissue (n = 8 ) were obtained by percutaneous biopsy or after nephrectomy of the renal graft. Indirect immunofluorescence analysis was performed as described (35) on 2-ju.m human kidney cryostat sections. The antibodies used were a mouse IgM mAb against the E. coli K5 capsular polysac charide, which has been described before (23), and the mouse IgG2b mAb 3G-10, reacting with HS stubs generated by heparitinase digestion (36) (a gift from Dr. G. David, University of Leuven, Belgium). Hep aritinase (Sigma, EC 4.2.2.8) digestion of the sections was done for 1 h at 37 °C, 0.25 units/ml in 10 mM HEPES buffer containing 1 mM Ca2+, pH 7.0. As secondary antibodies, fluorescein isothiocyanate-labeled goat anti-mouse IgM (Fc) (Nordic, Tilburg, The Netherlands), and flu orescein isothiocyanate-labeled F(ab)2 fragments of sheep anti-mouse IgG (Organon Teknika, Turnhout, Belgium) were used. Control exper iments in which the fluorescein isothiocyanate-labeled secondary anti bodies were applied to the sections without prior primary antibody incubation were consistently negative. mAb 3G10 was completely neg ative without pretreatment of the sections with heparitinase. Sections were embedded in Vectashield (Vector Laboratories Inc., Burlingame, CA) and examined on a Zeiss Axioskop microscope equipped for fluo rescence microscopy.

RESULTS
Expression of the Anti-K5 mAb 865 Epitope in H eparan S u l fates and Heparan Sulfate Proteoglycans -The binding of the anti-K5 mAb 865 to HS was tested in ELISA with a low sulfated HS preparation (-0.6 sulfate groups/disaccharide; iso lated from human aorta, designated as HS-II), a high sulfated HS preparation (-1.5 sulfate groups/disaccharide; isolated from porcine intestine), and heparin (-2.5 sulfate groups/disaccharide), and compared with the binding to the nonsulfated K5 polysaccharide. From Fig. 1A it becomes clear that HSs are recognized by the mAb, especially the low sulfated HS-II, al though to a lesser extent than the K5 polysaccharide. The mAb did not bind to heparin. Since the coating efficiency of these polysaccharides might be unequal due to differences in nega tive charge, we tested the same preparations in a fluid phase inhibition ELISA. HS-II was used to coat the ELISA plates. Results are shown in Fig. IB and are essentially the same as found in the direct ELISA (Fig. 1A). The inhibitory activity of HS-II was ± 3000-fold lower than that of K5. Since high sul fated HS demonstrated only weak inhibition and heparin did not inhibit at all, it is suggested that the extent of chain modification is inversely correlated with antibody binding.. In control experiments mAb 865 was tested for its binding to other glycosaminoglycans such as chondroitin sulfate A and C, dermatan sulfate, and hyaluronic acid and to dextran sulfate and DNA, which all were completely negative. These control exper iments exclude the possibility that the binding of mAb 865 to HS is due to nonspecific, charge-based interactions.
Next to glycosaminoglycans, the mAb was also tested for binding to isolated HSPG from mouse Engelbreth-Holm-Swarm sarcoma (perlecan) and from rat glomerular basement membranes. Both HSPGs were recognized by mAh 865 in the direct ELISA and as fluid phase inhibitor (Fig. 1 , C and D), Engelbreth-Holm-Swarm HSPG expressing more mAb 865 epitopes/mg core protein than glomerular basement membrane HSPG.
Heparitinase digestion of the HSPGs abolished all binding of mAb 865, whereas the binding of antibodies against the core proteins of both HSPGs were not affected by this treatment (not shown), thereby confirming HS specificity of mAb 865.  (Table I). Complete iV-sulfation of K5 abolished all binding to the mAb. To evaluate the effect of C-5 epimerization we tested completely (N-and 0-) desulfated, AT-reacetylated heparin. This preparation differs from K5 in one major regard, i.e. the occurrence of IdceA units (±80% of the total hexuronic acid contents). From Table I it is inferred that such units completely prevent antibody binding. We also tested an O-sulfated K5 polysaccharide preparation containing an average of -1.2 O-sulfate (but no JV-sulfate) groups/disaccharide unit. The locations of the O-sulfate groups were not defined but would presumably primarily involve C-6 of the GlcN units, along with C-2 and/or C-3 of the GlcUA units. Table I clearly show that O-sulfation of K5 polysaccharide completely prevents mAb 865 binding. From these experiments we concluded that each of the modifications known to occur during HS synthesis (iV-sulfation, C-5 epimer ization, and O-sulfation) prevents recognition by anti~K5 mAb 865.

R e q u ire m e n ts o f the m A b 865 E p ito p e s in th e H S C h a in -
In order to define the requirements of the mAb 865 epitopes in the HS chain more precisely, samples of HS-II were selectively degraded to isolate separately N-sulfated and N-acetylated block structures of the molecule (see "Materials and Methods"). These oligosaccharides were then tested in the mAb 865 inhi bition ELISA using HS-II as coated antigen. The results in Fig.  2 demonstrate complete loss of reactivity for the N -sulfated oligosaccharides. In contrast to this, the Af-acetylated oligosac charides are still recognized by mAb 865, although to a consid erably lower extent. Nitrous acid degradation at pH 3.9 alone, i.e. not preceded by iV-acetylation, had no influence on antibody binding, which indicates that iV-unsubstituted GlcN units are not located within the epitopes. In this respect, anti-K5 mAb 865 clearly differs from another anti-HS mAb (JM-403) that we described recently, whose epitope is dependent on the presence of N-unsubstituted glucosamine units in HS (37). The fact that the heavily modified, heparin-like A^-sulfated block sequences of the HS chains (obtained after nitrous acid deamination at pH 3.9 of N-deacetylated HS-II) lacked all mAb 865 binding sug gests to us that the epitope is located in the N-acetylated regions of HS. If this is true, why is there a considerable loss of  inhibitory capacity of the iV~acetylated domains, which are obtained after nitrous acid pH 1.5 cleavage of HS? From the work of others we know that a size of five sugar residues is sufficient for monovalent antibody binding (38). Therefore, we analyzed the minimal binding size of iV-acetylated oligosaccha rides derived from HS-II, which still demonstrate binding to the mAb. To this end, a 3-mg sample of HS-II was deaminated by nitrous acid, pH 1,5. The resulting oligosaccharides were subsequently separated by gel filtration on a Bio-Gel P-10 column. From Fig. 3 it becomes clear that only large, A^acetylated oligosaccharides >18 residues bound to mAb 865, This finding suggests that bivalent epitope recognition is required for adequate binding of the mAb, Immunohistology with mAb 865 on Normal H uman Renal Tissue-The epitopes detected by mAb 865 in cryostat sections of human renal tissue were expressed in most but not all basement membranes in a linear fashion. Glomerular base ment membranes were moderately positive, while Bowman's capsule and mesangial matrix were more prominent (Fig. 4A). Basement membranes of endothelial cells of peritubular capil laries and other blood vessels, including the basal laminae surrounding vascular smooth muscle cells were strongly posi tive (Fig. 4C). Tubular basement membrane staining varied from strongly positive to completely negative (Fig. 4, A and C). In two kidney specimens of older patients moderate interstitial fibrosis and mesangial matrix accumulation were found, which were stained with mAb 865 (not shown). HS specificity of the staining was demonstrated by pretreatment of the sections with heparitinase, which completely prevented all staining (not shown). On the other hand, all renal basement membranes were stained by mAb 3G10, which reacts with the residual HS stubs remaining after enzymatic cleavage by heparitinase (Fig.  4, B and D). The resultant 3G10 epitope contains an essential, terminal, 4,5-unsaturated uronate residue and thus can serve as a general HS marker, of which staining intensity is inde pendent of HS modifications (36). These results demonstrate the presence of HS in all renal basement membranes, some of them being negative for the mAb 865 epitope. The most likely explanation points toward differences in modification/sulfation of HS in the various basement membranes, highly modified HS being negative and low sulfated HS being positive for mAb 865.
Distribution of mAb 865 Epitopes in Transplant Kidneys with Chronic Vascular Rejection-Since we observed in some normal kidney biopsies that interstitial and periglomerular fibrosis and mesangial sclerosis were associated with a higher binding of mAb 865, we analyzed renal transplant biopsies with chronic vascular rejection. Chronic vascular rejection in kidney transplants is morphologically characterized by severe narrowing of arteries due to intima fibrosis and interstitial fibrosis with tubular atrophy. Sometimes a glomerulopathy,

N-Acetylated Domains in Heparan Sulfates
value agrees with the calculated distance between both Fab arms of an immunoglobulin molecule (39). Simultaneous bind ing of both F(ab) antibody domains will strongly promote the interaction. It is noteworthy that a comparable type of bivalent binding has been described recently for the interaction of interferon-y dimer with HS (18). In this elegant study it was shown that the high affinity binding domain for interferon-7 in HS is composed of two terminal iV-sulfated domains, linked together by an intervening iV-acetylated domain. The interfer-on~y dimer bound to both iV-sulfated terminal regions, while a monovalent interferon-y-HS interaction was insufficient for stable binding. Our finding that deamination at pH 1.5 results in partial loss of antibody recognition suggests that in native HS, not only the large Af-acetylated domains (S:18 saccharide units) but also smaller N-acetylated sequences bridged by a short iV-sulfated domain, are recognized by mAb 865. Cleavage of the HS polysaccharide at iV-sulfated glucosamine residues (deamination at pH 1.5) thus abolishes bivalent antibody rec ognition of such small iV-acetylated sequences. Alternatively, the extended minimal polymer size of 18 saccharide units needed for antibody recognition might be explained by assum ing a conformational epitope. For example, in a helical polymer structure, a much longer primary sequence will be required for the maintenance of a conformational epitope. Immunofluorescence studies on human kidney sections with mAb 865 revealed th at the carbohydrate epitope is not evenly distributed among the HS subspecies of this tissue. All base ment membranes, as expected, were found to contain HS, as evidenced by the anti-HS stub mAb 3G10. However, the po lysaccharide present in some tubular basement membranes stained poorly with mAb 865, suggesting the existence of HS isoforms lacking the corresponding epitope. The most likely explanation for this is the existence of a high sulfated HS isoform in these particular tubular basement membranes. This is supported by the finding that another anti-HS mAb (JM-403), which recognizes epitopes with JV-unsubstituted glucosa mine units in low sulfated HS isoforms (37), shows the same staining pattern. Alternatively, the N-acetylated domains may be inappropriately spaced for antibody binding. Staining with mAb 865 was also observed in mild fibrosis and sclerosis, which is found in normal renal tissue, secondary to aging. This led us to the analysis of renal tissue with more extensive fibrosis. To this end we evaluated renal transplant biopsies with chronic vascular rejection. In these biopsies, fibrotic and sclerotic le sions were positive for both anti-HS stub mAb 3G10 and for mAb 865. This increased staining, however, is not specific for vascular chronic rejection, since preliminary experiments in our laboratory indicated that also in experimental models of renal diseases (Adriamycin nephropathy and active Heymann glomerulonephritis), fibrotic areas clearly express the mAb 865 epitope.2 Using anti-K5 mAb 865 as a probe to detect HS, our findings indicate that HS in these fibrotic/sclerotic areas rep resents a low sulfated HS isoform. We suggest that these HS chains are attached to perlecan, since anti-perlecan core pro tein mAbs also stain renal fibrotic/sclerotic areas (40), and mAb 865 binds to perlecan in ELISA (Fig. 1 , C and D ).
What is the biological significance of these Af-acetylated do mains in HS? As indicated in the Introduction, many growth factors, enzymes, and enzyme inhibitors can bind to HS. Gen erally such interactions involve variously sulfated regions along the polysaccharide chain; so far no proteins have been shown to specifically interact with the N-acetyl ate d domains in HS. Nevertheless, N-acetylated sequences in HS have been implicated in a variety of biologically important processes.