title: N-Acetylated domains in heparan sulfates revealed by a monoclonal antibody against the Escherichia coli K5 capsular polysaccharide. Distribution of the cognate epitope in normal human kidney and transplant kidney with chronic vascular rejection.
abstract: 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.
authors.affiliation: Division of Nephrology, University Hospital St. Radboud, 6500 HB Nijmegen, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Born
authors.forename: K
authors.initials: K
authors.lastname: Jann
authors.forename: K J
authors.initials: KJ
authors.lastname: Assmann
authors.forename: U
authors.initials: U
authors.lastname: Lindahl
authors.forename: J H
authors.initials: JH
authors.lastname: Berden
mesh_terms: D000107:Acetylation; D000293:Adolescent; D000328:Adult; D000911:Antibodies, Monoclonal; D001011:Aorta / Q000737:chemistry; D016667:Bacterial Capsules; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis / Q000276:immunology; D004926:Escherichia coli; D019084:Fluorescent Antibody Technique, Indirect; D006084:Graft Rejection; D006497:Heparan Sulfate / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000737:chemistry* / Q000276:immunology; D016030:Kidney Transplantation / Q000276:immunology; D008297:Male; D008875:Middle Aged; D011135:Polysaccharides, Bacterial / Q000276:immunology*; D014018:Tissue Distribution
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D006497:Heparan Sulfate; D011135:Polysaccharides, Bacterial; C050673:capsular polysaccharide K5