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

The Escherichia coli K5 capsular polysaccharide has the same (GlcUA--> GlcNAc)(n) structure as the nonsulfated heparan sulfate/heparin precur sor polysaccharide. A monoclonal antibody (mAb 865) against the K5 pol ysaccharide has been described (Peters, H., Jurs, 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 86 5 to N-acetylated sequences in heparan sulfates and heparan sulfate pr oteoglycans but not to heparin. This is shown by direct binding and fl uid phase inhibition of mAb 865 in an enzyme-linked immunosorbent assa y. In this system we found that the binding of the mAb decreased with increasing sulfate content of the polysaccharide. By testing chemicall y modified K5 and heparin polysaccharides, we found that each of the m odifications that occur during heparan sulfate (HS) synthesis (N-sulfa tion, C-5 epimerization, and O-sulfation) prevents recognition by mAb 865. Samples of heparan sulfate from human aorta (HS-II) were selectiv ely degraded so as to allow the separate isolation of N-sulfated and N -acetylated block structures. N-Sulfated oligosaccharides (obtained af ter N-deacetylation by hydrazinolysis followed by nitrous acid deamina tion at pH 3.9) were not recognized by mAb 865, in contrast to N-acety lated oligosaccharides (obtained after nitrous acid deamination at pH 1.5), although the reactivity was lower than for intact HS-II. Analysi s of the latter's pH 1.5 deamination products by gel filtration indica ted that a minimal size of 18 saccharide units was necessary for antib ody binding. These results lead us to propose bivalent antibody-hepara n sulfate interaction, in which both F(ab) domains of the mAb interact with their epitopes, both of which are present in a single large (gre ater than or equal to 18 saccharide units) N-acetylated domain and add itionally with single epitopes present in two N-acetylated sequences ( each < 18 saccharide units) bridged by a short N-sulfated domain. Immu nohistochemistry with mAb 865 on cryostat sections of normal human kid ney tissue, revealed its binding to most but not all renal basement me mbranes. However, all renal basement membranes contain heparan sulfate , as shown by a mAb against heparitinase-digested heparan sulfate stub s (mAb 3G10). This finding indicates that not all heparan sulfate chai ns present in basement membranes express the mAb 865 epitopes. Besides the normal distribution, mAb 865 staining was found in fibrotic and s clerotic 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 d emonstrated by anti-K5 mAb 865 in normal and diseased kidneys.