Epitope specificity of myeloperoxidase antibodies: identification of candidate human immunodominant epitopes.

Anti-neutrophil cytoplasmic autoantibodies (ANCA) are a common feature of systemic vasculitides and have been classified as autoimmune conditions based, in part, on these autoantibodies. ANCA are subdivided further based on their primary target: cytoplasm (c-ANCA) or perinuclear region (p-ANCA). p-ANCAs commonly target myeloperoxidase (MPO), an enzyme with microbicidal and degradative activity. MPO antibodies are non-specific for any single disease and found in a variety of vasculitides, most commonly microscopic polyangiitis. Despite their prevalence, their role in human disease pathogenesis remains undefined. We sought to characterize the sequential antigenic determinants of MPO in vasculitis patients with p-ANCA. Of 68 patients with significant levels of p-ANCA, 12 have significant levels of MPO antibodies and were selected for fine specificity epitope mapping. Sequential antigenic targets, including those containing amino acids (aa) 213-222 (WTPGVKRNGF) and aa 511-522 (RLDNRYQPMEPN), were commonly targeted with a prevalence ranging from 33% to 58%. Subsequent analysis of autoantibody binding to the RLDNRYQPMEPN peptide was assessed using a confirmatory enzyme-linked immunosorbent assay format, with six patients displaying significant binding using this method. Antibodies against this epitope, along with four others (aa 393-402, aa 437-446, aa 479-488 and aa 717-726), were reactive to the heavy chain structure of the MPO protein. One epitope, GSASPMELLS (aa 91-100), was within the pro-peptide structure of MPO. B cell epitope prediction algorithms identified all or part of the seven epitopes defined. These results provide major common human anti-MPO immunodominant antigenic targets which can be used to examine further the potential pathogenic mechanisms for these autoantibodies.

Anti-neutrophil cytoplasmic antibodies target sequential functional proteinase 3 epitopes in the sera of patients with Wegener's granulomatosis.

Many patients with Wegener's granulomatosis (WG) have anti-neutrophil cytoplasmic antibodies (c-ANCA). Aside from being a diagnostic marker, these autoantibodies may play roles in disease pathogenesis. Proteinase 3 (PR3) is the primary target of c-ANCA in WG patient sera. Of 60 c-ANCA-positive patients, 10 patients were selected for detailed humoral epitope analysis, contingent upon serum availability, using samples with positive levels of anti-PR3 by enzyme-linked immunosorbent assay (ELISA). Sequential epitope specificities of anti-PR3 antibodies detected by screening the maximally overlapping solid-phase octapeptides of PR3 showed seven major common antigenic targets bound by WG patient sera. These include novel and previously identified sequential PR3 epitopes bound by c-ANCA. B cell epitope prediction algorithms identified all or part of the seven defined epitopes. Several epitopes share sequence and structural proximity with functional sites, including the catalytic triad and proposed binding sites of other potential proteins [PR3 complementary peptide and soluble endothelial protein C receptor (sEPCR)]. Epitope 4 (VVLGAHNVRTQ) had the highest binding prevalence (90%) and epitope 2 (AQPHSRPYMAS) has the highest average reactivity of the antigenic regions. Epitope 4 includes the interaction site between sEPCR and PR3 which may serve as an important interaction to down-regulate inflammation. Epitopes 3, 5 and 7 are in direct proximity to amino acids that form the catalytic triad of the protein. c-ANCA targets both unique and previously known sequential PR3 peptides. This information may prove useful in understanding anti-PR3-mediated disease pathogenesis in systemic vasculitides.

Expression of mucin-associated sulfo-Lea carbohydrate epitopes on human colon carcinoma cells.

The level of sulfo-Lea (SO3-3Gal beta 1-3(Fuc alpha 1-4)GlcNAc) epitope recognized by monoclonal antibody (mAb) 91.9H in hepatic metastasis of colon carcinoma is known to be lower than at the primary sites. We examined 19 human colon carcinoma cell lines for their production of this epitope. Sixteen cell lines were found to produce high M(r) components that metabolically incorporated [35S]sulfate and were resistant to heparitinase I and chondroitinase ABC, and 8 of them were reactive with mAb 91.9H as shown by western blotting analysis. These were all of the 4 cell lines derived from well differentiated primary tumors (HCCP-2998, LS174T, GEO, and CBS), 2 of 10 cell lines (DLD-1 and HCT116) from moderately to poorly differentiated primary tumors, and 2 of 5 cell lines (SW480 and HCC-M1544) from metastases. Incubation of LS174T cells with benzyl-N-acetyl-alpha-D-galactosaminide abrogated the incorporation of [35S]sulfate and the reactivity of mAb 91.9H with high M(r) components in the cell lysates. Sodium chlorate, which inhibits the formation of 3'-phosphoadenosine 5'-phosphosulfate, also inhibited the [35S]sulfate incorporation and reactivity with mAb 91.9H. These treatments did not change the incorporation of [14C]threonine into high M(r) components. These results indicated that sulfo-Lea epitopes were expressed on O-linked carbohydrate chains in sulfomucins. Immunohistochemical studies of tumor tissues in nude mice indicated that sulfo-Lea was expressed at the site of orthotopic transplantation in the cecum. The expression appeared to be suppressed in liver metastatic foci in nude mice.

Serum and plasma M(r) 92,000 progelatinase levels correlate with spontaneous metastasis of rat 13762NF mammary adenocarcinoma.

The expression of metalloproteinases, such as type IV collagenase/gelatinase, enables tumor cells to degrade type IV collagen present in the basement membrane and correlates with metastatic potential of several tumor types. We found that increased levels of rat serum type IV collagenolytic activity are associated with increased 13762NF mammary adenocarcinoma metastases in lungs and lymph nodes of syngeneic rats. To investigate serum metalloproteinases responsible for type IV collagenolysis, we performed zymography and Western blot analysis of rat sera. A M(r) 92,000 progelatinase (progelatinase B, M(r) 92,000 type IV procollagenase, MMP-9) was detected on zymograms of rat sera within 16 days after intramammary fat pad inoculation of highly metastatic MTLn3 cells. The activated serum M(r) 92,000 progelatinase degraded sodium dodecyl sulfate-denatured type I and IV collagens but was not active on casein, albumin, hemoglobin, and immunoglobulin. Sera from rats with fat pad tumors and lung metastases formed from highly metastatic clones possessed greater than 7 times higher levels of serum M(r) 92,000 progelatinase than control sera or sera from rats bearing similar size fat-pad tumors of low or nonmetastatic clones. The results were confirmed by Western blot analysis of rat sera using rabbit anti-human M(r) 92,000 progelatinase antibodies. Similar results were obtained from the analysis of rat plasma samples. The serum and plasma M(r) 92,000 progelatinase levels correlated with the extent of metastases in the lung and lymph nodes. The results indicate that high levels of serum and plasma M(r) 92,000 progelatinase are associated with the presence of disseminated metastatic adenocarcinoma cells and suggest that this enzyme may facilitate the invasion of blood-borne tumor cells through vascular basement membranes.

Human colonic sulfomucin identified by a specific monoclonal antibody.

Since the 1960s, the loss of sulfomucin from colonic epithelium has been considered to be an indicator of an early stage of carcinogenesis; yet, the biochemical basis for this phenomenon has never been elucidated. We recently prepared a monoclonal antibody (mAb) 91.9H that immunoprecipitates the normal colonic mucins metabolically incorporating [35S]-sulfate. This mouse IgG1 antibody did not cross-react with colon carcinoma mucins that lack sulfate groups. Using normal colonic epithelia unlabeled or radiolabeled with [35S]sulfate and [3H]glucosamine, we purified a high molecular weight glycoprotein that reacts with mAb 91.9H. This was achieved by a combination of DEAE-cellulose anion-exchange chromatography, consecutive treatments with chondroitinase ABC plus heparitinase and with sodium dodecyl sulfate plus 2-mercaptoethanol, and gel filtration on Sepharose CL-2B in the presence of 8 M urea. Antibody reactivity was found in acidic but not neutral high molecular weight glycoproteins. After Sepharose CL-2B fractionation, the mAb 91.9H-reactive fractions consisted of a component with an approximate molecular weight of 500,000-900,000. A purified sulfomucin contained protein, neutral sugar, amino sugar, sialic acid, and sulfate in an approximate ratio of 2.5:1.0:1.1:0.4:0.5. The polypeptide portion was rich in hydrophilic amino acids, particularly threonine. Binding of mAb 91.9H in solid-phase assays was inhibited to 50% by purified normal colon acidic mucin at doses of 5-50 micrograms/ml, depending on different preparations. Various glycosaminoglycans or sulfatides did not show inhibitory activity. Sulfomucin reactivity with mAb 91.9H, as determined by solid-phase-binding inhibition and by dot blot assays, was significantly reduced by chemical desulfation of sulfomucins with anhydrous hydrochloric acid, suggesting that sulfate groups served as a portion of the immunochemical determinant for this antibody. Sulfate residues were apparently linked to alkaline-sensitive carbohydrate chains, but alkaline-released carbohydrate chains did not react with mAb 91.9H. Immunohistochemical examinations showed that mAb 91.9H bound normal colonic epithelial cells, which also stained with high-iron diamine, more strongly than it bound colon carcinoma cells.