Radiation inactivation of human intestinal mucin: determination of the size of the functional antigenic unit

Previously we have shown that the major antigenic determinant of human intestinal mucin is associated with its glycopeptide monomers and not the 118 kDa 'link' component. In the present study, the size and nature of the functional unit containing the antigenic determinant has been assessed by radiation inactivation and immunological assays. Increasing doses of radiation led to a monoexponential decay in antigenic reactivity due to a progressive loss of antigenic determinants. From three independent mucin preparations, a value of 78500 +/- 7000 was determined for the Mr of the functional antigenic unit. Prolonged pronase digestion of native mucin released large degraded glycopeptide monomers containing all the mucin carbohydrate, and low molecular weight peptides. The antigenicity of the glycopeptides decreased with digestion but could not be recovered in the peptide fractions, suggesting that determinants were released and destroyed by the enzyme. Treatment of native mucin with trifluoromethanesulphonic acid caused a major loss of carbohydrate (approx. 70%), but the protein component was unchanged in amino acid profile and remained antigenic. Subsequent thiol reduction, however, abolished the antigenicity of the deglycosylated mucin. We conclude that antigenicity is associated with a non-glycosylated segment of the peptide backbone of the glycopeptides and that a large functional unit of Mr 78500 which is stabilized by disulphide bonds is important for full antigenic activity.     

Biochemical characterization of the component parts of intestinal mucin from patients with cystic fibrosis.

Previous studies have shown that human small-intestinal mucin consists of high-Mr glycoproteins and a smaller S-S-bonded protein of 118 kDa. The major antigenic determinants of the mucin were associated with the large glycoproteins, but depended for stability on intact disulphide bonds, and were destroyed by digestion with Pronase. In the present study we isolated and analysed the component parts of mucin from patients with cystic fibrosis with special attention being paid to the peptide constituents. After reduction with 0.2 M-beta-mercaptoethanol [5 min, 100 degrees C in 1% SDS (sodium dodecyl sulphate)], the large glycoproteins and smaller peptide with an apparent molecular size of 118 kDa were separated by equilibrium density-gradient centrifugation in CsCl, Sepharose 4B chromatography or preparative SDS/polyacrylamide-gel electrophoresis. The large glycoproteins contained about 70% of the protein of the native mucin. Digestion with Pronase resulted in a further loss of 'naked' protein (10% of the native mucin protein) from the C-terminal end of the glycoprotein peptide core, and left behind highly glycosylated proteins comprised mainly (70 mol%) of threonine, serine and proline. The 118 kDa component, which contained about 30% of the native mucin protein, consisted mainly of aspartic acid, serine, glutamic acid and glycine (40 mol%), plus threonine, proline, alanine, valine and leucine (35 mol%). Together with the 'naked' protein segment, the 118 kDa component contained most of the cysteine residues of the native mucin. Surprisingly, the peptide also contained carbohydrate (less than or equal to 5% of the native mucin carbohydrate but 50% by weight of the 118 kDa component), which included 9 mol% mannose, suggesting the presence of N-linked oligosaccharides. The peptide exhibited strong non-covalent interactions with the high-Mr glycoproteins and a tendency to self-aggregate in the absence of dissociating agents. Our findings therefore suggest that native mucin consists of large glycoproteins capable of forming disulphide bridges from their C-terminal 'naked' (antigenic) regions to a smaller glycopeptide having an Mr of 118 000.     

Heterogeneity of rat goblet-cell mucin before and after reduction.

Goblet-cell mucin of rat small intestine was purified from mucosal scrapings by using centrifugation, Sepharose 4B and Sepharose 2B chromatography. The mucin was applied in low concentrations (1 microgram/track) to slab gels containing 0.5% agarose/2% (w/v) polyacrylamide, and bands were detected after electrophoresis by silver stain or by fluorography of 3H-labelled mucin. Before reduction the mucin contained three distinct components: a polymeric species at the top of the gel and two large glycoproteins of higher mobility. After reduction, the polymer disappeared, the two glycoproteins remained unchanged, and two glycopeptide bands of higher mobility appeared. In addition, a non-glycosylated, heavily stained peptide of mol.wt. 118000 was detected. The individual mucin components were partially separated on Sepharose 2B, 0.2M-NaCl/1% sodium dodecyl sulphate being used as eluant. Individual amino acid and carbohydrate analyses suggested that the glycosylated components, despite their differences in size, had identical profiles. The 118000-mol.wt. peptide had a very different amino acid profile, with much less serine, threonine and proline. Glycine and aspartic and glutamic acids comprised 34% of the total amino acids. Thus the 'native' mucin is a heterogeneous structure containing at least two non-covalently associated glycoproteins plus polymeric material. The latter is stabilized by disulphide bonds and consists of several glycopeptides of different size as well as a 'link' peptide of mol.wt. 118000.     

The putative ''link'' glycopeptide associated with mucus glycoproteins. Composition and properties of preparations from the gastrointestinal tracts of several mammals.

The existence of a discrete 'link' peptide in epithelial mucins has been debated for many years. There is evidence that at least some mucins contain a specific 'link' peptide (or glycopeptide) that enhances mucin polymerization by forming disulphide bridges to large mucin glycoprotein subunits. A major difficulty has been to know whether the reported differences in putative 'link' components represent artifacts generated by inter-laboratory differences in technical procedures used in mucin purification. The present paper outlines the results of a collaborative study involving five laboratories and 53 samples of purified gastrointestinal mucins (including salivary, gastric, small-intestinal and colonic mucins) prepared by five techniques from four different animal species. An early step in mucin purification in all cases was the addition of proteinase inhibitors. Representative mucins were analysed for their composition, electrophoretic mobility in SDS/polyacrylamide-gel electrophoresis before and after disulphide-bond reduction, and for their reactivity with monospecific antibodies developed against the 118 kDa putative 'link' glycopeptide isolated from either rat or human small-intestinal mucins. Our results indicate that, despite differences in laboratory techniques, preparative procedures, organs and species, each of the purified mucins contained a 'link' component that was released by disulphide-bond reduction and produced a band on SDS/polyacrylamide-gel electrophoresis at a position of approx. 118 kDa. After electroelution and analyses, the 118 kDa bands from the different mucins were found to have similar amino acid profiles and to contain carbohydrate. It would appear therefore that a 'link' glycopeptide of molecular mass approx. 118 kDa is common to all of the gastrointestinal mucins studied.     

Thyroid cell surface glycoproteins. Nature and disposition of carbohydrate units and evaluation of their blood group I activity

The distribution along the polypeptide of the carbohydrate units of two major calf thyroid cell surface glycoproteins, GP-1 and GP-3, was obtained from a study of their glycopeptides obtained after Pronase digestion. The GP-3 molecule (Mr = 20,000) yielded two large glycopeptides (Mr = 9,500 and 7,000) in equimolar amounts which each consisted of one N-linked (Mr = 5,400) and several small O-linked oligosaccharides accounting for a total of nine carbohydrate attachment sites in a 27-amino acid residue segment of the peptide chain. The Pronase treatment of GP-1 (Mr = 100,000) revealed the presence of a large protease-resistant fragment (Mr = 50,000) which contained 34 carbohydrate units (eight N-linked and 26 O-linked) in a segment of 105 amino acids. In addition to these densely glycosylated peptides (one glycosylation site/3 amino acid residues), small glycopeptides with polymannose saccharide units were found in the digests of both proteins. The occurrence of repeating N-acetyllactosamine sequences in the N-linked carbohydrate units of GP-1 and GP-3 was suggested by the composition and size of the oligosaccharides released by hydrazinolysis and was demonstrated by endo-beta-galactosidase treatment. The cleavage products from digestion with this enzyme were identified as NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----3Gal, Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----3Gal, Gal beta 1----4GlcNAc beta 1----3Gal, and GlcNAc beta 1----3Gal with the tetrasaccharides constituting the predominant species. The terminal alpha-D-Gal residues accounted for the binding of GP-1 and GP-3 glycopeptides to Bandeiraea simplicifolia I-agarose; concanavalin A-Sepharose affinity chromatography indicated that most of the N-linked carbohydrate units of both glycoproteins contained more than two branches. Difference in the branching on the poly-N-acetyllactosamine sequences of GP-1 and GP-3 was suggested by the finding that only the latter glycoprotein, as well as its glycopeptides, reacted with anti-blood group I antibodies; neither glycoprotein demonstrated blood group i antigenicity. Examination of cultured thyroid follicular cells revealed that both I and i determinants were present at the cell surface.     

Characterization and localization of the putative ''link'' component in rat small-intestinal mucin.

Rat intestinal mucin is polymerized by a putative 'link' component of Mr 118,000 that can be released from the native mucin by thiol reduction [Fahim, Forstner & Forstner (1983) Biochem. J. 209, 117-124]. To confirm that this component is an integral part of the mucin and independent of the mucin purification technique, rat mucin was purified in the present study by three independent techniques. In all cases, the 118,000-Mr component was released after reduction. The 118 kDa band was electroeluted from SDS/polyacrylamide gels and its composition shown to resemble closely that of the link component of human intestinal mucin [Mantle, Forstner & Forstner (1984) Biochem. J. 224, 345-354]. Carbohydrates were present, including significant (10 mol/100 mol) amounts of mannose, suggesting the presence of N-linked oligosaccharides. Monospecific antibodies prepared against the rat 118,000-Mr component established its tissue localization in intestinal goblet cells. Mucins subjected to SDS/polyacrylamide-gel electrophoresis and Western blots using the same antibody, established that the link components of rat and human intestinal mucin are similar antigenically. Brief exposure (10 min) of native rat mucin to trypsin or Pronase (enzyme/mucin protein, 1:500, w/w) also released a 118,000-Mr component that reacted with the monospecific antibody. Thus the 118,000-Mr component is an integral part of the mucin and, although linked to large glycopeptides by disulphide bonds, this component also has proteinase-sensitive peptide bonds, presumably at terminal locations such that brief treatment with proteinases releases the molecule in a reasonably intact form. Under physiological conditions, therefore, one might expect that, after mucin is secreted into the intestinal lumen, luminal proteinases would rapidly remove the link component, thereby causing the mucin to depolymerize.     

Products of trypsin digestion of haptoglobin beta (heavy) chain

Trypsin digestion of haptoglobin beta (heavy) chain resulted in five glycopeptides. The glycopeptides were characterized by carbohydrate and sulphydryl groups content; their molecular mass was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. None glycopeptide possessed hemoglobin-binding capacity. glycopeptide I did not form any precipitate with antihaptoglobin serum but was shown to inhibit strongly the reaction of haptoglobin or beta chain with the antiserum. Glycopeptide II showed dominant antigenic determinants in relation to native haptoglobin and to beta chain. Reaction of this glycopeptide with concanavalin A was almost twice higher than the corresponding reaction of haptoglobin. Glycopeptides IV and V were inactive in the reaction with the lectin. Glycopeptide III exhibited relatively the strongest cross-reactivity with the specific antihaptoglobin serum while its inhibitory activity in the immunoreaction was the lowest.     

The effects of limited proteolysis by trypsin on human haptoglobin

Trypsin digestion of haptoglobin resulted i four glycopeptides. The glycopeptides were characterized by amino acid composition and molecular weight, as determined by thin-layer chromatography, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. Hemoglobin-binding capacity and immunological properties were investigated. glycopeptides I and II did not form an active complex with hemoglobin and they inhibited the reaction of haptoglobin with specific antiserum by over 70%. Glycopeptides III and IV showed 11 and 4% of the hemoglobin-binding capacity and 82 and 67% of antigenic reactivity of native haptoglobin, respectively. Glycopeptide IV contained three antigenic determinants, whereas glycopeptides III contained four, one of them being exposed by trypsin digestion. In crossed two-dimensional immunoelectrophoresis, glycopeptide III showed at least four components reacting with antihaptoglobin serum, and glycopeptide IV, two components.     

The isolation and characterization of the glycopeptides from horseradish peroxidase isoenzyme C.

The purity of horseradish peroxidase isoenzyme C was demonstrated using isoelectric focusing, polyacrylamide gel electrophoresis at two pH values and cellulose acetate electrophoresis at two pH values. The glycopeptides obtained upon trypsin digestion were isolated using the plant lectin, concanavalin A, and were resolved using paper electrophoresis. The carbohydrate content of the native peroxidase was 86% accounted for by the carbohydrate content of the glycopeptides thus suggesting little loss of carbohydrate during glycopeptide isolation and purification. In each of the seven glycopeptides isolated glucosamine was associated with asparagine, thus suggesting the carbohydrate chains are covalently bound to the peptide chain through N-glycosidic linkages. The purity of each glycopeptide was demonstrated by the sequential release of single amino acid residues by Edman degradation. As six glycopeptides had unique amino acid sequences, it was concluded that the carbohydrate prosthetic group was distributed in at least six units along the protein backbone. Five glycopeptides possessed the amino acid sequence about the point of carbohydrate attachment of Asn-X-(Ser, Thr) where X is any amino acid. The size of the carbohydrate units ranged from 1600 to 3000 daltons. The predominant carbohydrate residues in each glycopeptide were mannose and glucosamine with lesser and varying amounts of fucose, xylose, and arabinose. There was no apparent correlation of the carbohydrate composition with the amino acid sequence.     

Structural and compositional differences between intracellular and secreted mucin of rat small intestine.

An investigation was undertaken to discover whether mucin purified from secretions in the lumen of rat small intestine differed in structure or composition from intracellular mucin purified from rat intestinal tissue. To do this, ligated loops were constructed in situ from previously washed intestinal segments and mucin purified separately from tissue homogenates or loop fluid. Secreted mucin (SM) differed from intracellular mucin (IM) by having a higher proportion of 'minor' mucin amino acids (aspartic acid, glutamic acid, glycine and alanine) and a lower proportion of 'major' amino acids (serine, proline and threonine). SM also contained less N-acetylgalactosamine and a small, but measureable, amount of mannose. Gel electrophoresis showed that SM penetrated the gel more readily and, unlike IM, gave a rather prominent, but diffuse, band having a midpoint position of Mr 200,000. After reduction both IM and SM gave rise to the putative 'link' component of Mr 118,000 and the 200,000-Mr band of SM disappeared. SM was included to a greater extent than IM on Sepharose CL-2B chromatography, suggesting a smaller size. With the use of CsCl-density-gradient ultracentrifugation of SM, a lighter species [buoyant density (rho) = 1.38 g/ml] enriched in the 200,000-Mr component, was separated from a heavier, more glycosylated, species (rho = 1.50 g/ml). Purified 200,000-Mr component had a composition identical with that of the 118,000-Mr 'link' component of IM, reacted in Western blots with an antibody specific for the 118,000-Mr 'link' component, and after reduction gave rise to a 118,000-Mr component on gel electrophoresis. Thus secreted mucin contains a 200,000-Mr component which appears to represent a disulphide-linked dimer of the previously described 118,000-Mr 'link' component of intracellular mucin.     

Short synthetic glycopeptides successfully induce antibody responses to carcinoma-associated Tn antigen.

Glycopeptides containing a tumor-associated carbohydrate antigen (mono-, tri- or hexa-Tn antigen) as a B-cell epitope and a CD4+ T-cell epitope (PV: poliovirus or TT: tetanus toxin) were prepared for immunological studies. Several Tn antigen residues [FmocSer/Thr (alpha-GalNAc)-OH] were successively incorporated into the peptide sequence with unprotected carbohydrate groups. The tri- and hexa-Tn glycopeptides were recognized by MLS128, a Tn-specific monoclonal antibody. The position of the tri-Tn motif in the peptide sequence and the peptide backbone itself do not alter its antigenicity. As demonstrated by both ELISA and FACS analysis, the glycopeptides induced high titers of anti-Tn antibodies in mice, in the absence of a carrier molecule. In addition, the generated antibodies recognized the native Tn antigen on cancer cells. The antibody response obtained with a D-(Tn3)-PV glycopeptide containing three alpha-GalNAc-D-serine residues is similar that obtained with the Tn6-PV glycopeptide. These results demonstrate that short synthetic glycopeptides are able to induce anticancer antibody responses.     

Structural and immunochemical analysis of the products of proteolysis of simian adenovirus hexons

Hexon capsomers of simian adenovirus sim16 (SA7) and of human adenoviruses h5 (Ad5) and h6 (Ad6) were proteolytically digested and the resulting products studied by SDS-polyacrylamide gel electrophoresis and by radioimmunoprecipitation analysis. The trypsinolysis of native SA7 hexon leads to a stable molecular "core" containing 4-5 fragment species of 10 to 65 kDa and resembling the intact capsomer in quarternary structure (trimer). Similar cores but consisting of smaller fragments (less than 40 kDa) were obtained after chymotryptic digestion of native SA7, Ad5 and Ad6 hexons. The chymotryptic hexon fragments were also held together in pseudotrimeric structures. The similarity of proteolytic hexon fragment patterns between different primate adenoviral hexons suggested a homology to exist in localisation of the exposed tryptic and chymotryptic cleavage sites in their respective hexon polypeptide chains. Papain caused a complete hydrolysis of native SA7 hexon (trimer) yielding small peptides, but at first stage of digestion a stable papain hexon core containing small fragments (less than 10 kDa) was observed. The tryptic SA7 hexon cores in native state retained their antigenicity in reactions with homo- and heterologous antibodies, but after core denaturation the resulting fragments had no antigenic activity of native capsomer. In contrast to the data previously published, chymotryptic cores of SA7, Ad5 and Ad6 hexons not only reacted with respective homologous antibodies but also retained (at least in part) cross-reactive antigenic determinants. The questions of formation and stability of native adenoviral hexon conformation are discussed as well as the possible nature of hexon antigenic determinants.     

Monoclonal antibodies as structural probes of surface residues in the regulatory subunit of cAMP-dependent protein kinase II from porcine heart

Two murine monoclonal antibodies (H5 and B6) generated against bovine heart type II regulatory subunit of cAMP-dependent protein kinase were shown to cross-react equally well with the homologous subunit from porcine heart. The antibodies demonstrated specificity for only the type II regulatory subunit and showed negligible cross-reactivity with the type I regulatory subunit, the catalytic subunit, and cGMP-dependent protein kinase. Following limited proteolysis of type II regulatory subunit with chymotrypsin, the H5 monoclonal antibody was shown to cross-react with the Mr = 37,000 cAMP-binding domain corresponding to the COOH-terminal region of the polypeptide chain. To more specifically localize the antigenic sites, the porcine type II regulatory subunit was carboxymethylated and cleaved with cyanogen bromide. Both monoclonal antibodies cross-reacted with the NH2-terminal CNBr peptide, and this peptide demonstrated affinities similar to native bovine type II regulatory subunit in competitive displacement radioimmunoassays. Tryptic cleavage of this CNBr fragment destroyed all antigenicity for both monoclonal antibodies, whereas antigenicity was retained following chymotryptic digestion. A single major immunoreactive chymotryptic fragment that cross-reacted with H5 was isolated by gel filtration and reverse phase high performance liquid chromatography. this peptide retained the complete antigenic site and had the following sequence: Asn-Pro-Asp-Glu-Glu-Glu-Glu-Asp-Thr-Asp-Pro-Arg-Val-Ile-His-Pro-Lys-Thr-Asp-Gl n. This antigenic site was localized just beyond the major site of autophosphorylation, approximately a third of the distance from the NH2-terminal end of the polypeptide chain.     

Isolation and preliminary characterization of a purified pig zona antigen (PPZA) from porcine oocytes

A protocol was developed for isolation of a purified pig zona antigen (PPZA) under nondissociating conditions. Heating of isolated zona-encased porcine oocytes at 73 degrees C for 20 min resulted in optimal solubilization of zona antigen activity (ZAA) as assessed by radioimmunoassay. Subsequent fractionation of solubilized proteins by ammonium sulfate precipitation, ultrafiltration, gel filtration, and ion exchange chromatography yielded PPZA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of PPZA yielded a major diffuse band at 58,000 Mr which stained for protein and carbohydrate and which possessed ZAA. Two-dimensional gel electrophoresis confirmed the identity of the 58,000 Mr glycoprotein and one of the three major charge heterogeneous glycoprotein families of the porcine zona pellucida. These experiments established that the principal macromolecular and antigenic component of PPZA is a 58,000 Mr glycoprotein originating from the zona pellucida. The nature of PPZA antigenic determinants recognized by a rabbit antiserum to PPZA was studied by radioimmunoassay. ZAA of PPZA was sensitive to the action of mercaptans and proteases, indicating a contribution of polypeptide chain to PPZA antigenicity. Loss of ZAA upon periodate oxidation of PPZA also implicated carbohydrate residues. PPZA retains antigenic determinants of the intact zona pellucida as assessed by reactivity with antiserum to intact porcine zonae. Likewise, rabbit antiserum to PPZA reacts avidly with intact porcine zonae. These results demonstrate that PPZA is a suitable target antigen for further studies on immunocontraception.     

The phenoloxidases of the ascomycete Podospora anserina. Structural differences between laccases of high and low molecular weight.

In order to investigate the extent of the relationship between the three copper-containing glycoproteins, laccases I, II and III (Mr70000, 80000 and 390000 respectively) of Podospora anserina, the following experiments were carried out on laccases II and III: (a) determination of amino acid composition; (b) determination of N-terminal and C-terminal amino acid; (c) determination of sugar composition; (d) dissociation studies on native and denatured laccases and also after removal of copper from the enzymes; (e) digestion of the carbohydrate moieties with the aid of glycosylhydrolases. A comparison between the results of these experiments and data previously obtained with laccase I allows the following conclusions to be drawn. 1. Laccases II and III are not identical. 2. Neither of these low molecular weight laccases are as complete molecules subunits of the oligomeric laccase I. 3. The possibility of partial identity of amino acid sequences of laccases I and III can not be excluded. 4. Laccase II possibly consists of subunits of Mr37000 whereas laccase III does not. 5. Digestion of 50% of the carbohydrate content leads to complete loss of serological specificity (serological reaction and cross reaction). This finding is discussed with regard to the possible role of the carbohydrate moiety as antigenic determinants and thus as the reason for the immunological relationship. As a consequence, at least three independent structural genes for laccases must be assumed.     

Tracheal carbohydrate antigens identified by monoclonal antibodies

In a previous study we described a family of monoclonal antibodies directed against tracheal antigens having a variety of cellular and subcellular distributions. In the present study, we have extended our findings on four representative antibodies to determine the periodate sensitivity, glycosidase sensitivity, and apparent molecular weight of the corresponding antigens. Since mild periodate oxidation selectively cleaves carbohydrate moiety leaving amino acids intact, loss of antigenicity following this treatment suggests the involvement of sugar residues in the antigenic determinant. This can be confirmed by testing the sensitivity of the antigens to specific glycosidases. By enzyme-linked immunosorbent assay (ELISA), all four antibodies were found to have highest affinity for void volume components isolated by Bio-Gel A15m chromatography of the total tracheal secretion. Further analysis of this void volume material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions followed by immunoblot analysis revealed that all antigens were carried by high-molecular-weight species (greater than 200,000) which were periodate-Schiff positive but reacted poorly with Coomassie blue. In parallel experiments using immunofluorescence and ELISA, antibody binding was compared under control conditions and following periodate treatment of antigens under varying intensities (10 mM IO4-, 10 min, 4 degrees C; 50 mM IO4-, 1 h, 4 degrees C; 100 mM IO4-, 12 h, 20 degrees C). Similar results were obtained with the two methods, indicating a partial loss of antigenicity for one of the four antigens following the mildest periodate treatment, and total loss of antigenicity for all four antigens following each of the two prolonged treatments. All four antigens showed marked sensitivity to digestion with mixed exoglycosidases and three antigens were also susceptible to endo-beta-galactosidase digestion. Antigenicity was not decreased during incubation with chondroitinase ABC, heparitinase, or heparinase. Immunofluorescence analysis of tracheal tissue sections showed that the four antibodies recognized determinants in different locations, including gland and goblet cell cytoplasmic granules and the apical epithelial membrane. The characteristic immunofluorescence patterns of all antibodies were abolished by periodate incubation of the tracheal sections. Thus, the four antibodies appear to recognize carbohydrate antigens carried by high-molecular-weight glycoproteins, each with different cellular origins.     

Alkaline phosphatase and 5'-nucleotide phosphodiesterase from bovine intestine are cross-reactive

Polyclonal antibodies to native alkaline phosphatase and to native 5'-nucleotide phosphodiesterase were found to strongly cross-react with both enzymes. The antibodies also cross-react with both denatured enzymes, with glycopeptides from 5'-nucleotide phosphodiesterase, and with the oligosaccharides remaining after Pronase E digestion of the phosphodiesterase. They do not cross-react with either enzyme after their oligosaccharides have been modified or removed by periodate or trifluoromethanesulfonic acid treatment. Antibodies to denatured 5'-nucleotide phosphodiesterase do not bind to the native phosphodiesterase or alkaline phosphatase but do cross-react with denatured alkaline phosphatase even after removal or modification of the carbohydrate moieties. These results suggest that antibodies to denatured 5'-nucleotide phosphodiesterase may recognize amino acid sequence homology between alkaline phosphatase and 5'-nucleotide phosphodiesterase. However, antibodies to native enzymes apparently recognize cross-reactive determinants of the native enzymes which are carbohydrate in nature. This is the first report of antimammalian alkaline phosphatase antibodies which recognize the carbohydrate moieties of the enzyme.     

Analysis of Tn antigenicity with a panel of new IgM and IgG1 monoclonal antibodies raised against leukemic cells

CD175 or Tn antigen is a carbohydrate moiety of N-acetylgalactosamine (GalNAc)α1-O- linked to the residue of amino acid serine or threonine in a polypeptide chain. Despite the chemical simplicity of the Tn antigen, its antigenic structure is considered to be complex and the clear determinants of Tn antigenicity remain poorly understood. As a consequence, a broad variety of anti-Tn monoclonal antibodies (mAbs) have been generated. To further investigate the nature and complexity of the Tn antigen, we generated seven different anti-Tn mAbs of IgM and IgG classes raised against human Jurkat T cells, which are Tn-positive due to the low activity of T-synthase and mutation in specific chaperone Cosmc. The binding analysis of anti-Tn mAbs with the array of synthetic saccharides, glycopeptides and O-glycoproteins revealed unexpected differences in specificities of anti-Tn mAbs. IgM mAbs bound the terminal GalNAc residue of the Tn antigen irrespective of the peptide context or with low selectivity to the glycoproteins. In contrast, IgG mAbs recognized the Tn antigen in the context of a specific peptide motif. Particularly, JA3 mAb reacted to the GSPP or GSPAPP, and JA5 mAb recognized specifically the GSP motif (glycosylation sites are underlined). The major O-glycan carrier proteins CD43 and CD162 and isoforms of CD45 expressed on Jurkat cells were precipitated by anti-Tn mAbs with different affinities. In summary, our data suggest that Tn antigen-Ab binding capacity is determined by the peptide context of the Tn antigen, antigenic specificity of the Ab and class of the immunoglobulin. The newly generated anti-Tn IgG mAbs with the strong specificity to glycoprotein CD43 can be particularly interesting for the application in leukemia diagnostics and therapy.     

A collagenous glycoprotein found in dissociative extracts of foetal bovine nuchal ligament. Evidence for a relationship with type VI collagen.

A collagenous glycoprotein (Mr 140000) was isolated from dissociative extracts of foetal bovine nuchal ligament and purified by a combination of ion-exchange and gel-filtration chromatography. This glycoprotein (designated MFPI) exists as a large-Mr disulphide-bonded aggregate in the absence of a reducing agent. The purified glycoprotein was shown to contain about 6% (w/w) carbohydrate, mostly as galactose, glucose and mannose. Amino acid analysis showed the presence of hydroxyproline and hydroxylysine, indicative of its collagenous nature. The collagenous nature of this glycoprotein was further investigated by enzyme digestion. Pepsin digestion produced three major fragments, which were identical with peptides of type VI collagen. Bacterial-collagenase digestion of the unreduced glycoprotein also produced several discrete peptides. However, reduction of the glycoprotein before bacterial-collagenase digestion resulted in the degradation of these discrete peptides. Glycoprotein MFPI extracted in dissociative conditions appears to be a larger-Mr form of type VI collagen, believed to originate from microfibrillar components in the intact tissue.     

Structure and interactions of cartilage proteoglycan binding region and link protein.

Binding region and link protein were prepared from pig laryngeal cartilage proteoglycans after chondroitinase ABC and trypsin digestion. Experiments on gel chromatography showed the purified binding region to interact reversibly with hyaluronate (HA), and this binding was also shown to be stabilized by native link protein. The trypsin-prepared link protein showed properties of self-association in solution that were partially inhibited by oligosaccharides (HA10-16) and abolished by modification of free amino groups (lysine residues) with 2-methylmaleic anhydride. The Mr (sedimentation equilibrium) of the modified link protein was 41 700. Analysis of binding region showed it to contain 25% (w/w) carbohydrate, mainly in galactose, glucosamine, mannose and galactosamine. It contained some keratan sulphate, as digestion with endo-beta-D-galactosidase (keratanase) removed 28% galactose and 25% glucosamine and the Mr (sedimentation equilibrium) decreased from 66 500 to 60 800. After keratanase digestion the interaction with polyclonal antibodies specific for binding region was unaffected, but the response in a radioimmunoassay with a monoclonal antibody to keratan sulphate was decreased by 47%. Preparation of a complex between binding region, link protein and HA approximately 34 showed a single component (5.5S) of Mr (sedimentation equilibrium) 133 500. In this complex the antigenic determinants of link protein appeared masked, as previously found with proteoglycan aggregates. The isolated binding region and link protein were thus shown to retain properties comparable with those involved in the structure and organization of proteoglycan aggregates.