The association of an HPV16 oncogene variant with HLA-B7 has implications for vaccine design in cervical cancer.

HLA-restricted cytotoxic T-lymphocyte (CTL) recognition of human papillomavirus (HPV) oncogene products may be important in the control of the HPV infections associated with the development of cervical cancer. We have identified, in HLA-B7 individuals, a consistent variation in the HPV16 E6 oncoprotein sequence, which alters an HLA-B7 peptide binding epitope in a way likely to influence immune recognition by CTLs. These results illustrate a biologically relevant mechanism for escape from immune surveillance of HPV16 in HLA-B7 individuals. Thus, both HLA type and HPV16 strain variation need to be considered in the screening of at-risk individuals and for the rational design of anti-HPV vaccines.

Direct demonstration of increased expression of Thomsen-Friedenreich (TF) antigen in colonic adenocarcinoma and ulcerative colitis mucin and its concealment in normal mucin.

Increased binding of the lectin peanut agglutinin is a common feature in epithelial malignancy and hyperplasia. This may have considerable functional importance in the intestine by allowing interaction between the epithelium and mitogenic lectins of dietary or microbial origin. Peanut agglutinin binds the disaccharide Thomsen-Friedenreich (TF, T or core 1) blood group antigen, Gal beta (1-3) GalNAc alpha-, but is not totally specific for this site. Consequently, there has been controversy about the presence of this structure in colon cancer; studies with anti-TF monoclonal antibodies have failed to detect it. We have examined the presence of TF antigen in colonic mucus glycoprotein (mucin) using endo-alpha-N-acetylgalactosaminidase (O-Glycanase), which specifically catalyzes the hydrolysis of TF antigen from glycoconjugates. Samples of adenocarcinoma, inflammatory bowel disease (ulcerative colitis), and normal mucin were treated with O-glycanase, the liberated disaccharide was separated from the glycoprotein and analyzed using dual CarboPac PA-100 column high performance anion-exchange chromatography coupled with pulsed amperometric detection. O-Glycanase treatment released increased amounts of TF antigen from both colonic adenocarcinoma (8.0 +/- 3.9 ng/micrograms protein, n = 11; P < 0.0001 ANOVA) and ulcerative colitis mucin (3.3 +/- 0.3 ng/micrograms protein, n = 5; P = 0.04) compared with mucin samples from histologically normal mucosa distant from carcinoma (1.5 +/- 1.1 ng/micrograms protein, n = 9). However, after mild acid treatment to remove sialic acids and fucose, releasable TF antigen was increased in all nine of these histologically normal mucin samples (5.5 +/- 2.6 ng/micrograms protein, P < 0.0002). We conclude that TF antigen is an oncofetal antigen which is expressed in colon cancer, but is concealed by further glycosylation (sialylation and/or fucosylation) in the normal colonic mucosa.

Further studies of the specificities of monoclonal anti-i and anti-I antibodies using chemically synthesized, linear oligosaccharides of the poly-N-acetyllactosamine series.

The I- and i-antigen activities of chemically synthesized, linear oligosaccharides of the neolacto series containing one, two or three N-acetyllactosamine (Gal beta 1----4GlcNAc) units have been tested by inhibition of binding of five anti-i and eight anti-I monoclonal antibodies to radioiodinated I- and i-active glycoproteins. The inhibitory activities of the milk oligosaccharides lacto-N-neotetraose (Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc) and lacto-N-tetraose (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc) have also been determined. The results clearly show that: (a) the determinants that best fit the combining sites of anti-i antibodies are at least hexasaccharides of the neolacto series, (b) linear tetra- and hexasaccharides of the neolacto series can strongly inhibit the binding of anti-I antibodies of group 2 which are known to be primarily directed at the repeating Gal beta 1----4GlcNAc beta 1----3 domains of branched neolacto sequences, (c) the beta- but not the alpha-methyl anomer of the glycoside Gal beta 1----4GlcNAc beta 1-O-Me inhibits the binding of anti-I antibodies of group 1 which recognise the branch point sequence Gal beta 1----4GlcNAc beta 1----6-, (d) the reactivity of the beta-methylglycoside is impaired if the sequence is further elongated as in Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta-O-Me, and (e) lacto-N-tetraose has no inhibitory activity with any of the anti-i or anti-I antibodies tested.

A marker of human foetal endoderm defined by a monoclonal antibody involves Type 1 blood group chains.

This report demonstrates that a marker of human embryonic endoderm and embryonal carcinoma cells recognized by a hybridoma antibody FC 10.2, involves Type 1 blood group chains with the sequence Gal beta 1 leads to 3G1cNAc beta 1 leads to 3Gal beta 1 leads to 4G1c. This conclusion has been reached from antigenic analyses of meconium, ovarian cyst glycoproteins, oligosaccharides and glycolipids having Type 1 or Type 2 blood group chains. From knowledge of saccharide sequences and blood group related antigens in gastrointestinal tissues of man, we deduce that the 'disappearance' of FC 10.2 antigen from the normal, differentiated cells of the adult may result from masking by additional glycosylations or other substitutions.

Monoclonal antibody (EGR/G49) reactive with the epidermal growth factor receptor of A431 cells recognizes the blood group ALeb and ALey structures.

The carbohydrate specificity of the monoclonal antibody EGR/G49, raised against the epidermal growth factor (EGF) receptor of A431 cells, has been investigated by assessing its interactions with glycoproteins and erythrocytes derived from individuals of known blood group ABH, Lewis and secretor types, and by inhibition of binding assays using structurally defined oligosaccharides. The results indicate that this antibody reacts with the difucosylated blood group structures ALeb and ALey: (formula; see text) This antibody differs from the previously described anti-EGF receptor antibody. TL5, which is directed at the terminal blood group A trisaccharide structure and reacts poorly with the ALeb/Ley structures. Since both antibodies were selected for their reactivities with the receptor for EGF, their specificities provide evidence for the presence of both the mono- and difucosylated blood group A structures on the receptor glycoprotein. These antibodies will be invaluable in the studies of the distribution and the roles of blood group related carbohydrate structures in the organisation and function of the EGF and other receptor systems.

A proposed molecular model for the carboxy terminus of HIV-1 gp120 showing structural features consistent with the presence of a T-cell alloepitope.

A computer graphics molecular model of the C terminus of gp120 of HIV has been constructed using predicted secondary structure based on homologies with proteins for which X-ray crystallographic data have been published. The model shows sequences known to be important in CD4 binding in close proximity to regions with a high probability of forming alpha helical and beta strand motifs. The orientation adopted by these domains approximates to the known 3D structure of HLA-A2 alpha 2 chain without constraints based on HLA-A2 as a template being introduced. The model may therefore represent an energetically favourable conformation for a part of gp120 which mimics the binding domain for the T-cell receptor on MHC molecules. Recognition of gp120 as an alloepitope in high affinity association with CD4 would explain many of the sequelae of acquired immune deficiency on HIV infection.

Quantitation of the oligosaccharides of human serum IgG from patients with rheumatoid arthritis: a critical evaluation of different methods.

Several different chromatographic methods and a lectin-based assay have been compared for the quantitation of oligosaccharides released from immunoglobulin G (IgG). The analysis of a series of IgG samples purified from the serum of rheumatoid arthritis patients was carried out by these methods to evaluate the percentage of the glycoforms having 0, 1 or 2 galactose residues (G0, G1 and G2) in order to (a) identify the method that can be most widely used for quantitation, (b) accurately define the range of G0 values found in patients with rheumatoid arthritis, and (c) make available a series of characterised standards for distribution to clinical chemistry laboratories. The chromatographic methods involved: release of oligosaccharides by glycoamidase A after protease digestion followed by HPLC analysis of aminopyridine derivatives on reverse phase and normal phase columns; hydrazinolysis treatment with exoglycosidases (G0 mix) and Biogel P4 chromatography of 2-aminobenzamide (2-AB) derivatives; hydrazinolysis and weak anion exchange or normal phase HPLC of 2-AB derivatives; release of oligosaccharides by PNGase F and either Biogel P4 chromatography of 2-AB derivatives or HPAEC-PAD analysis of native oligosaccharides. The G0 values given by these methods compared favourably with each other and a dot blot assay of denatured IgG interaction with Ricinus communis agglutinin and Bandeiraea simplicifolialectin II. The HPLC and HPAEC methods give additional information that may be important in less routine assays.

HIV type 1 envelope glycoprotein 120 carboxy-terminal peptide-induced human T cell lines selectively suppress heterogeneous proliferative T cell responses to soluble antigens.

It has been proposed that the highly conserved human immunodeficiency virus type 1 (HIV-1) envelope gp120 carboxy-terminal sequence, TKAKRRVVEREKR (CT120), may represent a functional mimic of the human leukocyte antigen (HLA) class II DR beta-chain third hypervariable region (HVR3) sequence motif located at position 69-81. Presentation of this potentially pathogenic fragment by HLA class I and/or II molecules, in a manner analogous to the indirect pathway of allorecognition, may induce both widespread cellular activation and also break self-tolerance, resulting in the selective and progressive anti-self HLA class II-directed immune suppression, which is a central feature of HIV-1 infection and the associated acquired immune deficiency syndrome (AIDS). To investigate the functional role of the HIV-1 gp120 C-terminal fragment T cell lines (TCLs) were raised from three healthy HIV-1-seronegative subjects at low risk of HIV-1 exposure, by repeated stimulation with a short synthetic 13-mer CT120 peptide in vitro. Graded concentrations (10[3] to 5 x 10[4]) of CT120 TCLs suppressed the primary 6-day proliferation of autologous PBMCs in response to the soluble antigens tetanus toxoid (TT) and purified protein derivative (PPD). In contrast, CT120 TCLs demonstrated no suppressive effect on 3-day phytohemagglutinin (PHA), concanavalin A (ConA), and pokeweed mitogen (PWM) mitogenic responses. Fractionation of CT120 TCLs into highly purified CD4+ and CD8+ T cell subsets demonstrated that the CD8+ T cell fraction mediated the suppressor effector function. HLA restriction analysis revealed a complex pattern as both anti-HLA class II DR and anti-HLA class I (A, B, C) MAbs inhibited proliferation of oligoclonal CD8+ CT120 TCLs. Strategies aimed at specifically inhibiting such putative immunopathogenic HIV-1-encoded T cell epitopes may be an important consideration for development of future HIV-1 immunotherapy.

Glycosylation of immunoglobulin light chains associated with amyloidosis.

AL amyloidosis is a fatal disease caused by deposition of immunoglobulin light chains in a fibrillarforin (AL) in various organs. By searching the Kabat database of immunoglobulin sequences using the KabatMan software, we have shown that there is a preponderance of the consensus glycosylation sequon (AsnXxxSer/Thr) in the framework regions of amyloid light chains. We have characterised by computer graphics simulations, NMR spectroscopy and carbohydrate biochemistry the structure and conformation of the oligosaccharide from amyloid protein AL MS (lamba1) and from the amyloid associated Bence Jones protein of patient MH (kappa1). These proteins have glycosylation in the hypervariable complementarity-determining region versus framework region, respectively. Both contained a 2-6 sialylated core fucosylated biantennary chain mostly with bisecting GIcNAc. Together our results suggest that light chain glycosylation may be one of several modifications which may render the protein more prone to amyloid formation.

Characterization of the glycosylation status of proteins.

Proteins are posttranslationally modified by a number of mechanisms, by far the most significant of which in terms of the molecular mass and functional diversity is glycosylation. The oligosaccharide chains added shortly after biosynthesis at the ribosome, and further modified while the protein is translocated through the intracellular membranes, have multiple affects on transport, localization, stability, plasma membrane expression activity, and antigenicity. Characterization of the glycosylation patterns at each site around the protein is essential for detailed understanding of structure/function relationships and the design of potential therapeutic agents. This can be achieved by a series of chromatographic and physicochemical procedures including HPLC, GC, MS, NMR, and computer graphics molecular modeling, which culminate in information on monosaccharide sequence and linkage, glycoprotein conformation, and oligosaccharide-to-protein interactions.

Microassay analyses of protein glycosylation.

To accurately characterize the carbohydrate moieties of oligosaccharide chains in glycosylated proteins, it is necessary to distinguish exactly which types of oligosaccharides are present at which site. We describe lectin overlay assays, which take advantage of the ability of lectins to distinguish between different types of glycoproteins via recognition of terminal sugars, thus allowing the chain type and peripheral antigenic components to be determined. Three microassays involving lectins are reported in this paper: non-protease-treated intact glycoproteins; glycopeptides released by prior digestion of the glycoprotein and then separated by HPLC; and release of sugars from glycoproteins by hydrazinolysis and then coupling them to a multivalent support.

Glycosylation of prions and its effects on protein conformation relevant to amino acid mutations.

The three-dimensional coordinates from a nuclear magnetic resonance (NMR)-averaged structure containing residues 121-226 of mouse prion were used as the starting geometry for MD of prion either with or without glycan in both mutant and wild-type forms. The following mutants were studied: Asp-178 to Asn, Thr-183 to Ala, Phe-198 to Ser, Glu-200 to Lys, and Gln-217 to Arg. NMR data vs structural models were compared to observe any major differences. Simulations of the change in protein structure with and without glycan were performed, as they cannot be tested by NMR analysis. Several mutants were expressed and analyzed for altered glycosylation and the results interpreted in terms of molecular modeling. N-linked glycosylation is likely to play an important role in prion biology as shown by visualization of glycoprotein conformation.

Comparison of separation modes of high-performance liquid chromatography for the analysis of glycoprotein- and proteoglycan-derived oligosaccharides.

Porous graphitised carbon (PGC) has been explored for high-performance liquid chromatography (HPLC) of mono- and di-saccharides released from proteoglycans and of fluorescently labelled oligosaccharide derivatives for high-sensitivity detection. Sulphated oligosaccharides show good retention and separation behaviour on PGC-HPLC, and compared to anion-exchange or reversed-phase ion-pair chromatography the chromatography is carried out in the absence of salt. Due to their poor retention on PGC-HPLC the analysis of single uronic acids has been optimised with high pH anion-exchange chromatography. Fluorescent labelled derivatives formed by reductive amination of neutral oligosaccharides with 2-aminobenzamide have been chromatographed on PGC-HPLC and by BioGel P4 gel filtration.

The carbohydrate specificities of the monoclonal antibodies 29.1, 455 and 3C1B12 to the epidermal growth factor receptor of A431 cells.

Sixteen hybridoma-derived antibodies to the epidermal growth factor receptor of A431 cells were studied with respect to their reactions with blood group-related carbohydrate structures. Twelve of these were assessed as recognizing carbohydrate determinants on the basis of their immunostaining of reference blood group substances on nitrocellulose paper. Three of these antibodies were further investigated by inhibition of binding assays with glycoproteins and structurally defined oligosaccharides or by haemagglutination of erythrocytes before and after treatment with endo-beta-galactosidase. Two of the antibodies, 29.1 and 455, were shown to have blood group A-related specificities which differed from one another and from those of monoclonal anti-A antibodies described previously. The third antibody, 3C1B12, which was shown to recognize a determinant based on alpha 1----3 fucosylated Type 2 chains on linear and branched backbone sequences, also differs from previously described monoclonal antibodies of 3-fucosyl-N-acetyllactosamine type, such as anti-SSEA-1 (anti-mouse embryo) and several antibodies to human myeloid cells. While these antibodies are invaluable in providing structural information on the carbohydrate chains of the receptor glycoprotein and should help to elucidate their functions, their use as 'anti-receptor' reagents in cell biology will be influenced by the knowledge that the determinants they recognize are shared by other glycoproteins and glycolipids of diverse cell types.

Novel glycolipids of Mycobacterium avium and related M. paratuberculosis strains of relevance to AIDS and Crohn's disease.

The polar glycolipid fractions of several mycobacterial strains of the closely related species M. avium and M. paratuberculosis have been analysed by thin layer chromatography (TLC), high pH anion exchange chromatography (HPAEC), gas-liquid chromatography (GC) and nuclear magnetic resonance (NMR) spectroscopy. The upper phase of a Folch partitioning (rather than the lower phase analysed by others) was subjected to TLC in solvent system chloroform-methanol-water 50:40:10 v/v/v. A major band was purified from each mycobacterial strain. Monosaccharide analysis of that from M. avium A14 (from an AIDS patient) contained Glc, Ara, Man, Gal in ratios 7:4:3:2. whereas one strain of M. paratuberculosis (316F) had low levels of Ara, Gal and Man with major monosaccharides being Glc and two unidentified monosaccharides. A second M. paratuberculosis strain (J10) had a single TLC band containing only Glc. These known strains were compared to two slow growing mycobacterial isolates, one from a Crohn's patient and one isolated from armadillo. These were similar to J10 in only having Glc present: the former also had a similar NMR spectrum to J10, whereas the latter had a different NMR spectrum from any of the other strains analysed. The results therefore indicate that M. paratuberculosis strain 316F is more closely related to M. avium (from an AIDS patient) than it is to the classical M. paratuberculosis strain J10 and a Crohn's isolate.

Computer-assisted interpretation of 1H-n.m.r. spectra in the analysis of the structure of oligosaccharides.

A computer program that can be implemented on IBM compatible microcomputers has been used to interpret the 1H-n.m.r. spectra of a series of mucin-derived alditols. The program compares the chemical shifts of resonances in the spectra of unknown oligosaccharides with those in a standard library constructed from data in the literature. The program then compiles the identified sequences into possible structures, which, together with a second component of the program from which the individual assignments of chemical shifts can be made, facilitates ready access to the literature data for final confirmation of the structures.

Complete 1H-n.m.r. assignments for two core-region oligosaccharides of human meconium glycoproteins, using 1D and 2D methods at 500 MHz.

The complete 1H-n.m.r. assignments for alpha-D-GalNAc-(1----3)-D-GalNAc-ol and beta-D-Gal-(1----4)-beta-D-GlcNAc-(1----6)-D-GalNAc-ol have been made using a combination of 2D correlation experiments (COSY, RELAYED-COSY, and F1-decoupled) and an analysis of the high-resolution 1D-n.m.r. spectra at 500 MHz.

Characterisation by LSI-MS and 1H NMR spectroscopy of tetra-, hexa-, and octa-saccharides of porcine intestinal heparin.

The characterisation of oligosaccharide fragments isolated from enzymatically depolymerised porcine intestinal heparin is required in order to probe structure/function relationships of heparin in anticoagulation, antiangiogenesis and antiviral activity. We have used both LSI-MS and 600-MHz 1H NMR with chemical shift assignment by comprehensive 1H-1H TOCSY experiments to fully characterise the major oligosaccharide components including 4 tetrasaccharides, 3 hexasaccharides, and 2 octasaccharides. One of the octasaccharides has not been identified previously and has the structure: delta UA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS(6S)-IdoA(2S)- GlcNS(6S)-GlcA-GlcNS(6S), where delta UA is 4,5-unsaturated uronic acid (4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid), GlcN is --> 4)-alpha-D-glucosamine, IdoA is --> 4)-alpha-L-iduronic acid, GlcA is --> 4)-beta-D-glucuronic acid, and 2-O-, 6-O-, and 2-N-sulfate are abbreviated to 2S, 6S, and NS, respectively. Nearly complete NMR proton chemical shifts are reported for this data set. In addition a novel approach involving oxymercuration-lipid conjugation was used to independently assign sulfate substitution on the delta UA residues.

Further definition of the size of the blood group-i antigenic determinant using a chemically synthesised octasaccharide of poly-N-acetyllactosamine type.

In earlier studies, the minimum structure which inhibited the binding of anti-i to an i-active glycoprotein was the linear trisaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-D-Gal. There was an increasing hierarchy of inhibitory activities in the linear tetrasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D -GlcNAc , its methyl beta-glycoside, and in the methyl beta-glycoside of the hexasaccharide. The linear octasaccharide methyl beta-glycoside in this series is approximately only half as active as the hexasaccharide methyl beta-glycoside. Analyses by high resolution 1H-n.m.r. of these two oligosaccharides indicated that they have similar conformations in solution, and there is no evidence for the occurrence of inter-molecular interactions which might partially hinder the binding of anti-i to the octasaccharide methyl beta-glycoside. These results are consistent with the size of the i antigen being in the region of a hexasaccharide. It is proposed that the methyl aglycon group of the hexasaccharide methyl beta-glycoside confers an above normal activity by presenting a hydrophobic area for additional contact in the vicinity of the antibody-combining site.

Analysis of pigeon intestinal mucin allergens using a novel dot blot assay.

Many glycoproteins contain multiple glycosylation sites that can present multi-valent epitopes for antigenic recognition. Release of the oligosaccharides results in loss of avidity of antibody binding, which has been overcome by reforming clustered ligands, usually by reductive amination of free reducing oligosaccharides to poly-amine groups. We have adapted this approach to hydrazinolytic release of O-linked chains of mucin glycoproteins and 'one-pot' microscale coupling to poly-L-lysine (PLL). The conjugated PLL adheres to nitrocellulose membranes through washing procedures required for antibody or lectin overlay and detection. We show evidence for the applicability of this technique using lectin and antibody reactivity to the oligosaccharides of pigeon intestinal mucins, which have been implicated in the allergic disease pigeon fanciers' lung.