Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17 kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.     

Tn antigen promotes human colorectal cancer metastasis via H‐Ras mediated epithelial‐mesenchymal transition activation

Tn antigen is a truncated O‐glycan, frequently detected in colorectal cancer (CRC), but its precise role in CRC metastasis is not well addressed. Here we investigated the effects of Core 1 β3Gal‐T specific molecular chaperone (Cosmc) deletion‐mediated Tn antigen exposure on CRC metastasis and its underlying mechanism. We first used CRISPR/Cas9 technology to knockout Cosmc, which is required for normal O‐glycosylation, and thereby obtained Tn‐positive CRC cells. We then investigated the biological consequences of Tn antigen expression in CRC. The results showed that Tn‐positive cells exhibited an enhanced metastatic capability both in vitro and in vivo. A further analysis indicated that Tn antigen expression induced typical activation of epithelial‐mesenchymal transition (EMT). Mechanistically, we found that H‐Ras, which is known to drive EMT, was markedly up‐regulated in Tn‐positive cells, whereas knockdown of H‐Ras suppressed Tn antigen induced activation of EMT. Furthermore, we confirmed that LS174T cells (Tn‐positive) transfected with wild‐type Cosmc, thus expressing no Tn antigen, had down‐regulation of H‐Ras expression and subsequent inhibition of EMT process. In addition, analysis of 438 samples in TCGA cohort demonstrated that Cosmc expression was reversely correlated with H‐Ras, underscoring the significance of Tn antigen‐H‐Ras signalling in CRC patients. These data demonstrated that Cosmc deletion‐mediated Tn antigen exposure promotes CRC metastasis, which is possibly mediated by H‐Ras‐induced EMT activation.     

High-resolution structure of a new Tn antigen-binding lectin from Vatairea macrocarpa and a comparative analysis of Tn-binding legume lectins

Plant lectins have been studied as histological markers and promising antineoplastic molecules for a long time, and structural characterization of different lectins bound to specific cancer epitopes has been carried out successfully. The crystal structures of Vatairea macrocarpa (VML) seed lectin in complex with GalNAc-α-O-Ser (Tn antigen) and GalNAc have been determined at the resolution of 1.4 Å and 1.7 Å, respectively. Molecular docking analysis of this new structure and other Tn-binding legume lectins to O-mucin fragments differently decorated with this antigen provides a comparative binding profile among these proteins, stressing that subtle alterations that may not influence monosaccharide binding can, nonetheless, directly impact the ability of these lectins to recognize naturally occurring antigens. In addition to the specific biological effects of VML, the structural and binding similarities between it and other lectins commonly used as histological markers (e.g., VVLB4 and SBA) strongly suggest VML as a candidate tool for cancer research.     

Inversions and deletions of the Salmonella chromosome generated by the translocatable tetracycline resistance element Tn10

Spontaneous tetraoyoline-sensitive derivatives of a Tn10 insertion in the hisG gene of Salmonella typhimurium were isolated and subjected to genetic analysis. All 123 of the drug-sensitive derivatives characterized have undergone stable alterations in the Tn10 element itself; over half of the derivatives have also undergone major alterations of neighboring regions of the Salmonella chromosome. These chromosomal rearrangements are of two types: inversions and deletions. Any single inversion or deletion affects a contiguous stretch of chromosomal material extending from the site of the original Tn10 element either leftward or rightward.The genetic properties of deletion and inversion derivatives suggest that these chromosomal alterations are promoted by the Tn10 element itself. The role of translocatable elements in promoting chromosomal deletions is well documented; the ability of an element to promote inversions of chromosomal material has not previously been reported. Possible analogies between the 1400-base-pair inverted repetition at the end of Tn10 and the small insertion sequence IS1 predict particular structures for Tn10-promoted deletions. A structural explanation or model for Tn10-promoted inversions is presented. The observation that Tn10 promotes the formation of inversions suggests that such elements could play a previously unanticipated role in promoting chromosomal inversions during evolution of prokaryotic organisms. Generally applicable genetic methods for the identification and characterization of chromosomal inversions are described.     

Tn glycosylation of the MUC6 protein modulates its immunogenicity and promotes the induction of Th17-biased T cell responses

The Tn antigen (α-GalNAc-O-Ser/Thr) is one of the most specific human cancer-associated structures. This antigen, together with mucins, the major carriers of O-glycosylated tumor antigens in adenocarcinomas, are being evaluated as anti-cancer immunotherapeutic targets. In particular, the MUC6 protein, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. To develop anti-cancer vaccines based on the Tn antigen, we produced MUC6 proteins with different Tn density by using mixtures of recombinant ppGalNAc-T1, -T2, and -T7. The obtained glycoproteins were characterized and analyzed for their immunological properties, as compared with the non-glycosylated MUC6. We show that these various MUC6:Tn glycoproteins were well recognized by both MUC6 and Tn-specific antibodies. However, Tn glycosylation of the MUC6 protein strongly affected their immunogenicity by partially abrogating Th1 cell responses, and promoting IL-17 responses. Moreover, the non-glycosylated MUC6 was more efficiently presented than MUC6:Tn glycoproteins to specific T CD4(+) hybridomas, suggesting that Tn glycosylation may affect MUC6 processing or MHC binding of the processed peptides. In conclusion, our results indicate that Tn glycosylation of the MUC6 protein strongly affects its B and T cell immunogenicity, and might favor immune escape of tumor cells.     

Riproximin is a recently discovered type II ribosome inactivating protein with potential for treating cancer

The development of new anticancer drugs is a salient problem and the traditional use of plants is a potentially rich source of information for detecting new molecules with antineoplastic activity. Riproximin is a recently detected cytotoxic type II ribosome inactivating protein with high selectivity for certain tumor cell lines. Its activity was recognized as the main component in a plant powder used by African healers for treating cancer. By ribulose bisphosphate carboxylase gene sequencing analysis, the powder was identified to be derived from the plant Ximenia americana. The cDNA sequence of riproximin was identified, the protein was modeled to contain one A- and a B-chain, respectively, and a reliable purification procedure from kernels of X. americana was established. Riproximin displays high but differential antiproliferative activity in a panel of human and rodent cancer cell lines, with concentrations inhibiting cell proliferation by 50% (IC₅₀ values) that diverge by a factor of 100. Consistent antineoplastic activity was detected in colorectal and pancreatic cancer liver metastasis models in rats. The cytotoxic mechanism of action was determined to be based on cellular uptake of riproximin followed by its A-chain prompted depurination of the 28S ribosomal RNA and induction of unfolded protein response. Riproximin's specificity depended on its B-chain connected binding to cell surface glycans, the presence of which is crucial for subsequent internalization into cells and cytotoxicity. These N- and O-glycans include bi- and tri-antennary NA structures (NA2/NA3) as well as Tn3 structures (clustered Tn antigen). Riproximin was found to crosslink proteins with N- and O-glycan structure, thus indicating both types of binding sites on its B chain. Due to this crosslinking ability, riproximin is expected to show prominent cytotoxicity towards cells expressing both, NA2/NA3 and clustered Tn structures. Apart from the properties of riproximin, the plant X. americana has been known for some medical uses in traditional African medicine, including various types of infections.     

Sialyl Lewis x expression in cervical scrapes of premalignant lesions

Sialylated oligosaccharides of glycoproteins and glycolipids have been implicated in tumour progression and metastases. Altered expression of glycosidic antigens has been reported in cervical cancer. In cervix premalignant lesions, an increased expression of sialic acid has been reported. In the present study we determined the expression profiles of the glycosidic antigens Tn, sialyl Tn (sTn), Lewis a (Le^a), sialyl Lewis a (sLe^a), Lewis x (Le^x) and sialyl Lewis x (sLe^x) in cervical scrapes with cytological diagnoses of normal, low-grade squamous intraepithelial lesions (LGSIL) and high-grade squamous intraepithelial lesions (HGSIL). Cervical scrapings were collected to detect tumour antigens expressions by flow cytometry using monoclonal antibodies. Cytometry analysis of Tn, sTn, Le^a and Le^x did not reveal differences at the expression level among groups. The number of positive cells to sLe^a antigen increased in the HGSIL group with respect to the normal group (p?=?0.0495). The number of positive cells to sLe^x antigen in the samples increased with respect to the grade of squamous intraepithelial lesion (SIL) (p?<?0.001, Mann–Whitney U test). The intensity of expression of this antigen increased in the HGSIL samples with respect to normal samples (p?<?0.0068). sLe^x antigen could be a candidate to be used as biomarker for the early diagnosis of cervical cancer.     

Hfr formation directed by tn10

The transposable drug-resistance element, Tn10, can serve as a region of homology to direct the insertion of an F'ts114 lac plasmid into the chromosome of Salmonella typhimurium. Derivatives of F'ts114 lac were constructed that carry Tn10 insertions; these plasmids were transferred to strains having a Tn10 insertion in the chromosome. Under these circumstances, Hfr formation requires homologous recombination between plasmid-borne and chromosomal Tn10 elements. The process is dependent on recA function and on the presence of both Tn10 elements. All Hfr's isolated from a given merodiploid show the same direction of transfer. Depending on the orientation of Tn10 in the F' plasmid, Hfr's transferring in either direction can be obtained from any chromosomal Tn10 insertion. Since Tn10 insertions can be generated in any region of the chromosome, this method permits the isolation of Hfr's with either direction of transfer having their origin at almost any predetermined site. The Hfr's constructed by this method are sufficiently stable for standard genetic mapping crosses, and they have also been used to generate new F' plasmids. Implicit in the results above is the possibility of determining the orientation of any chromosomal Tn10 insertion by constructing an Hfr using a standard F' Tn10 plasmid and determining the direction of chromosome transfer. The general approaches described here are applicable to other transposable elements and other bacterial systems.     

GALNT11 as a new molecular marker in chronic lymphocytic leukemia

Aberrant mucin O-glycosylation often occurs in different cancers and is characterized by immature expression of simple mucin-type carbohydrates. At present, there are some controversial reports about the Tn antigen (GalNAcα-O-Ser/Thr) expression and there is a great lack of information about the [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts)] expression in chronic lymphocytic leukemia (CLL). To gain insight in these issues we evaluated the Tn antigen expression in CLL patient samples using two Tn binding proteins with different fine specificity. We also studied the expression from 14 GalNAc-Ts genes in CLL patients by RT-PCR. Our results have provided additional information about the expression level of the Tn antigen, suggesting that a low density of Tn residues is expressed in CLL cells. We also found that GALNT11 was expressed in CLL cells and normal T cell whereas little or no expression was found in normal B cells. Based on these results, GALNT11 expression was assessed by qPCR in a cohort of 50 CLL patients. We found significant over-expression of GALNT11 in 96% of B–CLL cells when compared to normal B cells. Moreover, we confirmed the expression of this enzyme at the protein level. Finally we found that GALNT11 expression was significantly associated with the mutational status of the immunoglobulin heavy chain variable region (IGHV), [?²(1) = 18.26; P < 0.0001], lipoprotein lipase expression [?²(1) = 13.72; P = 0.0002] and disease prognosis [?²(1) = 15.49; P < 0.0001]. Our evidence suggests that CLL patient samples harbor aberrant O-glycosylation highlighted by Tn antigen expression and that the over-expression of GALNT11 constitutes a new molecular marker for CLL.     

Monoclonal antibodies toward different Tn-amino acid backbones display distinct recognition patterns on human cancer cells. Implications for effective immuno-targeting of cancer

The Tn antigen (GalNAcα-O-Ser/Thr) is a well-established tumor-associated marker which represents a good target for the design of anti-tumor vaccines. Several studies have established that the binding of some anti-Tn antibodies could be affected by the density of Tn determinant or/and by the amino acid residues neighboring O-glycosylation sites. In the present study, using synthetic Tn-based vaccines, we have generated a panel of anti-Tn monoclonal antibodies. Analysis of their binding to various synthetic glycopeptides, modifying the amino acid carrier of the GalNAc(*) (Ser* vs Thr*), showed subtle differences in their fine specificities. We found that the recognition of these glycopeptides by some of these MAbs was strongly affected by the Tn backbone, such as a S*S*S* specific MAb (15G9) which failed to recognize a S*T*T* or a T*T*T* structure. Different binding patterns of these antibodies were also observed in FACS and Western blot analysis using three human cancer cell lines (MCF-7, LS174T and Jurkat). Importantly, an immunohistochemical analysis of human tumors (72 breast cancer and 44 colon cancer) showed the existence of different recognition profiles among the five antibodies evaluated, demonstrating that the aglyconic part of the Tn structure (Ser vs Thr) plays a key role in the anti-Tn specificity for breast and colon cancer detection. This new structural feature of the Tn antigen could be of important clinical value, notably due to the increasing interest of this antigen in anticancer vaccine design as well as for the development of anti-Tn antibodies for in vivo diagnostic and therapeutic strategies.     

Existence in Escherichia coli of a mechanism that generates ''staggered'' head-to-head dimers of plasmid DNA; possible involvement of the Tn3 transposase.

The structures of two plasmids, one found in one of the transformants with putative products of an in vitro DNA rearrangement reaction containing the Tn3 transposase, and another found as a spontaneous tnpA negative derivative of an overproducer of the Tn3 transposase, have been elucidated. They both had shown non-conventional results in restriction enzyme analysis. For the determination of the structures, we used restriction enzyme analysis, denaturation and renaturation experiments, and DNA nucleotide sequencing. The structures turned out to be 'staggered' head-to-head dimers of the original monomer plasmids, containing gigantic inverted repeats separated by two identical spacers which are also in inverted orientations themselves. Two alternative models for the mode of origin of such a structure, a bimolecular model and a unimolecular one, are discussed. The circumstances in which these two plasmids occurred suggest possible involvement of the Tn3 transposase in their generation.     

Comparison of antigen constructs and carrier molecules for augmenting the immunogenicity of the monosaccharide epithelial cancer antigen Tn

We have demonstrated previously that the optimal method for inducing an antibody response against defined cancer antigens is covalent conjugation of the antigen to keyhole limpet hemocyanin (KLH) and use of the potent saponin adjuvant QS-21. Single molecules of glycolipids (tetrasaccharides, pentasaccharides, or hexasaccharides) and MUC1 peptides (containing between one and five MUC1 tandem repeats) conjugated to KLH have proven sufficient for antibody recognition and vaccine construction. However, cancer specificity of monoclonal antibodies against the monosaccharide Tn and disaccharide sTn comes largely from recognition of clusters (c) of these molecules on the cell surface. Tn consists of a monosaccharide (GalNAc) O-linked to serine or threonine on epithelial cancer mucins which are uniquely rich in serines and threonines. We test here several Tn constructs: Tn monosaccharide, Tn(c) prepared on a triple threonine backbone, and Tn prepared on a partially or fully glycosylated MUC1 backbone. We determine that Tn(c) is more effective than Tn, and conjugation to KLH is more effective than conjugation to BSA or polystyrene beads for inducing ELISA reactivity against Tn, and FACS reactivity against Tn-positive tumor cells. Surprisingly, MUC1 glycosylated with Tn at three or five sites per 20 amino acid MUC1 tandem repeat and conjugated to KLH, induced the strongest antibody response against Tn and tumor cells expressing Tn, and had the additional advantage of inducing antibodies against MUC1.     

Solid-phase synthesis of a pentavalent GalNAc-containing glycopeptide (Tn antigen) representing the nephropathy-associated IgA hinge region

Incomplete or aberrant glycosylation leading to Tn antigen (GalNAcα1-Ser/Thr) expression on human glycoproteins is strongly associated with human pathological conditions, including tumors, certain autoimmune diseases, such as the idiopathic IgA nephropathy, and may modulate immune homeostasis. In addition, the Tn antigen is highly expressed by certain pathogens and plays a role in host-pathogen interactions. To enable experimental approaches to study interactions of the Tn antigen with the immune system and analyze anti-Tn antibody responses in infection or disorders, we generated a Tn-expressing resource that can be used for high-throughput screening. In consideration of IgA nephropathy in which the hinge region is incompletely glycosylated, we used this hinge sequence that encodes five potential glycosylation sites as the ideal template for the synthesis of a Tn antigen-expressing glycopeptide. Inclusion of an N-terminal biotin in the peptide enabled binding to streptavidin-coated ELISA plates as monitored using Helix pomatia agglutinin or anti-Tn monoclonal antibody. We also found that the biotinylated IgA-Tn peptide is a functional acceptor for β1-3-galactosylation using recombinant T-synthase (β1-3-galactosyltransferase). Besides its immunochemical functionality as a possible diagnostic tool for IgA nephropathy, the peptide is an excellent substrate for glycan elongation and represents a novel template applicable for glycan-antigen-associated diseases.     

Expression of the Tn antigen on T-lymphoid cell line Jurkat.

Expression of the Tn antigen on a T-lymphoid cell line, Jurkat, was investigated using an anti-Tn monoclonal antibody, MLS 128. Immunoprecipitation or immunoaffinity chromatography of a lysate of Jurkat cells led to the isolation of a 120 kDa glycoprotein carrying the Tn antigen. This glycoprotein and leukosialin (CD43) were indistinguishable on SDS-PAGE and as to immunoreactivity with MLS 128. Leukosialin from an erythroid cell line, K562, exhibited no reactivity with MLS 128 despite that this leukosialin has several GalNAc alpha-Ser(Thr) structures. Pulse-chase experiments with the Jurkat leukosialin showed that newly synthesized leukosialin acquired the antigenecity after a lag of about 30 min, whereas incorporation of GalNAc into the leukosialin occurred earlier. These results indicate that the Tn antigen is expressed on leukosialin and that its epitopic structure is more complex than GalNAc alpha-Ser(Thr).     

Tn5-FISH,a novel cytogenetic method to image chromatin interactions with sub-kilobase resolution

There is an increasing interest in understanding how three-dimensional (3D) organization of the genome is regulated. Different strategies have been employed to identify genome-wide chromatin interactions. However, due to current limitations in resolving genomic contacts, visualization and validation of these genomic loci with sub-kilobase resolution remain unsolved to date. Here, we describe Tn5 transposase-based Fluorescencein situhybridization (Tn5-FISH), a PCR-based, cost-effective imaging method, which can co-localize the genomic loci with sub-kilobase resolution, dissect genome architecture, and verify chromatin interactions detected by chromatin configuration capture (3C)-derived methods. To validate this method, short-range interactions in keratin-encoding gene (KRT) locus in topologically associated domain (TAD) were imaged by triple-color Tn5-FISH, indicating that Tn5-FISH is very useful to verify short-range chromatin interactions inside the contact domain and TAD. Therefore, Tn5-FISH can be a powerful molecular tool for the clinical detection of cytogenetic changes in numerous genetic diseases such as cancers.     

Mucin-associated T antigens in benign and malignant epithelium of the gall bladder,extrahepatic bile ducts and ampulla of Vater

The mucin-associated antigens Tn, sialosyl-Tn (STn), T and sialosyl-T (STAg) antigens accumulate through aberrant and incomplete glycosylation in malignant epithelial cells. Their diagnostic and prognostic significance in tumours of the colon and cervix has been described, and a possible role for Tn antigen in cell-to-cell adhesion has been suggested. These antigens have been demonstrated through peanut agglutinin (PNA) lectin binding and more recently using specific monoclonal antisera. Differences between the two methods have been described, which may be due to fixation schedules and/or specificity. We have investigated the effect of fixation on the binding of biotinylated PNA lectin and compared its reactivity with the immunoreactivity of monoclonal antisera to Tn, STn, T and STAg antigens in benign and malignant epithelium of the gall bladder, extrahepatic bile ducts and ampulla of Vater. We found that short-term fixation in formol sublimate resulted in poor PNA binding. All other tested fixation schedules showed strong perinuclear binding, similar to that found on cryostat sections. When compared with monoclonal antisera, PNA binding demonstrated the lowest specificity in benign epithelium. In both benign and malignant epithelium, the two methods cannot substitute for each other. STn and STAg antigens were found to be oncodevelopmental throughout the extrahepatic biliary tract. When used in a panel, they are useful as diagnostic markers of malignancy in gall bladder epithelium.