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The project of my PhD course has been focused on the characterization of glycoconjugate vaccines to develop a largely applicable methodology to identify their glycosylation sites. The saccharide antigens are covalently attached to the carrier protein by a spacer or using specific functionalities available in both components. The general analytical strategy has involved a tryptic digestion and an enzymatic or acid hydrolysis of carbohydrate antigens in glycoconjugates. The resulting mixture of peptides and glycopeptides with well-defined mass increment has been analyzed by liquid chromatography interfaced with a mass spectrometer (LC-MS) technique to qualitatively understand which lysine residues have been involved in the glycosylation process. Several conjugate vaccines against Candida albicans, Group A Streptococcus (GAS), Group B Streptococcus (GBS) and Neisseria meningitidis group A (MenA) have been analyzed to estimate the distribution of glycosylated lysine residues. Among those, GBS resulted the less glycosylated in comparison to Candida and MenA ones. This aspect it is probably due to the different conjugation chemistries and GBS polysaccharide size. The second part of my PhD project has been the synthesis of two β(1,3)-glucans antigens, a trisaccharide and a hexasaccharide and their conjugation to CRM197. The hexasaccharide has also been linked on a multimeric structure PAMAM4 and then conjugated to CRM197. The last part of this work has been the conjugation to CRM197 of a modified MenB LPS, provided from Institute of Biological Science (IBS) laboratory (National Research Council - Ottawa Canada). The target sites of the carrier protein (CRM197) and LPS have been activated by two different reactans, and the final glycoconjugate has been obtained by the reaction of these two. The resulting conjugate has been characterized (saccharide and protein content, MALDI-TOF mass spectrometry and NMR) using an accurate analytical panel.

Several glycoconjugates are involved in the immune response. Sialic acid is frequently the glycan terminal sugar and it may modulate immune interactions. Dendritic cells (DCs) are antigen-presenting cells with high endocytic capacity and a central role in immune regulation. On this basis, DCs derived from monocytes (mo-DC) are utilised in immunotherapy, though many features are ignored and their use is still limited. We analyzed the surface sialylated glycans expressed during human mo-DC generation. This was monitored by lectin binding and analysis of sialyltransferases (ST) at the mRNA level and by specific enzymatic assays. We showed that alpha 2-3-sialylated O-glycans and alpha 2-6- and alpha 2-3-sialylated N-glycans are present in monocytes and their expression increases during mo-DC differentiation. Three main ST genes are committed with this rearrangement: ST6Gal1 is specifically involved in the augmented alpha 2-6-sialylated N-glycans; ST3Gal1 contributes for the alpha2-3-sialylation of O-glycans, particularly T antigens; and ST3Gal4 may contribute for the increased alpha2-3-sialylated N-glycans. Upon mo-DC maturation, ST6Gal1 and ST3Gal4 are downregulated and ST3Gal1 is altered in a stimulus-dependent manner. We also observed that removing surface sialic acid of immature mo-DC by neuraminidase significantly decreased its endocytic capacity, while it increased in monocytes. Our results indicate the STs expression modulates the increased expression of surface sialylated structures during mo-DC generation, which is probably related with changes in cell mechanisms. The ST downregulation after mo-DC maturation probably results in a decreased sialylation or sialylated glycoconjugates involved in the endocytosis, contributing to the downregulation of one or more antigen-uptake mechanisms specific of mo-DC.

Sialyl Lewis X (sLex) is a well known carbohydrate antigen whose overexpression in cancer correlates with metastasis. In colon cancer, the molecular bases of sLex overexpression remain elusive. We have investigated sLex expression in normal and cancer colonic tissues and in cell lines as a function of the expression of fucosyltransferases and beta4GalNAcT-II. This enzyme, which is downregulated in colon cancers, is responsible for the biosynthesis of the Sda antigen [Siaalpha2,3(GalNAcbeta1,4)Galbeta1,4GlcNAc], whose biosynthesis competes with that of sLex. In colon cancers and cell lines, sLex expression correlates with a fucosyltransferase activity able to fucosylate 3'sialyllactosamine at low (0.5 mM) concentration. Transfection experiments with FucT-III, IV, V, VI and VII cDNAs in COS-7 cells indicate that only FucT-VI displays this property. In gastrointestinal cell lines, high levels of this fucosyltransferase activity are shown by cell lines expressing high levels of the FucT-VI transcript. In normal colon, sLex antigen is detectable upon de-acetylation but its expression does not correlate with any fucosyltransferase activity or transcript. Rather, we observed a significant correlation between sLex and the ratio between fucosyltransferase activity with 0.5 mM 3'sialyllactosamine and beta4GalNAcT-II activity. These data suggest that in both normal and cancer colonic tissues the biosynthesis of sLex is mainly due to FucT-VI or to a fucosyltransferase with similar kinetic properties, unknown at present. In normal colon, but not in colon cancers, the fucosyltransferase activity above described is counteracted by competing glycosyltransferases, as the high beta4GalNAcT-II activity that synthesizes the alternative Sda antigen.

The plant toxin ricin is synthesized in castor bean seeds as an endoplasmic reticulum (ER)-targeted precursor. Removal of the signal peptide generates proricin in which the mature A- and B-chains are joined by an intervening propeptide and a 9-residue propeptide persists at the N terminus. The two propeptides are ultimately removed in protein storage vacuoles, where ricin accumulates. Here we have demonstrated that the N-terminal propeptide of proricin acts as a nonspecific spacer to ensure efficient ER import and glycosylation. Indeed, when absent from the N terminus of ricin A-chain, the non-imported material remained tethered to the cytosolic face of the ER membrane, presumably by the signal peptide. This species appeared toxic to ribosomes. The propeptide does not, however, influence catalytic activity per se or the vacuolar targeting of proricin or the rate of retrotranslocation/degradation of A-chain in the cytosol. The likely implications of these findings to the survival of the toxin-producing tissue are discussed.

Beta-galactoside alpha2,6 sialyltransferase (ST6Gal.I), the enzyme which adds sialic acid in alpha2,6-linkage on lactosaminic termini of glycoproteins, is frequently overexpressed in cancer, but its relationship with malignancy remains unclear. In this study, we have investigated the phenotypic changes induced by the expression of alpha2,6-sialylated lactosaminic chains in the human colon cancer cell line SW948 which was originally devoid of ST6Gal.I. Clones derived from transfection with the ST6Gal.I cDNA were compared with untransfected cells and mock transfectants. The ST6Gal.I-expressing clones show (1) increased adherence to fibronectin and collagen IV but not to hyaluronic acid. Treatment with Clostridium perfrigens neuraminidase reduces the binding to fibronectin and collagen IV of ST6Gal.I-expressing cells but not that of ST6Gal.I-negative cells; (2) accumulation and more focal distribution of beta1 integrins on the cell surface; (3) different distribution of actin fibers; (4) flatter morphology and reduced tendency to multilayer growth; (5) improved ability to heal a scratch wound; (6) reduced ability to grow at the subcutaneous site of injection in nude mice. Our data suggest that the presence of alpha2,6-linked sialic acid on membrane glycoconjugates increases the binding to extracellular matrix components, resulting in a membrane stabilization of beta1 integrins, further strengthening the binding. This mechanism can provide a basis for the flatter morphology and the reduced tendency to multilayer growth, resulting in a more ordered tissue organization. These data indicate that in the cell line SW948, the effect of ST6Gal.I expression is consistent with the attenuation of the neoplastic phenotype.

The biosynthesis of the Sda histo-blood group antigen [Siaalpha2,3(GalNAcbeta1,4)Galbeta1,4GlcNAc-R], is catalyzed by Sda beta1,4GalNAc transferase (beta1,4GalNAc-T). This enzyme, which catalyzes the addition of GalNAc, the immunodominant sugar, to the Gal residue of an alpha2,3-sialylated type 2 chain, is highly expressed in normal colon but it is dramatically downregulated in colon cancer. The acceptor structure of beta1,4GalNAc-T is also a precursor of the biosynthesis of the sialyl Lewis x (sLex) antigen [Siaalpha2,3Galbeta1,4(Fuca1,3)GlcNAc-R], a major ligand for adhesion molecules of the selectin family whose ectopic expression in cancers favours metastasis formation. The fact that the precursor structures of the Sda and the sLex antigens are identical suggests a competition in the biosynthesis of the two structures and that the downregulation of beta1,4GalNAc-T opens the way to the biosynthesis of sLex, favouring the metastatic progression of colon cancer. The two beta1,4GalNAc-T mRNA species cloned to date differ in the first exon and predict two polypeptides with a different cytoplasmic tail: one encodes a polypeptide with an exceptionally long cytoplasmic sequence of 67 aminoacids, the other encodes a polypeptide with a cytoplasmic tail of 7 residues. To investigate the relationship between beta1,4GalNAc-T and sLex expression, we have stably expressed the two forms in the human colorectal cancer cell line LS174T, which expresses good level of sLex. Compared with mock-transfectants, the clones expressing either form of beta1,4GalNAc-T show a dramatic downregulation of sLex expression in FACS analysis, demonstrating that the level of expression of beta1,4GalNAc-T modulates the biosynthesis of sLex, in vitro.

The addition of sialic acid in alpha2,6-linkage to lactosaminic termini of glycoproteins is mainly mediated by beta-galactoside alpha2,6 sialyltransferase (ST6Gal.I). This enzyme and its cognate oligosaccharide structure are frequently overexpressed in cancer and are associated with increased malignancy but the cellular and molecular bases of this relationship are not clear. In this study, we have investigated the role of ST6Gal.I in the in vivo growth. First, we have xenografted in nude mice colon cancer cell lines and analyzed the expression of ST6Gal.I and of alpha2,6-sialylated sugar chains in the xenograft-derived (XD) cell lines. Compared with parental cell lines, the XD cell lines express dramatically increased levels of ST6Gal.I mRNA, detected by RT-PCR analysis, and enzyme activity, as well as an increased reactivity with the alpha2,6-sialyl-specific lectin from Sambucus nigra (SNA) suggesting either a selection of the cells expressing higher levels of ST6Gal.I. Then, we investigated the in vivo growth of clones derived from transfection with the ST6Gal.I cDNA of the colon cancer cell line SW48, which was originally devoid of ST6Gal.I expression. Compared with mock-transfectants, ST6Gal.I-transfectants form more rapidly growing tumours. Moreover, when an identical number of mock- and ST6Gal.I-transfected cells was mixed and injected in the nude mice, the xenografts and the xenograft-derived cell lines were comprised only of SNA-positive ST6Gal.I-transfectants, while mock-transfected cells were usually not present in the xenograft. These results indicate that during the growth of the tumour the cells expressing high levels of alpha2,6-sialylated glycoconjugates are positively selected because of a growth advantage.

beta-galactoside alpha2,6 sialyltransferase (ST6Gal.I), the enzyme which adds sialic acid in alpha2,6-linkage on lactosaminic termini of glycoproteins, is frequently overexpressed in cancer. This change is associated with increased malignancy but the molecular bases of this relationship remain elusive. In this study, we have investigated the phenotypic changes related with overexpression of alpha2,6-sialylated lactosaminic chains by using clones derived from transfection of the human colon cancer cell line SW948 with the ST6Gal.I cDNA or mock transfected. Compared with untransfected cells and mock-transfectants, the ST6Gal.I-expressing clones show increased adherence to fibronectin and collagen IV but not to hyaluronic acid. Neuraminidase treatment resulted in reduced binding to fibronectin and collagen IV of a ST6Gal.I-expressing clone but in no effect on a mock-transfectant. While untransfected and mock-transfected cells tend to form multistratified tissues, ST6Gal.I-expressing clones show a flatter morphology and the tendency to grow as a monolayer. FACS analysis revealed that all ST6Gal.I-expressing clones display higher amounts of beta1 integrins on the cell surface; this difference is not supported by differences at the beta1 integrin mRNA level and is lost when cells are left in suspension for several hours, suggesting a mechanism of membrane stabilization of beta1 integrins dependent on the presence of alpha2,6-linked sialic acid. Our data support a model in which the presence of alpha2,6-linked sialic acid on membrane glycoconjugates increases the binding to extracellular matrix components, resulting in membrane stabilization of beta1 integrins, further strengthening the binding. This mechanism provides a basis for the altered phenotype associated with ST6Gal.I overexpression.