Littérature scientifique sur le sujet « Glicosilazione »

Introduzione: Le catene N-linked associate al principale sito di N-glicosilazione (Asn297) delle IgG sono di tipo bi-antennario e presentano una grande microeterogeneità in quanto una o entrambe le antenne possono terminare con uno o due residui di acido sialico, galattosio o N-acetilglucosammina ed essere core-fucosilate. Nell’invecchiamento e in malattie infiammatorie aumenta la percentuale di glicani associati alle catene pesanti delle IgG privi del galattosio terminale (IgG-G0). La glicosilazione enzimatica delle proteine è classicamente un processo intracellulare, sebbene recenti studi abbiano messo in evidenza la possibilità di una glicosilazione ecto-cellulare in quanto le piastrine sono ottimi donatori di nucleotidi-zuccheri. Scopo: Misurare le attività delle glicosiltrasferasi ST6Gal1 e B4GalT plasmatiche (potenzialmente responsabili della glicosilazione di proteine plasmatiche) in soggetti di entrambi i sessi e di età compresa tra 5 e 105 anni e correlarle con lo stato di glicosilazione di IgG circolanti (analizzato mediante lectin-blot) e il GlycoAge test, un noto marcatore di invecchiamento, espresso come il logaritmo del rapporto tra gli N-glicani agalattosilati e di-galattosilati associati a glicoproteine plasmatiche. Risultati e conclusioni: I dati ottenuti indicano che: 1) l’attività B4GalT si propone come nuovo marcatore di invecchiamento perché aumenta linearmente con l’età; 2) la ST6Gal1 è maggiormente espressa solo nei bambini e negli over 80; 3) le attività delle due glicosilatransferasi non risultano correlate in modo significativo né tra loro né con il GlycoAge test, indicando che questi tre marcatori siano espressioni di diversi quadri fisio-patologici legati all’invecchiamento; 4) con l’età si ha una predominanza di glicoforme di IgG pro-infiammatorie, ovvero prive dell’acido sialico, del galattosio terminali e del core fucose; 5) l’attività della ST6Gal1 e B4GalT risultano in controtendenza con il grado di sialilazione e galattosilazione delle IgG, indicando quindi che la loro glicosilazione non avviene a livello extracellulare.
Background: The N-linked chains associated with the main N- glycosylation site (Asn297) of IgG are bi- antennary type and present a great microheterogeneity because one or both antennas may end with one or two residues of sialic acid , galactose or N -acetilglucosammine and can be core – fucosilated. In aging and inflammatory diseases the percentage of glycans associated with heavy chains of IgG that lack terminal galactose (IgG - G0) increases. The enzymatic glycosylation of proteins is classically an intracellular process although recent studies have highlighted the possibility of extracellular glycosylation because the platelets are an excellent source of the sugar nucleotide – donors. Aim of study: To measure the activities of plasma glycosyltransferases ST6Gal1 and B4GalT (potentially responsible for the glycosylation of plasma proteins ) in individuals of both sexes and aged between 5 and 105 years, and correlate them with the state of glycosylation of circulating IgG (determined by lectin immunoblot analysis) and GlycoAge test, a know marker of aging that is the logarithm of the ratio between agalactosylated and di-galactosylated N-linked chains associated to plasma glycoprotein. Results and conclusions: Own results indicated that: 1) B4GalT acitvity is a new marker of aging because it increase linearly with age ; 2) ST6Gal1 activity is mostly expressed only in children and over 80; 3) The activity of the two glycosyltransferases does not relate each other and with the GlycoAge test indicating that all three of these markers are expressions of different physio- pathological frameworks related to aging; 4) during aging there is an abundance of pro-inflammatory glycoforms of IgG; 5) the activity of the ST6Gal1 and B4GalT are in contrast with the degree of sialylation and galactosylation of IgG , indicating that their glycosylation does not occur at the extracellular level .

Introduzione: Le catene N-linked associate al principale sito di N-glicosilazione (Asn297) delle IgG sono di tipo bi-antennario e presentano una grande microeterogeneità in quanto una o entrambe le antenne possono terminare con uno o due residui di acido sialico, galattosio o N-acetilglucosammina ed essere core-fucosilate. Nell’invecchiamento e in malattie infiammatorie aumenta la percentuale di glicani associati alle catene pesanti delle IgG privi del galattosio terminale (IgG-G0). La glicosilazione enzimatica delle proteine è classicamente un processo intracellulare, sebbene recenti studi abbiano messo in evidenza la possibilità di una glicosilazione ecto-cellulare in quanto le piastrine sono ottimi donatori di nucleotidi-zuccheri. Scopo: Misurare le attività delle glicosiltrasferasi ST6Gal1 e B4GalT plasmatiche (potenzialmente responsabili della glicosilazione di proteine plasmatiche) in soggetti di entrambi i sessi e di età compresa tra 5 e 105 anni e correlarle con lo stato di glicosilazione di IgG circolanti (analizzato mediante lectin-blot) e il GlycoAge test, un noto marcatore di invecchiamento, espresso come il logaritmo del rapporto tra gli N-glicani agalattosilati e di-galattosilati associati a glicoproteine plasmatiche. Risultati e conclusioni: I dati ottenuti indicano che: 1) l’attività B4GalT si propone come nuovo marcatore di invecchiamento perché aumenta linearmente con l’età; 2) la ST6Gal1 è maggiormente espressa solo nei bambini e negli over 80; 3) le attività delle due glicosilatransferasi non risultano correlate in modo significativo né tra loro né con il GlycoAge test, indicando che questi tre marcatori siano espressioni di diversi quadri fisio-patologici legati all’invecchiamento; 4) con l’età si ha una predominanza di glicoforme di IgG pro-infiammatorie, ovvero prive dell’acido sialico, del galattosio terminali e del core fucose; 5) l’attività della ST6Gal1 e B4GalT risultano in controtendenza con il grado di sialilazione e galattosilazione delle IgG, indicando quindi che la loro glicosilazione non avviene a livello extracellulare.
Background: The N-linked chains associated with the main N- glycosylation site (Asn297) of IgG are bi- antennary type and present a great microheterogeneity because one or both antennas may end with one or two residues of sialic acid , galactose or N -acetilglucosammine and can be core – fucosilated. In aging and inflammatory diseases the percentage of glycans associated with heavy chains of IgG that lack terminal galactose (IgG - G0) increases. The enzymatic glycosylation of proteins is classically an intracellular process although recent studies have highlighted the possibility of extracellular glycosylation because the platelets are an excellent source of the sugar nucleotide – donors. Aim of study: To measure the activities of plasma glycosyltransferases ST6Gal1 and B4GalT (potentially responsible for the glycosylation of plasma proteins ) in individuals of both sexes and aged between 5 and 105 years, and correlate them with the state of glycosylation of circulating IgG (determined by lectin immunoblot analysis) and GlycoAge test, a know marker of aging that is the logarithm of the ratio between agalactosylated and di-galactosylated N-linked chains associated to plasma glycoprotein. Results and conclusions: Own results indicated that: 1) B4GalT acitvity is a new marker of aging because it increase linearly with age ; 2) ST6Gal1 activity is mostly expressed only in children and over 80; 3) The activity of the two glycosyltransferases does not relate each other and with the GlycoAge test indicating that all three of these markers are expressions of different physio- pathological frameworks related to aging; 4) during aging there is an abundance of pro-inflammatory glycoforms of IgG; 5) the activity of the ST6Gal1 and B4GalT are in contrast with the degree of sialylation and galactosylation of IgG , indicating that their glycosylation does not occur at the extracellular level .

Muscular dystrophies are a group of diseases genotypically and clinically heterogeneous, characterized by progressive muscular weakness. The histopathologic profile can vary from mild to severe myopathy, with degeneration, regeneration and adipose-fiber substitution. Muscular dystrophy etiopathogenesis involves basal membrane elements and muscle fiber cytoskeleton alterations. A prominent role is played by the dystrophin glycoprotein complex (DGC), which provides a strong stabilization link between the intracellular cytoskeleton and extracellular matrix. The DGC is composed by dystrophin, sarcoglycans, distrobrevins, sintrophins, sarcospan and dystroglycan. Dystroglycan is a glycoprotein, encoded by one gene DAG1, that is post-translationally cleaved into 2 subunits, ?-dystroglycan and ?-dystroglycan. The dystroglycan ensure connection and stability and is expressed in a large variety of tissue, including skeletal and cardiac muscle, central and peripheral nervous system tissue and epithelia. ?-DG is a highly glycosylated peripheral membrane protein, which binds to several molecules (laminin, agrin, perlecan, neurexin, biglycan) and tightly interacts with the extracellular portion of ?-DG. ?-DG has the ability to connect to the cytoplasmic domain of dystrophin, to caveolin-3 and other proteins implicated in signal trasduction. The predicted molecular weight of ?-dystroglycan is about 72 kDa, but can vary due to glycosylation. In fact ?-dystroglycan has an apparent molecular weight of 156 kDa in skeletal muscle. So far no pathogenic mutations have been identified in genes encoding for dystroglycan. It is clear that the pathogenesis of in muscular dystrophy involves post-translational processing events that are important for the interaction of dystroglycan with its ligands. In particular ?-dystroglycan glycosylation has a critical role in maintaining integrity of extracellurar matrix proteins. Alterations in ?-dystroglycan glycosylation lead to a particular group of recessive autosomic muscular dystrophy called dystroglycanopathies. The dystroglycanopathies show strong phenotypic heterogeneity: in fact at the most severe end of the clinical spectrum are Walker-Warburg Syndrome (WWS), Muscle-Eye-Brain (MEB), Fukuyama congenital muscular dystrophy (FCMD), MDC1C. At the mild end there is Limb-Girdle muscular dystrophy (LGMD). Genetically, dystroglycanopathies are caused by mutations in 6 known genes: POMT1, POMT2, POMGnT1, Fukutin, LARGE, FKRP. All these genes encode for putative or known glycosyltransferases. This present work intends to characterize a large group of patients affected by congenital or limb-girdle muscular dystrophy with unknown etiopathogenesis, trying to extend genotype and phenotype correlations. In fact a precise molecular diagnosis will provide an essential basis for further studies leading to identification of new therapeutic strategies. Selection criteria Patient groups are sorted from the muscular biopsy database “Centro delle Malattie Neuromuscolari”, Department of Neuroscience, University of Padova, which comprises over 8000 samples. In order to be selected, each patient must meet one of these criteria: a) be affected by congenital muscular dystrophy or limb-girdle muscular dystrophy with unknown etiopathogenesis, b) have floating CK values, weakness during fever or steroid responsiveness. 234 patients were selected for screening. Immunohistochemistry Immunohistochemistry was performed on 234 muscular biopsies. Cryosections were incubated with an antibody directed to the ?-dystroglycan epitope (IIH6C4 - Upstate Biotechnology). Immunoblot Immunoblot was performed on 94 samples, with reduced ?-dystroglycan glycosylation in immunohistochemistry. The analysis shows exact congruence between immunohistochemistry and immunoblot data. Mutation analysis Of 94 analyzed patients, 26 were idenfied as having pathogenic mutations. 73% of patients showed reduced ?-dystroglycan glycosylation, while 27% of patients exhibited total absence of ?-dystroglycan glycosylation (in immunohistochemistry and immunoblot). The data set obtained by mutation analysis lead to a general mutation rate of 28% (26 patients out of 94 patients with ?-dystroglycan deficit) and can be subdivided as a) 24% if mutation rate is examined inside partially deficit patient group, or in b) 46% if mutation rate is considered inside totally deficit patient group. In conclusion, in the last few years it has become more and more evident that ?-dystroglycan play a pivotal role in the dystrophin glycoprotein complex (DGC), permitting connection and assuring stability between the intracellular cytoskeleton and extracellular matrix. Moreover a better understanding of ?-dystroglycan O-glycosylation could lead to the development of new therapeutic approaches. In fact, post-translational processes that alter dystroglycan glycosylation are the basis of muscular dystrophy pathogenesis, principally dystroglycanopathy. The mutation rate found in our patients (with ?-dystroglycan deficit) is about 26%, which an incidence of 50% in patients with total absence of ?-dystroglycan. This work confirms high genotypic and phenotypic heterogeneity of dystroglycanopathy and the wide phenotypic spectrum related to these disorders.

Muscular dystrophies are a group of diseases genotypically and clinically heterogeneous, characterized by progressive muscular weakness. The histopathologic profile can vary from mild to severe myopathy, with degeneration, regeneration and adipose-fiber substitution. Muscular dystrophy etiopathogenesis involves basal membrane elements and muscle fiber cytoskeleton alterations. A prominent role is played by the dystrophin glycoprotein complex (DGC), which provides a strong stabilization link between the intracellular cytoskeleton and extracellular matrix. The DGC is composed by dystrophin, sarcoglycans, distrobrevins, sintrophins, sarcospan and dystroglycan. Dystroglycan is a glycoprotein, encoded by one gene DAG1, that is post-translationally cleaved into 2 subunits, ?-dystroglycan and ?-dystroglycan. The dystroglycan ensure connection and stability and is expressed in a large variety of tissue, including skeletal and cardiac muscle, central and peripheral nervous system tissue and epithelia. ?-DG is a highly glycosylated peripheral membrane protein, which binds to several molecules (laminin, agrin, perlecan, neurexin, biglycan) and tightly interacts with the extracellular portion of ?-DG. ?-DG has the ability to connect to the cytoplasmic domain of dystrophin, to caveolin-3 and other proteins implicated in signal trasduction. The predicted molecular weight of ?-dystroglycan is about 72 kDa, but can vary due to glycosylation. In fact ?-dystroglycan has an apparent molecular weight of 156 kDa in skeletal muscle. So far no pathogenic mutations have been identified in genes encoding for dystroglycan. It is clear that the pathogenesis of in muscular dystrophy involves post-translational processing events that are important for the interaction of dystroglycan with its ligands. In particular ?-dystroglycan glycosylation has a critical role in maintaining integrity of extracellurar matrix proteins. Alterations in ?-dystroglycan glycosylation lead to a particular group of recessive autosomic muscular dystrophy called dystroglycanopathies. The dystroglycanopathies show strong phenotypic heterogeneity: in fact at the most severe end of the clinical spectrum are Walker-Warburg Syndrome (WWS), Muscle-Eye-Brain (MEB), Fukuyama congenital muscular dystrophy (FCMD), MDC1C. At the mild end there is Limb-Girdle muscular dystrophy (LGMD). Genetically, dystroglycanopathies are caused by mutations in 6 known genes: POMT1, POMT2, POMGnT1, Fukutin, LARGE, FKRP. All these genes encode for putative or known glycosyltransferases. This present work intends to characterize a large group of patients affected by congenital or limb-girdle muscular dystrophy with unknown etiopathogenesis, trying to extend genotype and phenotype correlations. In fact a precise molecular diagnosis will provide an essential basis for further studies leading to identification of new therapeutic strategies. Selection criteria Patient groups are sorted from the muscular biopsy database “Centro delle Malattie Neuromuscolari”, Department of Neuroscience, University of Padova, which comprises over 8000 samples. In order to be selected, each patient must meet one of these criteria: a) be affected by congenital muscular dystrophy or limb-girdle muscular dystrophy with unknown etiopathogenesis, b) have floating CK values, weakness during fever or steroid responsiveness. 234 patients were selected for screening. Immunohistochemistry Immunohistochemistry was performed on 234 muscular biopsies. Cryosections were incubated with an antibody directed to the ?-dystroglycan epitope (IIH6C4 - Upstate Biotechnology). Immunoblot Immunoblot was performed on 94 samples, with reduced ?-dystroglycan glycosylation in immunohistochemistry. The analysis shows exact congruence between immunohistochemistry and immunoblot data. Mutation analysis Of 94 analyzed patients, 26 were idenfied as having pathogenic mutations. 73% of patients showed reduced ?-dystroglycan glycosylation, while 27% of patients exhibited total absence of ?-dystroglycan glycosylation (in immunohistochemistry and immunoblot). The data set obtained by mutation analysis lead to a general mutation rate of 28% (26 patients out of 94 patients with ?-dystroglycan deficit) and can be subdivided as a) 24% if mutation rate is examined inside partially deficit patient group, or in b) 46% if mutation rate is considered inside totally deficit patient group. In conclusion, in the last few years it has become more and more evident that ?-dystroglycan play a pivotal role in the dystrophin glycoprotein complex (DGC), permitting connection and assuring stability between the intracellular cytoskeleton and extracellular matrix. Moreover a better understanding of ?-dystroglycan O-glycosylation could lead to the development of new therapeutic approaches. In fact, post-translational processes that alter dystroglycan glycosylation are the basis of muscular dystrophy pathogenesis, principally dystroglycanopathy. The mutation rate found in our patients (with ?-dystroglycan deficit) is about 26%, which an incidence of 50% in patients with total absence of ?-dystroglycan. This work confirms high genotypic and phenotypic heterogeneity of dystroglycanopathy and the wide phenotypic spectrum related to these disorders.

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.

Study of lysozyme in the context of diabetic nephropathy Introduction Lysozyme (LZ), enzyme known for its antimicrobial and immunomodulant activity, has recently shown to be effective in the context of diabetic nephropathy, one of the major complications associated to diabetes and one of the major cause of end stage kidney disease in Western countries. Experimental evidences strictly link the development and progression of the diabetic nephropathy to the enormous production and accumulation of Advanced Glycation Endproducts (AGEs). AGEs are a chemically heterogeneous group of macromolecules which formation is dramatically increased in diabetic patient mainly due to chronic hyperglicaemia. Nevertheless, several factor, such oxidative and carbonyl stress and reduced renal clereance seem to be related to the increase of the production and accumulation of AGEs. The first step of AGEs formation starts in presence of reducing sugars, as glucose, which are able to interact with free amino group (Maillard reaction), forming Schiff base. The rearrangements from the Schiff base leads to the formation of Amadori products, that are eventually oxidazed, leading to the formation of AGEs. AGEs binding to structural proteins and deposition in tissues alter functions, leading to stiffening, enhanced local cytokine production and is a marker of cumulative metabolic stress. At cellular level, AGEs-induced events, depend mainly by their interaction with specific receptor, among which RAGE, is the most well characterized. AGE-RAGE interactions determine a number of events, such as an increased production of reactive oxygen species (ROS), induction of pro-inflammatory mediators, upregulation of RAGE receptor. In the last decades, it has been shown that hen egg white LZ is able to act effectively as AGEs scavenger (Kd= 50 nM). In in vivo models it was demonstrated that orally administered microencapsulated LZ, can act prevent the insurgence of a number of relevant early manifestations of diabetic nephropathy, such as microalbuminuria and glomerular hypertrophy. Aim The aim of the present work is to develop an in vitro model for studying the molecular mechanisms responsible for the nephroprotective activity of LZ, through the identification and validation of an adequate cell line and a marker. In order contribute to understand the higher in vivo effectiveness of microencapsulated LZ compared to an equidose of “free” LZ, a sophisticated model of incannulated rat will be developed. Results and discussion An adequate cell model was selected among three different cell lines: a primary culture of endothelial cells (ADMEC), due to the implication of vascular tissue in diabetes and two proximal tubular cell lines, LLC-PK1 (porcine cells) and HK-2 (human cells) due to the tubular involvement in the context of diabetic nephropathy. HK-2 cell line was selected on the basis of its dose-dependent sensibility to the AGE treatments, quantified, in terms of viability, by means of MTT test. One of the most relevant AGE-induced effect is the induction of inflammatory response. A pivotal event in the context of inflammation is represented by macrophage recruitment. For this reason, it was performed a migration assay, using monocyte U937 differentiated in macrophage, toward a stimulus represented by supernatants obtained treating HK-2 cells with AGE e LZ. AGE treatment induced a significant increased (+50%) of the migrated macrophage. The co-treatment significantly prevented the migration induction. LZ treatment, as expected, did not modify migratory ratio. It is known that cytokine play a crucial role in macrophage recruitment. Interleukin-6 (IL-6), for what diabetic nephropathy is concerned, has a pivotal role. On the basis of this evidence, the IL-6 mRNA levels after AGE and LZ treatments were measured. According with expectations, AGE induced a significant IL-6 mRNA increase after 24 h of treatment. LZ exposition did not induced any modifications, whereas the co-treatment with AGE and LZ showed that LZ prevented IL-6 mRNA increase. In order to confirm the effectiveness of LZ toward AGE-induced IL-6 increase, an ELISA assay was performed. LZ was able to significantly and dose-dependently reduce the AGE-induced release of IL-6 in the supernatants. Several mechanisms have been reported to be capable to induce IL-6 release. The mechanisms that determine IL-6 release are several. On the basis of the recognized crucial role attributed to RAGE receptor, it was tested the capacity of LZ to influence RAGE mRNA levels, by means of RT-PCR technique. The results showed no variations of RAGE levels after exposition to AGE and LZ for 24 up to 96 h. The induction of ROS represents one of the events attributable to AGEs. Nevertheless, on the cellular model employed, this parameter did not show any significant increase. Considering that LZ activity is associated also to its AGE scavenging action, it was evaluated by means of confocal microscopy, if the presence of LZ could influence the AGE capacity to enter inside the cell. The images obtained by means of this technique showed that AGEs and LZ are both able to enter in the cells. In order to investigate the co-treatment effect a cytometry analysis was performed. The results suggest that LZ is not able, in this cellular model, to prevent the entrance on AGEs inside the cells, excluding that LZ scavenging action could be the cause of preventive action of in vitro IL-6 production. Subsequent studies were performed on pathways potentially involved in the action of LZ toward AGEs, such as MAPK cascade (p38) and lysosomial degradation. Preliminary data showed a possible involvement of p38. In fact, AGEs showed the capacity to increase phosphorylation level of this protein, effect reduced by the co-treatment with an equimolar dose of LZ. Promising are also the first results obtained from the lysosomial localization study of AGE, in presence of LZ. In fact, the qualitative analysis of the obtained figures confirms the hypothesis that LZ is able to bring AGEs in lysosomes, supporting their degradation. Taken together these data do not exclude stochastically that LZ action is mainly antidotic. However, the LZ capacity to act toward pivotal mediators of AGE-induced effects, such as IL-6 and p38, open new work perspectives for what LZ action is concerned, allowing to speculate its use as cooperating drug in the control of inflammation in the context of diabetic nephropathy. In order to investigate if the higher effectiveness of orally administered microencapsulated LZ was due to the capacity of microparticles to deliver more efficiently the LZ in the blood stream an in vivo test was performed. The results showed that microparticles can contribute significantly to deliver LZ more efficiently to serum
Studio del lisozima nel contesto della nefropatia diabetica Introduzione Il lisozima (LZ), enzima noto per la sua attività antibatterica ed immunomodulante, è stato recentemente identificato come principio attivo con potenzialità terapeutica nel contesto della nefropatia diabetica, una delle principali complicanze associate al diabete e principale causa di patologie renali terminali nell’Occidente sviluppato. Evidenze sperimentali correlano strettamente l’insorgenza e la progressione della nefropatia diabetica all’abnorme formazione ed accumulo di molecole note come prodotti finali della glicosilazione avanzata (Advanced Glycation Endproducts, AGE). Gli AGE sono un gruppo chimicamente eterogeneo di macromolecole la cui formazione risulta drammaticamente incrementata nel paziente diabetico a causa dell’iperglicemia cronicizzata. La formazione degli AGE ha inizio in presenza di zuccheri riducenti, come il glucosio, i quali nella loro forma aperta sono in grado di interagire con gruppo amminici liberi (reazione di Maillard) formando le basi di Schiff. Tali composti si riarrangiano in prodotti di Amadori, dai quali derivano direttamente gli AGE. Gli effetti patologici AGE-indotti derivano dal loro deposito tissutale e dal legame con proteine a lento turnover, con conseguente riduzione della funzionalità e/o dell’elasticità degli stessi. Gli eventi AGE-indotti, a livello cellulare, dipendono principalmente dalla loro interazione con i propri recettori specifici, tra i quali il meglio caratterizzato è RAGE. L’interazione AGE-RAGE è alla base di numerosi eventi tra i quali un ruolo centrale spetta all’aumento di specie reattive dell’ossigeno (ROS), all’induzione del rilascio di stimoli pro-infiammatori e all’upregolazione del recettore RAGE stesso. Recentemente, è stato dimostrato che il LZ da bianco d’uovo è in grado di fungere efficacemente da scavenger degli AGE (Kd= 50 nM). In modelli in vivo con diabete indotto, è stato dimostrato che il LZ microincapsulato, somministrato oralmente, è in grado di prevenire l’insorgenza di alcuni dei principali sintomi precoci della nefropatia diabetica, quali microalbuminuria ed ipertrofia glomerulare. Scopo L’obiettivo di questo lavoro di dottorato è quello di sviluppare un modello in vitro per lo studio dei meccanismi molecolari alla base dell’attività nefroprotettiva del LZ attraverso l’individuazione e la validazione di una linea cellulare e di un marker adeguati. Per chiarire alcuni aspetti di ordine farmacocinetico è stata anche quantificata la concentrazione plasmatica di LZ, in seguito a somministrazione orale in forma microincapsulata, rispetto alla somministrazione in forma libera. Risultati e discussione La selezione di un modello cellulare adeguato ai successivi studi in vitro è avvenuta nell’ambito di tre linee cellulari potenzialmente adatte, ovvero: una coltura primaria di cellule endoteliali del microcircolo dermico (ADMEC), per via dell’implicazione del sistema vascolare nel diabete e due linee cellulare di tubulo prossimale renale, LLC-PK1 (di origine suina) e HK-2 (di origine umana), per via del coinvolgimento tubulare nel contesto della nefropatia diabetica. Una serie di esperimenti ha permesso di far ricadere la scelta sulle cellule HK-2, le quali hanno mostrato la maggior sensibilità ai trattamenti con AGE (decremento dose-dipendente e tempo-indipendente) in termini di vitalità misurata mediante MTT test e per questioni di ordine pratico e logistico. Uno dei principali effetti degli AGE nell’organismo è l’induzione della risposta infiammatoria. Un evento cruciale dell’infiammazione è rappresentato dal reclutamento macrofagico. Sulla base di questi presupposti è stato effettuato un saggio di migrazione dei monociti U937, differenziati a macrofagi, verso uno stimolo rappresentato da sovranatanti ottenuti trattando le cellule HK-2 con AGE e LZ. Il trattamento con AGE ha indotto un incremento significativo (+ 50%) dei macrofagi migrati. Il trattamento con il LZ, come atteso, non ha modificato il tasso migratorio mentre, in presenza di AGE, è stato in grado di prevenire significativamente l’induzione della migrazione. È noto che le citochine svolgono un ruolo fondamentale nel reclutamento macrofagico. Nell’ambito della nefropatia diabetica, l’interleuchina-6 (IL-6) risulta rivestire un ruolo rilevante. Si è quindi deciso di misurare i livelli di mRNA di IL-6 in seguito a trattamenti con AGE e LZ. In conformità alle nostre aspettative, gli AGE hanno indotto un incremento statisticamente significativo dei livelli di mRNA di IL-6 dopo 24 h di trattamento. L’esposizione al LZ non ha indotto modificazione, mentre il co-trattamento con AGE e LZ ha evidenziato la capacità del LZ di prevenire l’incremento dei livelli di IL-6, mantenendoli a valori comparabili a quelli dei controlli. Per confermare l’attività del LZ nei confronti di IL-6 AGE-indotto, è stato condotto un saggio ELISA che ha rafforzato i dati ottenuti mediante RT-PCR: il LZ è in grado di ridurre, in maniera dose-dipendente e statisticamente significativa, la quantità di IL-6 rilasciata AGE-indotta. I meccanismi che portano al rilascio di IL-6 sono molteplici. Nell’arco di questa ricerca, ci si è focalizzati su alcuni eventi direttamente correlati all’incremento del rilascio di IL-6. Visto il ruolo chiave attribuito al recettore RAGE negli effetti AGE-indotti, si è deciso di testare la capacità del LZ di influenzare i livelli di mRNA di RAGE, mediante RT-PCR. I risultati non hanno evidenziato variazioni dei livelli di RAGE dopo esposizione al LZ a 24 e 96 h. L’induzione delle ROS rappresenta uno degli eventi notoriamente attribuibili ad un’azione AGE-indotta. Tuttavia, nel modello cellulare da noi utilizzato, questo parametro non ha mostrato innalzamenti rispetto al controllo negativo, pur mostrandosi sensibile al trattamento con ABAP (induttore di ROS di riferimento). Dal momento che l’attività del LZ è associata anche alla sua azione di scavenger degli AGE si è deciso di investigare, mediante microscopia confocale, se la presenza del LZ potesse influenzare la capacità degli AGE di penetrare all’interno della cellula. Le immagini acquisite con questa tecnica hanno permesso di stabilire che gli AGE e il LZ sono in grado di penetrare attraverso la membrana cellulare. Per quel che concerne il co-trattamento sono stati effettuati alcuni studi di citometria a flusso. I risultati suggeriscono che il LZ non è in grado, in questo modello cellulare, di ridurre l’ingresso degli AGE nella cellula, escludendo che l’azione di scavenger sia alla base della prevenzione della produzione di IL-6 in vitro. Successivi studi, sono stati effettuati su due possibile vie coinvolte negli effetti AGE-indotti, quali la cascata delle MAPk, mediante p38, e la via di degradazione lisosomiale. I dati preliminari ottenuti hanno evidenziato un possibile ruolo i p38. Gli AGE si sono dimostrati capaci di incrementare i livelli di fosforilazione di questa proteina, effetto ridotto dalla contemporanea presenza di una dose equimolare di LZ. Promettenti sono anche i primi risultati ottenuti dallo studio di co-localizzazione degli AGE, in presenza di LZ, nei lisosomi. Le immagini acquisite confermano, infatti, l’ipotesi che il LZ sia in grado di veicolare gli AGE nei lisosomi, favorendone la degradazione. Nel loro insieme i risultati ottenuti non escludono che l’azione del LZ nei confronti degli AGE e dei suoi effetti sia prettamente di tipo antidotico e di scavenging. Tuttavia, la capacità del LZ di agire su un mediatore chiave degli effetti AGE-indotti come IL-6 e p38, aprono una nuova finestra di studio per quel che concerne il LZ, permettendo di ipotizzare un suo possibile impiego come co-adiuvante per il controllo dell’infiammazione nella nefropatia diabetica. Al fine di investigare se la maggior efficacia del lisozima microincapsulato sia dovuta alla capacità dei microsistemi di veicolare il LZ, nel torrente circolatorio di quanto non avvenga con la forma libera è stato messo a punto un modello in vivo nell’ambito del quale il LZ è stato somministrato oralmente microincapsulato oppure libero. Nel corso di tale prova è stato dimostrato che i microsistemi sono capaci di veicolare il lisozima a livelli serici superiori di quanto non avvenga dopo somministrazione di un’equidose di LZ in forma libera. Questi risultati dimostrano l’efficacia dei microsistemi orali per la veicolazione in circolo di principi biologicamente attivi

It is well established that protein glycosylation, a bioorthogonal process, is a posttranslational modification that profoundly affects protein folding, stability, immunogenicity, and biological properties and activities. In this regard, while native O- and N-glycosidic bonds of glycopeptides are prone to hydrolytic cleavage by O- and N-glycosidases, synthetic C- and S-analogs are expected to be stable toward such enzymatic degradation. Therefore, much effort has been devoted in the last decades to synthesizing C- and S-glycosyl amino acids and their assembly in glycopeptides. They with these non-native linkages can be used as probes for biochemical studies and leads in drug discovery, such as, for example, vaccines. In the first part of this thesis project we developed two routes to synthesis of S-glycopeptides. One route consists of S-glycosyl amino acid synthesis from photoinduced addition of sugar thiols to alkenyl glycine (TEC) followed by incorporation of this amino acid into a peptide. The second route, that is, specific for a cysteine containing peptide such as glutathione, involves peptide S-homoallylation followed by TEC with sugar thiol. We also demonstrated the selective propargylation of cysteine-containing peptides followed by photoinduced thiol-yne coupling with glycosyl thiols as an effective one-pot two-step platform for the dual glycosylation of peptides. By this strategy, we also performed the sequential glycosylation and biotinylation of peptides. In recent years, N-heterocyclic carbenes (NHCs) have attracted considerable interest due to their unique features, which allow them to be used as ligands for organometallic catalysis, reagents in the synthesis of heterocycles, and efficient organocatalysts in umpolung transformations. In the latter sub-area of research many efforts have been devoted to the realization of highly stereoselective versions of the classical benzoin and Stetter reactions through optimal pre-catalyst design, to the discovery of new transformations, and to the umpolung of electrophiles alternative to aldehydes and pyruvates, mainly acylsilanes and Michael acceptors. In this thesis, our group has recently demonstrated the capability of linear and cyclic dialkyl α-diketones to undergo polarity reversal under thiazolium carbene catalysis in benzoin-type and Stetter reactions, and thus act as a novel class of acyl anion precursors. Contrarily, it has been observed that diaryl α-diketones do not undergo polarity reversal in the presence of (benzo)thiazolium carbenes but are engaged in a novel multicomponent reaction with water to efficiently give medicinally relevant 1,4-thiazin-3-one heterocycles. The umpolung reactivity of diaryl 1,2-diones, however, can be effectively triggered by different NHCs.

The main purpose of this thesis was to develop peptide probes to detect and isolate specific and high affinity antibodies from sera of patients suffering from multiple sclerosis (MS). We selected a di-glucosylated adhesin HMW1 peptide as the shortest sequence up to now able to compete with the highest affinity with anti-N(Glc) IgM binding. 40 kDa dextran was modified with propargyl groups and used as a scaffold to conjugate by CuAAC the di-glucosylated peptide. This novel polymeric structure was proven to dramatically increase binding potency of IgGs and IgMs in MS sera. Abs from a representative MS serum, were successfully purified on a sepharose resin specifically modified with the adhesin peptide-dextran conjugate, as confirmed by ELISA. This result appears promising as a proof-of-concept of the selective removal of circulating autoantibodies (possibly perpetuating nonself recognition) that could likely lead to develop a specific apheresis-based device.