Assembly of neuronal networks with distinct patterns of connectivity during nervous system development involves the growth, extension and branching of axons and dendrites. Over the years genetic and biochemical studies in model organisms have contributed significantly in identifying mechanisms regulating axon growth and extension. However the molecular mechanisms underlying axon branching remain unclear. The egg-laying neuronal circuitry in C. elegans has proven to be a robust system for identifying and characterizing novel genes involved in neuronal morphology. This circuitry which mediates egg-laying behavior in nematodes is composed of two families of motorneurons, HSNs and VCs, which are among the most branched neurons in C. elegans. A genetic screen for axon branch defects in the egg-laying neurons identified png-1 to disrupt neuronal morphology including axon branching. png-1 encodes a Peptide: N-glycanase (PNGase), a conserved cytosolic enzyme that removes N-linked sugar moieties from glycoproteins. In this thesis I present my work characterizing and examining png-1 and its role in mediating axon branching. Mutations in png-1 resulted in excessive ectopic axon branching in the VC4 and VC5 egg-laying neurons as well as branching in the normally unbranched AVL and DVB neurons. Behavioral analysis in these mutants revealed defects in egg-laying behavior and mild in-utero egg retention phenotypes. Cellular characterization shows ubiquitous expression of png-1 in many tissues including vulva cells, muscles, gonads, and neurons. My analysis also shows that png-1 acts both cell-autonomously and cell non-autonomously from neurons and epithelial cells to restrict axon branching around the vulva. Using a candidate gene approach I identified a deletion allele of the DNA repair gene, rad-23, to display axon branching defects and interact with png-1 within a common pathway to regulate axon branching. Additionally, through a genetic modifier screen for enhancers and suppressors of VC4-5 branching defects in png-1, I identified a new allele of sax-2 as an enhancer mutation. sax-2 encodes a scaffolding protein that regulates the activity and localization of sax-1, an NDR kinase. Examination of neuronal phenotypes in sax-1 and sax-2 mutants revealed similar png-1 like defects in VC4-5. Genetic analysis of the double mutants png-1;sax-1 and png-1;sax-2 revealed strong synergistic phenotypes suggesting that png-1 and sax-1/sax-2 function in parallel pathways to regulate axon branching. In summary, this thesis reveals novel components and pathways in the regulation of neuronal branching.

Este trabalho procurou avaliar algumas isozimas envolvidas com o processo de degradação de polissacarídeos da parede celular vegetal, encontradas em Aspergillus versicolor, fungo pertencente a um gênero onde a multiplicidade de componentes enzimáticos com a mesma atividade oscila de uma a três. Duas ß-xilosidases foram purificadas por meio de DEAE-celulose e precipitação com sulfato de amônia. Ambas mostraram-se enzimas que, quando deglicosiladas por PNGaseF, apresentam mesmos mapas trípticos. A glicosilação mostrou-se importante para a manifestação de diferenças bioquímicas relacionadas às interações com ambiente eletrolítico adjacente, visto as mudanças das curvas de pH e alterações de comportamento frente a sais de íons metálicos, tão bem como para a manutenção do funcionamento das enzimas. Também foi verificado que as diferentes formas glicosiladas periplásmicas são produzidas em meios distintos (xilana e xilose), cujos pH finais corresponderam aos pH das enzimas encontradas. Por meio de zimogramas, verificou-se que estas não eram produzidas de imediato, mas selecionadas ao longo do tempo de cultivo. Esta seleção foi inicialmente independente do pH, visto este, mesmo tamponado, ser corrigido pelo fungo, de modo a se apresentar, ao final de 48h, correlato com o da enzima selecionada. Exame cromatográfico em Concanavalina-A das enzimas deglicosiladas por EndoH mostraram que a oriunda de xilana tem aporte maior de ramificações biantenárias, que são ricas em galactose. Ambas apresentaram mapas trípticos iguais. A ß-xilosidase induzida por xilana apresentou proporções de manose, galactose e glucose iguais a 69,68 : 30,25 : 0,056 %, respectivamente, enquanto que ß-xilosidase induzida por xilose apresentou proporções iguais a 85,35 : 14,54 : 0,094%, respectivamente, resultado que corrobora com a resultado anterior. Duas ß-glucosidases de superfície micelial foram purificadas por meio de DEAE-Sephacel e DEAE-celulose. Ambas mostraram-se mais semelhantes entre si que as ß-xilosidases, e independentes de fonte de carbono ou pH. No entanto, apresentaram diferenças frente à inibição por celobiose (acentuada a partir de 10mM para ß-glucosidase I e 20mM para B-glucosidase II) e, embora sutis, a pH. Após serem submetidas a 45 ºC por 30min (temperatura que induziu segunda conformação estável) mostraram curvas de pH muito distintas, que foram eliminadas nas enzimas submetidas ao mesmo experimento após deglicosilação por PNGaseF. Ambas apresentaram mapas trípticos iguais. A ß-glucosidase I mostrou-se constituída de Man:Gal:Glu em proporções iguais a 78,36%:21,61%:0,033% do carboidrato total, respectivamente, e a ß-glucosidase II, Man:Gal:Glu iguais a 83,59%:16,38%:0,028%, respectivamente. Ambas também apresentaram sinergismo quando juntas. Sobre celobiose, foi verificado apenas em pH acima de 5.5. Sobre celooligossacarídeos, manifestou-se em pH 5,25. A ß-glucosidase I foi menos ativa que a ß-glucosidase II quando em ausência de glucose e celobiose na solução de reação inicial, situação na qual o contrário foi verdadeiro. O sinergismo foi drasticamente eliminado após deglicosilação por PNGaseF. Não se sabe se este resultado foi oriundo de mudanças cinéticas provocadas pela deglicosilação ou de uma possível eliminação de agregação anteriormente existente entre glicosiladas. O componente ß-glicosidásico extracelular foi purificado por meio de DEAE-Sephacel e Octyl-Sepharose, e se revelou um heteroagregado de fosfatase ácida com ß-glicosidase, que se mostrou estável e ativo em ampla gama de temperaturas e pH, com ótimos de 55ºC e 5,5, respectivamente. Entretanto, os perfis das curvas foram distintos entre as enzimas componentes. Somente concentrações superiores a 0,8mM de cloreto de cobalto apresentaram efeito desagregador, podendo ser empregado em conjunto com DEAE-celulose para purificação das enzimas isoladas. Quando separadas, fosfatase mostrou-se 260% mais ativa e ß-glicosidase, 50% menos ativa. Nesta condição, as faixas de pH se restringiram, acidificando-se, localizando-se entre pH 4,0 e 5,5. Aparentemente, a agregação reforça a atividade celobiásica. Fosfatase foi ativa sobre farelo de trigo e fitato de sódio e adsorveu fortemente em farelo de trigo. ß-glicosidase apresentou atividade sobre farelo de trigo, xilana e Avicel (liberando apenas glicose desta última), revelando-se enzima com atividade celobioidrolásica inespecífica, embora ávida por celobiose. As atividades foram determinadas como oriundas do mesmo sítio catalítico. ß-glicosidase não foi seqüestrada por farelo de trigo quando desagregada, resultado invertido quando reagregada. O seqüestro por farelo de trigo, aparentemente, deveu-se a interações entre sítio catalítico da fosfatase e substrato. / This study explored enzyme multiplicity on hemicelllulose and cellulose degrading systems. It first demonstrates the differences of PNGaseF deglycosylation and EndoH deglycosylation on forms of two ß-glicosidase activities present on surface of mycelia from Aspergillus versicolor grown on several carbon sources. Aspergillus versicolor produces ß-xylosidases with different biochemical properties and different degree of glycosylation, when grown on xylan or xylose. Were investigated the biochemical properties of these ß-xylosidases after deglycosylation. The purified enzymes were deglycosylated with endo-H or PNGase F. After this treatment both enzymes migrated faster in PAGE exhibiting the same Rf. On SDS-PAGE both enzymes showed similar migration. The optima temperature of xylan-induced and xylose-induced ß-xylosidases was 45 ºC and 40 ºC, respectively, and of 35 ºC after deglycosylation. The xylan-induced enzyme was more active at acidic pH than the xylose-induced enzyme. After deglycosylation the optimum pH of both enzymes was 6.0. The thermal resistance of the enzymes at 55 ºC showed a half-life of 15 min and 9 min for xylose-and xylan-induced enzymes, respectively. After deglycosylation both exhibited half-lives of 7.5. Native enzymes exhibited different response to ions, while deglycosylated enzymes exhibited identical sensitivity to ions. Limited proteolysis yielded coincident profiles in SDS-PAGE for both deglycosylated enzymes. All data suggest that the two A.versicolor ß-xylosidases share a common polypeptide core with differential glycosylation, apparently responsible for their biochemical and biophysical differences. Aspergillus versicolor also produced ß-glucosidases with different biochemical properties and different degree of glycosylation independently of carbon source. ß-Glucosidase I differed from ß-glucosidase II principally considering the amount and composition of carbohydrate, sensitivity to ions and pH. The purified enzymes shared the same tripitic maps and molecular masses after deglycosylations. All results showed that the biochemical differences observed for two enzymes were directly linked to PNGaseF- deglycosylation. Considering that Rfs, elution profiles on Con-A and residual glycosylation of both enzymes treated with EndoH or PNGaseF were the same, but differed on the mannose/galactose ratio, we inferred differences on proportion of hybrid-type/high-mannose-type glycans. The significance of this glycoform diversity was stressed in analysis of the action of mixture of both ß-glucosidases on celooligosoccharides and on cellobiose. This synergism was abolished after PNGaseF deglycosylation. These results are the first to show synergism between glycoforms of glycosil-hydrolases, representing a new class of synergistic type. The work also described a new form of aggregation between enzymes. Generally, ß-glycosidases are described as soluble components, attached to cell wall or free in the culture medium. This work verified that it could be extracelular adsorbed to wheat straw when aggregated with an acid phosphatase. The results strongly suggested that phosphatase is the component responsible for the process of adsorption on the substrate. The disaggregation was cobalt mediated, being not observed for another ions. The aggregation state has positive effects on glycosidase activity, extending pH ratio and increasing hydrolysis velocity. The opposite was found to phosphatase activity.

Este trabalho procurou avaliar algumas isozimas envolvidas com o processo de degradação de polissacarídeos da parede celular vegetal, encontradas em Aspergillus versicolor, fungo pertencente a um gênero onde a multiplicidade de componentes enzimáticos com a mesma atividade oscila de uma a três. Duas ß-xilosidases foram purificadas por meio de DEAE-celulose e precipitação com sulfato de amônia. Ambas mostraram-se enzimas que, quando deglicosiladas por PNGaseF, apresentam mesmos mapas trípticos. A glicosilação mostrou-se importante para a manifestação de diferenças bioquímicas relacionadas às interações com ambiente eletrolítico adjacente, visto as mudanças das curvas de pH e alterações de comportamento frente a sais de íons metálicos, tão bem como para a manutenção do funcionamento das enzimas. Também foi verificado que as diferentes formas glicosiladas periplásmicas são produzidas em meios distintos (xilana e xilose), cujos pH finais corresponderam aos pH das enzimas encontradas. Por meio de zimogramas, verificou-se que estas não eram produzidas de imediato, mas selecionadas ao longo do tempo de cultivo. Esta seleção foi inicialmente independente do pH, visto este, mesmo tamponado, ser corrigido pelo fungo, de modo a se apresentar, ao final de 48h, correlato com o da enzima selecionada. Exame cromatográfico em Concanavalina-A das enzimas deglicosiladas por EndoH mostraram que a oriunda de xilana tem aporte maior de ramificações biantenárias, que são ricas em galactose. Ambas apresentaram mapas trípticos iguais. A ß-xilosidase induzida por xilana apresentou proporções de manose, galactose e glucose iguais a 69,68 : 30,25 : 0,056 %, respectivamente, enquanto que ß-xilosidase induzida por xilose apresentou proporções iguais a 85,35 : 14,54 : 0,094%, respectivamente, resultado que corrobora com a resultado anterior. Duas ß-glucosidases de superfície micelial foram purificadas por meio de DEAE-Sephacel e DEAE-celulose. Ambas mostraram-se mais semelhantes entre si que as ß-xilosidases, e independentes de fonte de carbono ou pH. No entanto, apresentaram diferenças frente à inibição por celobiose (acentuada a partir de 10mM para ß-glucosidase I e 20mM para B-glucosidase II) e, embora sutis, a pH. Após serem submetidas a 45 ºC por 30min (temperatura que induziu segunda conformação estável) mostraram curvas de pH muito distintas, que foram eliminadas nas enzimas submetidas ao mesmo experimento após deglicosilação por PNGaseF. Ambas apresentaram mapas trípticos iguais. A ß-glucosidase I mostrou-se constituída de Man:Gal:Glu em proporções iguais a 78,36%:21,61%:0,033% do carboidrato total, respectivamente, e a ß-glucosidase II, Man:Gal:Glu iguais a 83,59%:16,38%:0,028%, respectivamente. Ambas também apresentaram sinergismo quando juntas. Sobre celobiose, foi verificado apenas em pH acima de 5.5. Sobre celooligossacarídeos, manifestou-se em pH 5,25. A ß-glucosidase I foi menos ativa que a ß-glucosidase II quando em ausência de glucose e celobiose na solução de reação inicial, situação na qual o contrário foi verdadeiro. O sinergismo foi drasticamente eliminado após deglicosilação por PNGaseF. Não se sabe se este resultado foi oriundo de mudanças cinéticas provocadas pela deglicosilação ou de uma possível eliminação de agregação anteriormente existente entre glicosiladas. O componente ß-glicosidásico extracelular foi purificado por meio de DEAE-Sephacel e Octyl-Sepharose, e se revelou um heteroagregado de fosfatase ácida com ß-glicosidase, que se mostrou estável e ativo em ampla gama de temperaturas e pH, com ótimos de 55ºC e 5,5, respectivamente. Entretanto, os perfis das curvas foram distintos entre as enzimas componentes. Somente concentrações superiores a 0,8mM de cloreto de cobalto apresentaram efeito desagregador, podendo ser empregado em conjunto com DEAE-celulose para purificação das enzimas isoladas. Quando separadas, fosfatase mostrou-se 260% mais ativa e ß-glicosidase, 50% menos ativa. Nesta condição, as faixas de pH se restringiram, acidificando-se, localizando-se entre pH 4,0 e 5,5. Aparentemente, a agregação reforça a atividade celobiásica. Fosfatase foi ativa sobre farelo de trigo e fitato de sódio e adsorveu fortemente em farelo de trigo. ß-glicosidase apresentou atividade sobre farelo de trigo, xilana e Avicel (liberando apenas glicose desta última), revelando-se enzima com atividade celobioidrolásica inespecífica, embora ávida por celobiose. As atividades foram determinadas como oriundas do mesmo sítio catalítico. ß-glicosidase não foi seqüestrada por farelo de trigo quando desagregada, resultado invertido quando reagregada. O seqüestro por farelo de trigo, aparentemente, deveu-se a interações entre sítio catalítico da fosfatase e substrato. / This study explored enzyme multiplicity on hemicelllulose and cellulose degrading systems. It first demonstrates the differences of PNGaseF deglycosylation and EndoH deglycosylation on forms of two ß-glicosidase activities present on surface of mycelia from Aspergillus versicolor grown on several carbon sources. Aspergillus versicolor produces ß-xylosidases with different biochemical properties and different degree of glycosylation, when grown on xylan or xylose. Were investigated the biochemical properties of these ß-xylosidases after deglycosylation. The purified enzymes were deglycosylated with endo-H or PNGase F. After this treatment both enzymes migrated faster in PAGE exhibiting the same Rf. On SDS-PAGE both enzymes showed similar migration. The optima temperature of xylan-induced and xylose-induced ß-xylosidases was 45 ºC and 40 ºC, respectively, and of 35 ºC after deglycosylation. The xylan-induced enzyme was more active at acidic pH than the xylose-induced enzyme. After deglycosylation the optimum pH of both enzymes was 6.0. The thermal resistance of the enzymes at 55 ºC showed a half-life of 15 min and 9 min for xylose-and xylan-induced enzymes, respectively. After deglycosylation both exhibited half-lives of 7.5. Native enzymes exhibited different response to ions, while deglycosylated enzymes exhibited identical sensitivity to ions. Limited proteolysis yielded coincident profiles in SDS-PAGE for both deglycosylated enzymes. All data suggest that the two A.versicolor ß-xylosidases share a common polypeptide core with differential glycosylation, apparently responsible for their biochemical and biophysical differences. Aspergillus versicolor also produced ß-glucosidases with different biochemical properties and different degree of glycosylation independently of carbon source. ß-Glucosidase I differed from ß-glucosidase II principally considering the amount and composition of carbohydrate, sensitivity to ions and pH. The purified enzymes shared the same tripitic maps and molecular masses after deglycosylations. All results showed that the biochemical differences observed for two enzymes were directly linked to PNGaseF- deglycosylation. Considering that Rfs, elution profiles on Con-A and residual glycosylation of both enzymes treated with EndoH or PNGaseF were the same, but differed on the mannose/galactose ratio, we inferred differences on proportion of hybrid-type/high-mannose-type glycans. The significance of this glycoform diversity was stressed in analysis of the action of mixture of both ß-glucosidases on celooligosoccharides and on cellobiose. This synergism was abolished after PNGaseF deglycosylation. These results are the first to show synergism between glycoforms of glycosil-hydrolases, representing a new class of synergistic type. The work also described a new form of aggregation between enzymes. Generally, ß-glycosidases are described as soluble components, attached to cell wall or free in the culture medium. This work verified that it could be extracelular adsorbed to wheat straw when aggregated with an acid phosphatase. The results strongly suggested that phosphatase is the component responsible for the process of adsorption on the substrate. The disaggregation was cobalt mediated, being not observed for another ions. The aggregation state has positive effects on glycosidase activity, extending pH ratio and increasing hydrolysis velocity. The opposite was found to phosphatase activity.

For a long time, the scientific community underestimated the value of carbohydrates and the approach of most scientists to the complex world of glycans was apprehensive. The scenario, however, has changed today. With the development of new research tools and methodologies the study of carbohydrates and glycoconjugates has progressed rapidly, increasing our understanding of these molecules. Carbohydrates are most abundant amongst biological polymers in nature and vital for life processes. In their simplest form, they serve as a primary source of energy to most living organisms. In generalis, they exist as complex structures (glycans), and as conjugates of protein (glycoproteins, proteoglycans), lipids (glycolipids) and nucleosides (UDP-Glucose). Defined in the broadest sense, the study of glycans in all their forms and their interacting partners is termed “Glycobiology”. Glycans are ubiquitously found in nature decorating cells of almost all types with a “sugar coat”. They are also present within the cytoplasm, as well as in the extra-cellular matrix. They have key roles in a broad range of biological processes, including signal transduction, cell development and immune responses. All living organisms have evolved to express proteins that recognize discrete glycans and mediate specific physiological or pathological processes. One major class of such proteins is “Lectins”. Found in all forms of life, they are characterized by their ability to recognize carbohydrates. They are proteins of non-immune origin that bind glycans reversibly with a high degree of stereo-specificity in a non-catalytic manner. It must be emphasized that they are a different class from glycan-specific antibodies. Lectins were first discovered in plants and a large amount of work has been carried out on plant lectins to decipher their structural organization, mode of interaction with substrate and as models to study protein stability and folding. Study on animal and microbial lectins, on the other hand, gathered momentum only recently. In spite of this, more is known about their function in animals and micro-organisms rather than in plants. Lectin-glycan binding is implicated in several important biological processes such as protein folding, trafficking, host-pathogen interactions, immune cell responses and in malignancy and metastasis. Most lectins have one or more carbohydrate recognition domains (CRDs) which often share either 3-D structural features or amino acid sequence. New members of a family can be identified using either sequence or structural homology. Interestingly, it turns out that several plant and microbial lectins have structural or sequential similarity with animal lectins , revealing that these CRDs are evolutionarily related. This thesis, entitled “Structural, Biophysical and Biochemical Studies on Mannose-specific Lectins”, focuses on three lectins, Banana lectin (Banlec), Calreticulin (CRT) and Peptide-N-Glycanase (PNGase). Although all three lectins have distinct biological functions, they share a common ligand specificity at the monosaccharide level i.e. mannose. This thesis, besides characterizing these lectins, studies in detail, the difference in the mode of interaction with their ligands. Chapter 1 is a general introduction on lectins, glycan-lectin interactions and the various techniques that are employed to characterize these interactions. Several principles have emerged about the nature of glycan–lectin interactions. It has been observed that the binding sites for low molecular weight glycans are of relatively low affinity (Kd values in the high micromolar to low millimolar range). Selectivity is mostly achieved via a combination of hydrogen bonds and by van der Waals packing of the hydrophobic faces of monosaccharide rings against aromatic amino acid side chains. Further selectivity and enhanced affinity can be achieved by additional contacts between the glycan and the protein. It is notable that the actual region of contact between the saccharide and the polypeptide typically involves only one to three monosaccharide residues. As a consequence of all of the above, these lectin-binding sites tend to be of relatively low affinity, although they can exhibit high specificity. It is intriguing to observe that such low-affinity sites have the ability to mediate biologically relevant interactions. There are many different ways to study binding of glycans to proteins, and each approach has its advantages and disadvantages in terms of thermodynamic rigor, amounts of protein and glycan needed, and the speed of analysis. In examining these interactions, two broad categories of techniques are applied: (1) kinetic and near-equilibrium methods, such as titration calorimetry; and (2) non-equilibrium methods such as glycan microarray screening and ELISA-based approaches. Two of the most widely used biophysical approaches for examining glycan-lectin interactions at the molecular level are X-ray crystallography and nuclear magnetic resonance (NMR). However, as small molecules often co-crystallize with a lectin better than large molecules, a lot of our knowledge about glycan–lectin interactions at the atomic level is based on co-crystals of lectins with unnatural ligands. Thus, a great challenge exists in attempting to understand glycan–lectin interactions in the context of natural glycans present as glycoproteins, glycolipids, or proteoglycans. Chapter 2 introduces Banana lectin and describes the stability studies carried out. The unfolding pathway of Banlec was determined using GdnCl induced denaturation. Analysis of isothermal denaturation provided information on its conformational stability and the high values of ΔG of unfolding at various temperatures indicated the strength of inter-subunit interactions. It was found that Banlec is a very stable protein and denatures only at high chaotrope concentrations. The basis of the stability may be attributed to strong hydrogen bonds at the dimeric interface along with the presence of water bridges. This is a very unique example in proteins where subunit association is not a consequence of the predominance of hydrophobic interactions. High temperature molecular dynamics simulations have been utilized to monitor and understand early stages of thermally induced unfolding of Banlec. The present study investigates the behavior of the dimeric protein at four different temperatures. The process of unfolding was monitored by monitoring the radius of gyration, the rms deviation of each residue, change in relative solvent accessibility and the pattern of inter- and intra-subunit interactions. The overall study demonstrates that the Banlec dimer is a highly stable structure, the stability in most part contributed by interfacial interactions. The pattern of hydrogen bonding within the subunits and at the interface across different stages has been analyzed and has provided the rationale for its intrinsic high stability. In Chapter 3 the conformational and dynamic behaviour of three mannose containing oligosaccharides, a tetrasaccharide with α1→2, and α1→3, and a penta- and a heptasaccharide with α1→2, α1→3, and α1→6 linkages has been evaluated. Molecular mechanics, molecular dynamics simulations and NMR spectroscopy methods were used for evaluation. It is found that they display a fair amount of conformational freedom, with one major and one minor conformation per glycosidic linkage. The evaluation of their recognition by Banlec has been performed by STD NMR methods and a preliminary view of their putative interaction mode has been carried out by means of docking procedures. In Chapter 4 the conformational behaviour of three mannose containing oligosaccharides, namely, the α1→3[α1→6] trisaccharide, the heptasaccharide with α1→2, α1→3, and α1→6 linkages and the tetrasaccharide consisting of α1→3 and α1→2 linkages, when bound to Banlec has been evaluated by trNOE NMR methods and docking calculations. It is found that the molecular recognition event involves a conformational selection process, with only one of the conformations, among those available to the sugar in free state, being recognised at the lectin binding site. It is known that many proteins, including members of the Jacalin-related lectin family (of which Banlec is a member), bind the high-mannose saccharides found on the surface of the HIV-associated envelope glycoprotein, gp120, thus interfering with the viral life cycle, potentially providing a manner of controlling a variety of infections, including HIV. These proteins are thought to recognize the high-mannose type glycans with subtly different structures, although the precise specificities are yet to be clarified. This study was carried out to gain a better understanding of these protein-carbohydrate recognition events. Chapter 5 reports interactions of Calreticulin (CRT) with the trisaccharide Glcα1-3Manα1-2Man. Previously in our laboratory it was established using modeling studies the residues in CRT important for sugar binding. Here, the relative roles of Trp-319, Asp-317 and Asp-160 for sugar binding have been explored by using site-directed mutagenesis and isothermal titration calorimetry (ITC). Residues corresponding to Asp-160 and Asp-317 in calnexin (CNX) are known to play important roles in sugar binding. The present study demonstrates that Asp-160 is not involved in sugar binding, while Asp-317 plays a crucial role. Further, it is also validated that hydroxyl-pi interactions of the sugar with Trp-319 dictate sugar binding in CRT. This study defines further the binding site of CRT and also highlights its subtle differences with that of CNX. Additionally, mono-deoxy analogues of the trisaccharide unit Glcα1-3Manα1-2Man have been used to determine the role of various hydroxyl groups of the sugar substrate in sugar-CRT interactions. Using the thermodynamic data obtained by carrying out ITC of CRT with these analogues, it is demonstrated that the 3-OH group of Glc1 plays an important role in sugar-CRT binding, whereas the 6-OH group does not. Also, the 4-OH, 6-OH of Man2 and 3-OH, 4-OH of Man3 in the trisaccharide are involved in binding, of which 6-OH of Man2 and 4-OH of Man3 have a more significant role to play. Therefore, the interactions between the substrate sugar of glycoproteins and the lectin chaperone CRT are further delineated. Chapter 6 introduces Peptide-N-Glycanase (PNGase) and delineates the various interactions involved in the binding of oligomannose structures of glycoproteins to the C-terminal domain (the carbohydrate recognition module) of PNGase. ITC is used to characterize the interaction to oligosaccharides in terms of affinity, stoichiometry, enthalpy, entropy and heat capacity changes with the mouse PNGase C-terminal domain. Using the thermodynamic data obtained, it was determined that PNGase requires the tri-mannoside moiety of the native glycan on glycoproteins as the basic minimum entity for recognition and binding. Additional mannose moieties on the glycan do not significantly interact with PNGase and therefore no enhancement in binding affinity is observed (unlike CRT) which is in concordance with its role of stripping glycans from misfolded glycoproteins targeted for degradation via the ERAD (Endoplasmic reticulum assisted degradation) pathway. Chapter 7 briefly summarizes all the findings of the research carried out and presents a comparative analysis of the three lectins studied. Appendix A: Protein folding in the ER is assisted by molecular chaperones. Lectin chaperones such as CRT and CNX assist the folding of glycoproteins by their N-linked oligosaccharide chains. Dynamic processing of the original glycan chain of (GlcNAc)2(Man)9(Glc)3 to remove the terminal glucose moieties is essential for accurate folding. Proteins that attain their native conformation are then transported to the Golgi complex for further glycan modifications. In case of aberrant folding the proteins are retrotranslocated into the cytosol, ubiquitinated, deglycosylated and degraded by the proteasome. Peptide-N-glycanase is a cytosolic enzyme that releases N-glycans from glycoproteins and glycopeptides. PNGase is now widely recognized as a major participant in protein quality control machinery for ERAD or the proteasomal degradation of retrotranslocated glycoproteins. It is therefore desirable to synthesize fluorescently labeled glycoprotein substrates which will provide direct understanding of how, when and where, the interaction between the substrate and the enzyme occurs. Towards this goal, cloning of GFP and RFP tagged full length mouse and human PNGase and CRT was carried out which is described in this section.