L'AE1 (SLC4A1) est un échangeur chlorure/bicarbonate. Cette protéine est la protéine membranaire la plus abondante à la surface des globules rouges des vertébrés. Elle est participe au transport du CO2 et à l'ancrage du cytosquelette à la membrane plasmique. Des mutations ponctuelles dans la partie membranaire de l'AE1, liées à des pathologies humaines, convertissent l'échange électroneutre en voie de conductance pour le sodium et le potassium ou induisent une fuite de cations dans un échangeur d'anions toujours fonctionnel. Les déterminants moléculaires qui induisent les mouvements d'ions au travers de cet échangeur sont encore inconnus. Le travail présenté a eu pour but d'identifier et de cartographier le site de transport de la protéine normale ou " pathologique ". Nous avons adapté à l'AE1 des outils basés sur la chimie des sulfhydriles capable de donner des informations sur le rôle d'acides aminés dans le site de transport de la protéine. Cette stratégie combinée à l'élaboration d'un modèle tridimensionnel de la protéine in silico basé sur le symporteur uracile/proton nous a permis de définir le site de transport de l'AE1. Nos résultats démontrent qu'un site de transport unique dans l'AE1 peut basculer entre 3 conformations différentes : échange chlorure/bicarbonate, fuite de cation et échange anionique ou fuite de cation uniquement. Ce site met en jeu les segments transmembranaires (TM) 3, 5 et 8 ainsi qu'une boucle intracellulaire très conservée située entre les TM 8 et 9. Le site de transport se structure autour des acides aminés L468, F471, L530, I533 et L673 se terminant au niveau du E681. La boucle intracellulaire 690 à 705 agissant comme un filtre à cations.

Milk derived ingredients from the production of cheese and butter can be used as vehicles for nutrients. Buttermilk is a nutritious product of milk that comes from the churning of cream into butter. One of the advantages of buttermilk is that it is enriched in milk fat globule components, such as phospholipids and forms emulsions with fat when treated with high shear. The objective of this work was to explore the effects of shear on regular buttermilk and whey buttermilk in terms of liposomal aggregate size and chemical composition. The effects of microfluidization at 2000 psi and homogenization at 2000 psi/500 psi on the particle size distribution of liposomal aggregates between whey buttermilk (WBM) at pH 4.6 and 6.8 and commercial sweet buttermilk (SBM) at pH 4.60 were compared with whey protein isolate (WPI) at pH 4.6. At pH 6.80, WPI and SBM are too soluble in water to measure particle size but WBM is not as soluble. From this investigation, the mean particle diameter of the SBM aggregates at pH 4.6 decreased after the first pass through the microfluidizer and the same is true, after homogenization. SBM aggregates at pH 4.6 had a significantly larger mean particle diameter before treatments in both shear processes compared to WPI at pH 4.6 and WBM at pH 4.6 and WBM at pH 6.8 (p < 0.0001). WPI at pH 4.6 and WBM at both pH showed no significant differences in their mean particle size in both homogenized and microfluidized treatments. WPI and SBM samples resulted in significant particle diameter differences vi from before to after homogenizing at pH 4.6. SBM at pH 4.6 had significantly larger average particle diameter than WBM at pH 4.6 (p < 0.0002), WPI at pH 4.6 (p < 0.0002) and WBM at pH 6.8 (p < 0.0045) before microfluidization at pass 0. WBM and WPI across all treatments showed very similar tendencies in small particle size attributes and some similarities in protein composition. In addition, the small aggregate size of WBM is suggested to be influenced by the presence of phospholipids and thus, creating significantly smaller mean particles compared to SBM even before inducing high shear. In contrast, treated and untreated SBM differed from WBM in phospholipid composition in both homogenization and microfluidization techniques. WBM samples contained more phospholipids than SBM, whereas WPI samples contained very low concentrations of phospholipids. Through HPLC analysis, WPI, SBM, and WBM showed different profiling of the phospholipid classes. These differences may be due structural changes of the aggregates from shearing, initial thermal treatments or hydrophobic and/or protein-phospholipid interactions between the aggregates. SBM samples also exhibited different protein profiling than WBM and WPI samples. This study suggests that high shear and presence of phospholipids impact the size distribution of liposomal aggregates through structural alterations. The aggregates can be utilized as a novel ingredient and in the processing of dairy foods to deliver nutrition.

La toxine diphtérique (DT) une toxine bactérienne sécrétée par Corynebacterium diphtheriae. Lors de l'intoxication d'une cellule, le domaine de translocation (T) de la DT s'insère dans la membrane à pH acide et assiste la translocation du domaine catalytique (C) dans le cytosol. Le domaine T adopte une conformation en molten globule (MG) et devient compétent pour l'interaction membranaire. Nous avons identifié par mutagenèse les résidus du domaine T dont la protonation favorise la formation de l'état MG en solution et l'interaction membranaire. Les résultats montrent que la protonation concertée des six histidines du domaine T est impliquée dans la formation de l'état MG en solution. La paire His223-257 et l'His251 ont un effet prépondérant dans la formation de l'état molten globule en solution, alors que la paire His322-323 (mais également His251) est davantage impliquée dans l'interaction avec la membrane, et en particulier la liaison à la membrane.<br />Nous avons étudié les changements de conformation des deux domaines C et T dans une protéine CT correspondant à la DT tronquée de son domaine R, afin de comprendre les effets du lien covalent sur les conformations respectives de C et T en fonction du pH. Les mutants CTW50/153F et CTW206/281F ont permis de suivre la fluorescence des Trp de chaque domaine dans la protéine CT. Les<br />résultats montrent que le domaine T dirige la translocation de C dans les premières étapes de la translocation (formation de l'état MG, liaison et insertion membranaire), et qu'il pourrait jouer un rôle de chaperon en stabilisant l'état MG du domaine C.

Vanderghem Caroline. (2009). La membrane du globule gras du lait : études physico-chimiques et sa valorisation technofonctionnelle dans les babeurres (Thèse de doctorat en Anglais). Gembloux, Belgique, Faculté Universitaire des Sciences Agronomiques de Gembloux. 198p., 14 tabl., 59 fig. Résumé La membrane du globule gras du lait (MGGL) est une structure complexe qui enveloppe, protège et délivre des composants bioactifs et des nutriments dune façon efficace au nouveau-né. Sa structure est unique par rapport à dautres systèmes biologiques de transport de lipides. Lobjectif général de ce travail a été de contribuer à augmenter les connaissances et la compréhension de cet émulsifiant naturel. La première partie de cette thèse concerne létude de la structure de la MGGL. Limportance des protéines membranaires concernant la stabilité et leur disposition dans la MGGL ont été étudiés. Les protéines de la MGGL sont présentes en petite quantité dans le lait et une technique de pré-fractionnement simpose afin de les isoler par rapport aux autres protéines du lait (caséines et protéines du lactosérum). Une technique dextraction qui a été développée dans notre laboratoire a été testée afin disoler la MGGL de la crème laitière. Une analyse détaillée a révélé que cette procédure est très bien adaptée pour lélimination dun maximum de protéines du lait écrémé. Une approche protéomique a été établie et a permis lidentification de protéines majeures de la MFGM ainsi que dautres protéines mineures (GTP-binding proteins, annexins, actin) afin de compléter le protéome. Par la suite, différentes protéases ont été testées en vue dobtenir différents degrés et/ou sélectivité dhydrolyse des protéines de la MGGL. La distribution asymétrique des protéines de la MGGL a été étudiée par une approche basée sur lattaque protéolytique du globule gras natif. Sur base de nos résultats et des données bibliographiques récentes, un nouveau modèle de la structure de la MGGL est proposé. La deuxième partie de cette thèse est consacrée à évaluer les propriétés technofonctionnelles de la MGGL dans les babeurres. Les propriétés interfaciales, moussantes et émulsifiantes du babeurre sont comparées à dautres ingrédients laitiers comme le lait écrémé et le concentré protéique de lactosérum. Nos résultats montrent le bon pouvoir émulsifiant du babeurre en comparaison des autres ingrédients laitiers. Dans les émulsions recombinées, la stabilité envers le crémage a été améliorée et les membranes ont présenté une meilleure résistance face à la coalescence. Les résultats sur la tension interfaciale et les propriétés rhéologiques interfaciales ont été mis en relation avec certaines propriétés des mousses et des émulsions. Vanderghem Caroline. (2009). The milk fat globule membrane: physico-chemical studies and its techno-functional valorisation in buttermilk (Thèse de doctorat). Gembloux, Belgium, Gembloux Agricultural University, 198p., 14 tabl., 59 fig. Summary The milk fat globule membrane (MFGM) is a complex structure that surrounds, protects and delivers bioactive compounds and nutrients in an efficient manner to the neonate. Its structure is unique in relation with any other biological lipid transport system. The overall aim of this work was to contribute to increase the knowledge and comprehension of this natural emulsifier. The first part of this thesis concerned the study of the structure of the MFGM. Membrane proteins were targeted and their importance regarding stability and disposition in the MFGM was studied. MFGM proteins are present at low concentrations in milk and a pre-fractionation of the sample is required in order of MFGM proteins to be visible among other milk proteins (caseins and whey proteins). A mild procedure developed in our laboratory was tested in order to isolate MFGM from cream. Detailed analysis revealed that this procedure is very well suited for the elimination of a maximum of skim milk proteins. A proteomic approach was established and allowed the identification of the most important MFGM proteins and additional minor MFGM proteins for additional completion of the proteome (GTP-binding proteins, annexins, actin). Subsequently, different proteases were screened in an attempt to obtain different degrees and/or selective proteolysis of MFGM proteins. Asymmetric arrangement of MFGM proteins was studied with an approach based on the proteolytic attack on the native fat globule. Based on our results and recent bibliographic data, an updated model of the MFGM structure was proposed. The second part of this thesis was devoted to assess the techno-functional properties of the MFGM in buttermilks. Interfacial, foaming and emulsifying properties of buttermilk were assessed and compared to classic milk ingredients such as skim milk and whey protein concentrate. Our results highlighted that buttermilk possesses good emulsifying power compared to other milk ingredients. In buttermilk recombined emulsions stability towards creaming was improved and the recombined membrane presented a great resistance to coalescence. Results of the interfacial pressure and the interfacial rheological properties were related to some foams and emulsions properties.

F4 positive enterotoxigenic Escherichia coli (ETEC) is the most common pathogen causing neonatal diarrhea in piglets. The pathogenesis requires the attachment of ETEC to the intestinal brush border, mediated by F4 fimbria. Colostral anti-F4 antibodies and some non-immunoglobulin porcine skim milk proteins can bind F4 and prevent colonization and infection by F4-positive ETEC. Little is known, however, about the F4-binding ability of porcine milk fat globule membrane (MFGM) proteins. In addition, the knowledge of the absorption of porcine colostral proteins into the blood of neonatal piglets is limited, despite the well accepted concept that in neonatal piglets, protein absorption from the intestine is non-selective.<p> In this study, the ability of porcine MFGM proteins to bind purified F4ac (one of the three subtypes of F4 fimbriae) was investigated. Porcine MFGM proteins were first separated by 2D SDS-PAGE and subsequently identified by mass spectrometry. Overlay western Blot was then employed to demonstrate the interaction between porcine MFGM proteins and purified F4ac. Several proteins from porcine MFGM reacted with F4ac, and of these, lactadherin, butyrophilin, adipophilin, and acyl-CoA synthetase 3 reacted strongly. The biological function of these proteins in vivo was not investigated but it is possible that their interaction with F4ac positive ETEC interferes with bacterial attachment and colonization. In order to investigate protein absorption by neonatal piglets after natural suckling, the protein profiles of the plasma of pre-suckling and 24 h post-suckling neonatal piglets were studied by 2D SDS-PAGE. Those plasma proteins that increased prominently after suckling were then identified by mass spectrometry. Only immunoglobulins were unequivocally determined to be absorbed, because they were absent before suckling and present in large quantity in plasma 24 h after suckling. The absorption of other colostral proteins was either equivocal or not detectable by our detection methods. These results suggest that, unlike immunoglobulins, major non-immunoglobulin proteins in porcine colostrum may not be absorbed into systemic circulation in substantial amounts.

F4 positive enterotoxigenic Escherichia coli (ETEC) is the most common pathogen causing neonatal diarrhea in piglets. The pathogenesis requires the attachment of ETEC to the intestinal brush border, mediated by F4 fimbria. Colostral anti-F4 antibodies and some non-immunoglobulin porcine skim milk proteins can bind F4 and prevent colonization and infection by F4-positive ETEC. Little is known, however, about the F4-binding ability of porcine milk fat globule membrane (MFGM) proteins. In addition, the knowledge of the absorption of porcine colostral proteins into the blood of neonatal piglets is limited, despite the well accepted concept that in neonatal piglets, protein absorption from the intestine is non-selective.<p> In this study, the ability of porcine MFGM proteins to bind purified F4ac (one of the three subtypes of F4 fimbriae) was investigated. Porcine MFGM proteins were first separated by 2D SDS-PAGE and subsequently identified by mass spectrometry. Overlay western Blot was then employed to demonstrate the interaction between porcine MFGM proteins and purified F4ac. Several proteins from porcine MFGM reacted with F4ac, and of these, lactadherin, butyrophilin, adipophilin, and acyl-CoA synthetase 3 reacted strongly. The biological function of these proteins in vivo was not investigated but it is possible that their interaction with F4ac positive ETEC interferes with bacterial attachment and colonization. In order to investigate protein absorption by neonatal piglets after natural suckling, the protein profiles of the plasma of pre-suckling and 24 h post-suckling neonatal piglets were studied by 2D SDS-PAGE. Those plasma proteins that increased prominently after suckling were then identified by mass spectrometry. Only immunoglobulins were unequivocally determined to be absorbed, because they were absent before suckling and present in large quantity in plasma 24 h after suckling. The absorption of other colostral proteins was either equivocal or not detectable by our detection methods. These results suggest that, unlike immunoglobulins, major non-immunoglobulin proteins in porcine colostrum may not be absorbed into systemic circulation in substantial amounts.

Les gouttes, les vésicules et les globules rouges sont des objets mous et déformables, structurés et de taille micrométrique (de 1 à 100 microns de diamètre). L'objectif de ce travail est de déterminer la participation de chaque paramètre mécanique des objets étudiés (tension de surface, élasticité, viscosité...) à partir de leur comportement sous écoulement. Nous avons choisi de nous intéresser tout particulierement à la réponse de ces objets sous écoulement confiné (ou semi-confiné) en utilisant une approche de type « microfluidique ». <br />La production de tensio-actif à la surface d'une goutte en mouvement influence sa forme et sa dynamique. L'étude détaille notamment les effets de la concentration et de la géométrie.<br />La mise au point de vésicules aux propriétés complexes modulables par l'action de la température est exposée. Nous avons aussi étudié l'impact de la transition sol/gel de la membrane lipidique de vésicules DMPC sur leur comportement sous champs externes (pression osmotique, écoulement...). <br />Dans le cas des globules rouges soumis à un cisaillement, deux types de mouvements sont connus : un mouvement de bascule et un mouvement de chenille de char. Nous avons mis en évidence l'existence d'un nouveau régime d'oscillations superposé au mouvement de chenille de char. Notre étude a également porté sur le comportement de globules rouges s'écoulant dans des canaux de dimension comparable a leur taille. Un diagramme de forme a été établi en fonction de la vitesse de l'objet, de la viscosité externe et de la section du capillaire. Nous avons développé un système mesurant la chute de pression associée au passage d'une cellule unique dans un canal de même dimension. Cette mesure permet notament de corréler le signal obtenu avec les propriétés physiques et mécaniques des objets étudiés. Nous avons illustré cette approche avec des globules blancs et des globules rouges. Nous nous sommes enfin intéressés au cas d'une suspension concentrée de cellules sanguines dans un écoulement pathologique: la thrombose.

The milk fat globule membrane (MFGM) obtained from bovine milk is a source of bioactive compounds. In this research, the hypothesis that the MFGM possesses anticarcinogenic capacity and immune modulatory properties was tested, along with the hypothesis that processing history of the fat globules will affect the bio-functionality of the MFGM. Throughout all experiments, lipopolysaccharide (LPS) was minimized by extracting the milk using a catheter, working under aseptic conditions and avoiding contamination via reagents. To study the anticarcinogenic capacity, native MFGM was tested in vitro using two colon adenocarcinoma cell lines (HT-29, Caco-2). Stimulated cells showed a dose-dependent decrease in cell proliferation, starting at very low concentrations (1 μg/mL based on protein) and its efficiency was comparable or even superior to the commercial anticarcinogenic drugs melphalan and C2-ceramide. Cytotoxicity and apoptosis were found to be mechanisms responsible for this bioactivity, as shown by the lactate dehydrogenase enzyme activity and the increase in caspase-3 production. MFGM- immune modulatory capacity was also tested by studying its effects on splenocyte proliferation, apoptosis and cytokine production. While splenocyte proliferation was not affected when the MFGM isolate was used alone, it suppressed cell division in the presence of polyclonal activators (LPS, Concanavalin A) used to simulate inflammatory conditions. Cytokine production suggests inhibition of the splenocytes’ activation process as the mechanism behind its bioactivity. Milk heating caused a significant decrease in bioactivity, indicating that protein interactions and denaturation as well as the structural changes caused by this treatment, directly affect the MFGM bio-functionality. Partial hydrolysis (by trypsin and phospholipase-A2) caused a similar effect, suggesting that not only the phospholipids are involved in the anticarcinogenic capacity, but also, the complex mixture of proteins which form part of the MFGM complete system. In summary, the bioactivity of the MFGM extracted from milk is not limited to its anticarcinogenic but also to its immune regulatory ability, by helping control the response of the immune system when inflammatory conditions arise. However, pre-treatment of the raw material greatly affects this bioactivity, suggesting that greater care may be needed in processing of fat globules to maintain such important characteristics of their components. / Canadian Dairy Council (CDC), Ontario Dairy Council (ODC), Natural Sciences and Engineering Research Council of Canada (NSERC).

Background Observational studies have shown that early nutrition influences short- and long-term health of infants. Formula-fed infants have higher protein and energy intakes and lower intakes of several biologically active components present in human milk. Some of these are present in the milk fat globule membrane (MFGM). The aim of the present study was to examine the effects of feeding term infants an experimental low energy low protein formula supplemented with bovine milk fat globule membranes. Our hypothesis was that infants fed experimental formula (EF), compared to infants fed standard formula (SF), would have outcomes more similar to a breast-fed reference (BFR) group. Methods In a double-blinded randomized controlled trial, 160 exclusively formula-fed, healthy, term infants were randomized to receive EF or SF from &lt;2 to 6 months of age. A BFR group consisted of 80 breast-fed infants. Measurements were made at baseline, 4, 6 and 12 months of age. The EF had lower energy (60 vs. 66 kcal/100 mL) and protein (1.20 vs. 1.27 g/100 mL) concentrations, and was supplemented with a bovine MFGM concentrate. Results At 12 months of age, the EF group performed better than the SF group in the cognitive domain of Bayley Scales of Infant Development, 3rd Ed. During the intervention, the EF group had a lower incidence of acute otitis media than the SF group, less use of antipyretics and the EF and SF groups differed in concentrations of s-IgG against pneumococci. The formula-fed infants regulated their intakes by increasing meal volumes. Thus, there were no differences between the EF and SF groups in energy or protein intakes, blood urea nitrogen, insulin or growth including body fat percent until 12 months of age. Pressure-to-eat score at 12 months of age was reported lower by parents of formula-fed infants than by parents of breast-fed infants, indicating a low level of parental control of feeding in the formula-fed groups. Neither high pressure-to-eat score nor high restrictive score was associated with formula feeding. During the intervention, the EF group gradually reached higher serum cholesterol concentrations than the SF group, and closer to the BFR group. At 4 months of age, there was no significant difference in the prevalence of lactobacilli in saliva between the EF and SF groups. Conclusions Supplementation of infant formula with a bovine MFGM fraction enhanced both cognitive and immunological development in formula-fed infants. Further, the intervention narrowed the gap in serum cholesterol concentrations between formula-fed and breast-fed infants. The lower energy and protein concentrations of the EF were totally compensated for by a high level of self-regulation of intake which might, at least partly, be explained by a low level of parental control of feeding in the study population. The findings are of importance for further development of infant formulas and may contribute to improved short- and long-term health outcomes for formula-fed infants.

L’AE1 (SLC4A1) est un échangeur chlorure/bicarbonate. Cette protéine est la protéine membranaire la plus abondante à la surface des globules rouges des vertébrés. Elle est participe au transport du CO2 et à l’ancrage du cytosquelette à la membrane plasmique. Des mutations ponctuelles dans la partie membranaire de l’AE1, liées à des pathologies humaines, convertissent l’échange électroneutre en voie de conductance pour le sodium et le potassium ou induisent une fuite de cations dans un échangeur d’anions toujours fonctionnel. Les déterminants moléculaires qui induisent les mouvements d’ions au travers de cet échangeur sont encore inconnus. Le travail présenté a eu pour but d’identifier et de cartographier le site de transport de la protéine normale ou « pathologique ». Nous avons adapté à l’AE1 des outils basés sur la chimie des sulfhydriles capable de donner des informations sur le rôle d’acides aminés dans le site de transport de la protéine. Cette stratégie combinée à l’élaboration d’un modèle tridimensionnel de la protéine in silico basé sur le symporteur uracile/proton nous a permis de définir le site de transport de l’AE1. Nos résultats démontrent qu’un site de transport unique dans l’AE1 peut basculer entre 3 conformations différentes : échange chlorure/bicarbonate, fuite de cation et échange anionique ou fuite de cation uniquement. Ce site met en jeu les segments transmembranaires (TM) 3, 5 et 8 ainsi qu’une boucle intracellulaire très conservée située entre les TM 8 et 9. Le site de transport se structure autour des acides aminés L468, F471, L530, I533 et L673 se terminant au niveau du E681. La boucle intracellulaire 690 à 705 agissant comme un filtre à cations. / AE1 (SLC4A1, band 3) is a member of the SLC4 bicarbonate transporter family. This protein is the most abundant membrane protein on the surface of vertebrate red blood cells. The AE1 exchanges chloride and bicarbonate ions in physiological conditions. In red blood cells, it is essential to many tasks including CO2 transport and cytoskeleton anchoring in the plasma membrane. Point mutations in the membrane spanning domain of AE1 convert the electroneutral exchange into a conductance for sodium and potassium cations or induce a cation leak in a still functional anionic exchanger.The molecular determinants that induce the movement of ions through the exchanger are still unknown. This work aims at identifying and mapping the transport site of AE1 protein in normal and pathological conditions. We modified a sulfhydryl-based chemistry to AE1. This provided information on the role of amino acids in the transport site of the protein. This strategy combined with the development of a three-dimensional model of the protein in silico, based on the uracil/proton symporter, allowed us to define the transport site of AE1. Analysis of our results showed that a single transport site in AE1 can switch between three different conformations depending on protein mutation: classical chloride/bicarbonate exchange, cation leak and anion exchange, cation leak only. The transport site involves the transmembrane segments 3, 5 and 8 and a highly conserved intracellular loop between transmembrane segments 8 and 9. The transport site is centered around the amino acids L468, F471, L530, L673, I533 and ends at glutamic acid 681. The intracellular loop 690-705 acts as a cation filter.