The monotopic membrane protein alMGS, a glycosyltransferase catalyzing glucolipid synthesis in Acholeplasma laidlawii, was overexpressed in Escherichia coli. Optimization of basic growth parameters was performed, and a novel method for detergent and buffer screening using a small size-exclusion chromatography was developed. This resulted in a tremendous increase in protein yields, as well as the unexpected discovery that the protein induces intracellular vesicle formation in E. coli. This was confirmed by sucrose density separation and Cryo-TEM of membranes, and the properties of the vesicles were analyzed using SDS-PAGE, western blot and lipid composition analysis. It is concluded that both alMGS and alDGS, the next enzyme in glucolipid pathway, have the ability to make the membrane bend and eventually form vesicles. This is likely due to structural and electrostatic properties, such as the way the proteins penetrate the membrane interface and thereby expand one monolayer. The highly positively charged binding surfaces of the glycosyltransferases may bind negatively charged lipids, such as Phosphatidylglycerol (PG), in the membrane and withdraw it from the general pool of lipids. This would increase the overall lipid synthesis, since PG is a pace-keeper, and the local concentration of nonbilayer prone lipids, such as Phosphatidylethanolamine, can increase and also induce bending of the membrane. The formation of surplus membrane inside the E. coli cell was used to develop a generic method for overexpression of membrane proteins. A proof-of-principle experiment with a test set of twenty membrane proteins from E. coli resulted in elevated expression levels for about half of the set. Thus, we believe that this method will be a useful tool for overexpression of many membrane proteins. By engineering E. coli mutants with different lipid compositions, fine-tuning membrane properties for different proteins is also possible. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 3: Manuscript.

La spondylarthrite ankylosante (SA), la forme la plus commune des spondylarthrites (SpA), est fortement associée à la molécule du CMH de classe I HLA-B27 mais le rôle de cet antigène d'histocompatibilité dans le développement de ces pathologies reste encore inexpliqué. L’étude des rats transgéniques HLA-B27, développant une pathologie inflammatoire spontanée ressemblant aux SpA, a permis de confirmer l’implication directe du HLA-B27 et de corréler l’apparition des symptômes avec une forte expression de cette molécule. De plus, il a été montré que l’HLA-B27 présentait une propension particulière au mauvais repliement et à la formation d’oligomères de chaînes lourdes. L’objectif de mon travail de thèse était de déterminer si le trafic et/ou la formation d’oligomères du HLA-B27 étaient corrélés à sa surexpression. Pour cela, notre équipe a développé des protéines de fusion (HLA-BYFP et HLA-BRLuc) ainsi que la technique BRET afin d’étudier les interactions HLA-B/HLA-B. Au moyen de ce système expérimental, nous avons montré la formation de vésicules intracellulaires riches en protéines HLA-B mal repliées lorsqu’elles étaient fortement exprimées, qu'il s'agisse d'allèles associés à la SA (HLA-B*2702, -05, et -07) ou non (HLA-B*2706, et -09, HLA-B*0702). Cependant, ce phénomène était significativement plus prononcé pour les sous-types associés à la SA. Dans les conditions de forte expression, nous avons également observé que les sous-types associés à la SA formaient des oligomères qui se comportent différemment de ceux formés par la protéine HLA-B7. Ce phénomène ne semble pas être dû au déclenchement de la réponse cellulaire « Unfolded Protein Response » (UPR) et n’est pas abrogé par l’inhibition du protéasome. / Ankylosing spondylitis (AS), the most common form of spondyloarthritis (SpA), is strongly associated with the MHC class I HLA-B27 molecule. Although this association has been largely studied, mechanisms of pathology remain unclear. Development of a spontaneous inflammatory disease resembling human SpA in HLA-B27 transgenic rats confirmed the direct involvement of HLA-B27 and allowed to associate disease development with high expression levels of this molecule. Moreover, the HLA-B27 protein has an enhanced propensity to misfold and form aberrant disulfide linked heavy chain oligomers in the endoplasmic reticulum and at the cell surface. The goal of my thesis work was to determine if the HLA-B27 traffic and/or its ability to form oligomers are involved in this requirement of overexpression. For that, our team has developed fusion proteins ((HLA-BYFP and HLA-BRLuc) and the BRET technique to study, in vitro, the HLA-B/HLA-B interactions. Using this experimental system, we have shown the formation of intracellular vesicles, in which misfolded/unfolded HLA-B proteins accumulated when they were highly expressed, for both AS-associated alleles (HLA-B*2702, -05, et -07) or not (HLA-B*2706, et -09, HLA-B*0702). This phenomenon is strongly pronounced for AS-associated subtypes. For high-level expression, we also observed that the AS-associated subtypes form oligomers that behave differently from those formed by the HLA-B7 control protein. This phenomenon doesn’t appear to be due to unfolded protein response (UPR) triggering and is not abrogated by proteasome inhibition.

The interplay between lipids and membrane proteins is known to affect membrane protein topology and thus have significant effect (control) on their functions. In this PhD thesis, the influence of lipids on the membrane protein function was studied using three different membrane protein models. A monotopic membrane protein, monoglucosyldiacylglyecerol synthase (MGS) from Acholeplasma laidlawii is known to induce intracellular vesicles when expressed in Escherichia coli. The mechanism leading to this unusual phenomenon was investigated by various biochemical and biophysical techniques. The results indicated a doubling of lipid synthesis in the cell, which was triggered by the selective binding of MGS to anionic lipids. Multivariate data analysis revealed a good correlation with MGS production. Furthermore, preferential anionic lipid sequestering by MGS was shown to induce a different fatty acid modeling of E. coli membranes. The roles of specific lipid binding and the probable mechanism leading to intracellular vesicle formation were also investigated. As a second model, a MGS homolog from Synechocystis sp. PCC6803 was selected. MgdA is an integral membrane protein with multiple transmembrane helices and a unique membrane topology. The influence of different type of lipids on MgdA activity was tested with different membrane fractions of Synechocystis. Results indicated a very distinct profile compared to Acholeplasma laidlawii MGS. SQDG, an anionic lipid was found to be the species of the membrane that increased the MgdA activity 7-fold whereas two other lipids (PG and PE) had only minor effects on MgdA. Additionally, a working model of MgdA for the biosynthesis and flow of sugar lipids between Synechocystis membranes was proposed. The last model system was another integral membrane protein with a distinct structure but also a different function. The envelope stress sensor, CpxA and its interaction with E. coli membranes were studied. CpxA autophosphorylation activity was found to be positively regulated by phosphatidylethanolamine and negatively by anionic lipids. In contrast, phosphorylation of CpxR by CpxA revealed to be increased with PG but inhibited by CL. Non-bilayer lipids had a negative impact on CpxA phosphotransfer activity. Taken together, these studies provide a better understanding of the significance of the interplay of lipids and model membrane proteins discussed here.