%0 Generic
%A Roxlau, Thomas Johannes
%D 2020
%F heidok:27099
%R 10.11588/heidok.00027099
%T Investigation of the lipid environment of the mammalian transamidase complex
%U https://archiv.ub.uni-heidelberg.de/volltextserver/27099/
%X The glycosylphosphatidylinositol (GPI) anchor is a lipid moiety attached to over 150 human proteins  and plays a crucial role in cell surface display at the plasma membrane. The transamidase  complex, consisting of the subunits PIGK/S/T/U and GPAA1, is localised to the endoplasmic reticulum  (ER) and is responsible for attachment of the GPI-anchor to receiving proteins. Previously,  interactions between the transamidase complex subunit GPAA1 and a sphingolipid have been  shown. The aim of this thesis was to establish an experimental setup to analyse protein lipid interactions  of the transamidase complex.  These interactions were assessed via in-gel fluorescence and western blot quantification through  click chemistry with bifunctional sphingolipids combined with transient expression of transamidase  subunits. Thin layer chromatography was used to verify the metabolic status of exogenously  added photoactivatable and clickable sphingosine (pacSph) at given time points. Results  confirmed the previous reported interaction of GPAA1. The validity of this outcome was limited  to GPAA1 only due to uncertainty with regards to the transient expression, such as affecting  the stoichiometry of the transamidase complex and mislocalisation of the transiently overexpressed  GPAA1. To circumvent these limitations, CRISPR/Cas9-based gene editing was performed  to insert a C-terminal tag in the genomic region of GPAA1. This allowed to establish a cellular  model to study sphingolipid and cholesterol interactions with the endogenous transamidase  complex. Gene edited tagging of GPAA1 did not interfere with synthesis and trafficking of GPIanchor  proteins to the cell surface. The analysis of the composition of the transamidase complex  in this cell line by co-immunoprecipitation showed only PIGK/T/S to interact with GPAA1, but  not PIGU. When styrene maleic anhydride (SMA) co-polymer was used to extract the transamidase  complex and its native lipid environment, even PIGU was detected, suggesting that PIGU  might be only loosely attached to the transamidase complex. Furthermore, first studies on mass  spectrometric analysis of lipids extracted from an SMA-immunoprecipitation suggested that this  approach allows to determine lipids in direct proximity of the transamaidase complex. Finally,  GPAA1-pacSph metabolite interaction could be inhibited through the ceramide synthase inhibitor  FB1, while the glucosylceramide synthase inhibitor PPMP did not interfere with the interaction.  This suggested that ceramide or a metabolite upstream of this lipid is the interaction partner of  GPAA1.  In summary, the established cellular model verified previously published results in an endogenous  setting. For the future, this model lays the foundation for quantitative determination of the  lipid environment of the transamidase complex. Furthermore, this system might also provide  structural information via single particle cryo-electron microscopy of affinity purified GPAA1  complexes.