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Protein-sphingolipid interactions and their role in immunity and disease



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Shamin, Maria 


Sphingolipids are a diverse class of lipids involved in fundamental cellular processes. In addition to their role in membrane architecture, sphingolipids can act as bioactive molecules, regulate membrane protein function and be presented to the immune system as lipid antigens by CD1 family molecules. The critical importance of sphingolipids for cell function is highlighted by the catastrophic diseases that result from defects in sphingolipid metabolism.

Lipid loading onto CD1d is facilitated by the lysosomal lipid transfer protein Saposin B, but the lipid loading mechanism is unknown. I used a range of highly sensitive biochemical assays to probe the interaction between CD1d and SapB. Interestingly, no direct interaction was detectable. Importantly, this suggests that additional proteins may promote their interaction.

In addition to their role as antigens, sphingolipids have roles in immunity by regulating the trafficking and function of immune receptors. Krabbe disease, a neurodegenerative disease caused by loss of function of the lysosomal sphingolipid hydrolase GALC, is accompanied by immune defects possibly driven by phagocytic cell dysfunction. I investigated how GALC deficiency alters the phenotype and function of the phagocytic cell line THP-1. This study was compromised by substantial off-target effects caused by genetic manipulation of THP-1 cells. Characterising these off-target changes identified perturbations in sphingolipid metabolism pathways, confirming that these cell lines are not appropriate for further studies.

The lysosomal enzyme acid ceramidase (AC) hydrolyses ceramide to sphingosine, thus regulating the levels of pro-apoptotic and pro-survival lipids. This role in apoptosis means that regulation of AC activity is of interest for cancer treatment. Although the structure of AC was recently determined, it is still unclear how it accesses membrane-embedded lipid substrates. I attempted to co-crystallise AC with Saposin A lipoprotein nanodiscs to understand this process. Although this system did not prove suitable for this project, I was able to determine the crystal structure of a new saposin oligomer highlighting even greater diversity of tertiary arrangement of these important lipid transfer proteins. I next asked whether AC processes galactosylceramide to psychosine, the cytotoxic GALC substrate accumulating in Krabbe disease, contributing to Krabbe disease pathology by increasing oligodendrocyte cell death. Surprisingly, I found that GALC knockout oligodendrocytes were not more susceptible to cell death than wild-type oligodendrocytes, calling into question whether psychosine accumulation is the direct cause of oligodendrocyte cell death and demyelination in Krabbe disease.

The sphingolipid-interacting adhesion protein PTPRM is lost at the surface of GALC-deficient oligodendrocytes, suggesting that specific sphingolipid-PTPRM interactions cause the mistrafficking of this adhesion protein in Krabbe disease oligodendrocytes, potentially contributing to demyelination. I probed the interaction of PTPRM with sphingolipids through X-ray crystallography studies and fluorescence-based trafficking assays.





Deane, Janet


sphingolipids, saposins, CD1d, lipid presentation, sphingolipid degradation, galactosylceramidase, acid ceramidase


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Wellcome Trust (203984/Z/16/Z)