The roles of heparan sulfate in autophagy

Abstract: Background: Autophagy is a lysosomal degradation process to eliminate damaged components from the cells. It also provides a survival mechanism in stressed conditions and limits growth processes. Heparan sulfate (HS) is a polysaccharide attached to different proteoglycans. Besides other functions, it regulates growth factors' interactions with their receptors. The interplay of HS and autophagy in growth signaling demands studying the possible association between HS and autophagy. In the drosophila model, the HS biosynthesis genes knockdown displayed increased autophagy. However, the mammalian homologs of these genes are yet to be studied in the context of autophagy. Aims: To investigate the effects of HS on autophagy in mammalian cells Materials & Methods: Chinese hamster ovary (CHO) and Mouse embryonic fibroblasts (MEF) were used to evaluate autophagy in wild-type (Wt) and HS biosynthesis enzyme knockouts (KO). CHO-K1(Wt), CHO-677 (KO), MEF Wt, MEF Glucuronic Acid Epimerase (GLCE) knockout (Epi-KO), and MEF Exostosin Glycosyltransferase 1 knockout (Ext1-KO) cells were treated with 10 μM chloroquine to block autophagy steps downstream autophagosome formation. Autophagy biomarkers, light chain 3-II (LC3-II), sequestosome-1 (p62), Unc-51-like autophagy activating kinase 1 (ULK1), and autophagy-related 5 (Atg5), were assessed via immunoblotting. To validate the findings, MEF cells were transiently transfected with GFP-LC3 for direct fluorescence microscopy.  Results: Compared to the wild-type CHO and MEF cells, LC3-II was decreased in CHO -677 and Ext1-KO cells which have compromised HS synthesis.  This conclusion was further confirmed by the results from fluorescence microscopy of transfected cells.  GFP-LC3 puncta were fewer in Ext1-KO compared to Wt. In agreement with the downregulation of LC3-II, the levels of p62 were elevated in CHO-677 compared to CHO-K1 cells. However, the levels of ULK1 and Atg5 were inconclusive. Conclusions: The decreased LC3-II levels in KO suggest that HS might be associated with autophagy. It indicates a distinct role of HS in autophagy in mammals which differs from previous studies in drosophila.