Binding and uptake of triglyceride-rich lipoproteins (TRLs) in mice depends on heparan sulfate and hepatic proteoglycan, syndecan-1. Alteration of glucosamine N-sulfation by deletion of glucosamine N-deacetylase-N-sulfotransferase 1 (Ndst1) and 2-O-sulfation… Click to show full abstract
Binding and uptake of triglyceride-rich lipoproteins (TRLs) in mice depends on heparan sulfate and hepatic proteoglycan, syndecan-1. Alteration of glucosamine N-sulfation by deletion of glucosamine N-deacetylase-N-sulfotransferase 1 (Ndst1) and 2-O-sulfation of uronic acids by deletion of uronyl 2-O-sulfotransferase (Hs2st) led to diminished lipoprotein metabolism, whereas inactivation of glucosaminyl 6-O-sulfotransferase 1 (Hs6st1), which encodes one of the three 6-O-sulfotransferases, had little effect on lipoprotein binding. However, other studies have suggested that 6-O-sulfation may be important for TRL binding and uptake. In order to explain these discrepant findings, we used CRISPR/Cas9 gene editing to create a library of mutants in the human hepatoma cell line, Hep3B. Inactivation of EXT1 encoding the heparan sulfate copolymerase, NDST1 and HS2ST dramatically reduced binding of TRLs. Inactivation of HS6ST1 had no effect, but deletion of HS6ST2 reduced TRL binding. Compounding mutations in HS6ST1 and HS6ST2 did not exacerbate this effect indicating that HS6ST2 is the dominant 6-O-sulfotransferase and that binding of TRLs indeed depends on 6-O-sulfation of glucosamine residues. Uptake studies showed that TRL internalization was also affected in 6-O-sulfation deficient cells. Interestingly, genetic deletion of syndecan-1 only marginally impacted binding of TRLs, but reduced TRL uptake to the same extent as treating the cells with heparin lyases. These findings confirm that syndecan-1 is the dominant endocytic proteoglycan receptor for TRLs in human Hep3B cells and that binding and uptake of TRLs depends on syndecan-1 and N- and 2-O-sulfation as well as 6-O-sulfation of heparan sulfate chains catalyzed by HS6ST2.
               
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