👤 Md Ferdous Anower-E-Khuda

Binding and uptake of triglyceride-rich lipoproteins (TRLs) in mice depend on heparan sulfate and the hepatic proteoglycan, syndecan-1 (SDC1). 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 SDC1 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 SDC1 is the dominant endocytic proteoglycan receptor for TRLs in human Hep3B cells and that binding and uptake of TRLs depend on SDC1 and N- and 2-O-sulfation as well as 6-O-sulfation of heparan sulfate chains catalyzed by HS6ST2. Show less

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder with wide variation in clinical phenotype and is caused by heterogeneous germline mutations in two of the Ext genes, EXT-1 and EXT-2, which encode ubiquitously expressed glycosyltransferases involved in the polymerization of heparan sulfate (HS) chains. To examine whether the Ext mutation could affect HS structures and amounts in HME patients being heterozygous for the Ext genes, we collected blood from patients and healthy individuals, separated it into plasma and cellular fractions and then isolated glycosaminoglycans (GAGs) from those fractions. A newly established method consisting of a combination of selective ethanol precipitation of GAGs, digestion of GAGs recovered on the filter-cup by direct addition of heparitinase or chondroitinase reaction solution and subsequent high-performance liquid chromatography of the unsaturated disaccharide products enabled the analysis using the least amount of blood (200 µL). We found that HS structures of HME patients were almost similar to those of controls in both plasma and cellular fractions. However, interestingly, although both the amounts of HS and chondroitin sulfate (CS) varied depending on the different individuals, the amounts of HS in both the plasma and cellular fractions of HME patient samples were decreased and the ratios of HS to CS (HS/CS) of HME patient samples were almost half those of healthy individuals. The results suggest that HME patients' blood exhibited reduced HS amounts and HS/CS ratios, which could be used as a diagnostic biomarker for HME. Show less