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Triglyceride-rich lipoprotein binding and uptake by heparan sulfate proteoglycan receptors in a CRISPR/Cas9 library of Hep3B mutants.

ABSTRACT: 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.

SUBMITTER: Anower-E-Khuda F

PROVIDER: S-EPMC6639542 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Triglyceride-rich lipoprotein binding and uptake by heparan sulfate proteoglycan receptors in a CRISPR/Cas9 library of Hep3B mutants.

Anower-E-Khuda Ferdous F Singh Gagandeep G Deng Yiping Y Gordts Philip L S M PLSM Esko Jeffrey D JD

Glycobiology 20190701 8

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 ...[more]

PMID: 31094413

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Project description:Background and Aims: Inflammasome-mediated caspase-1 activity regulates the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1M-CM-^_ and IL-18. Recently, we showed that caspase-1 deficiency strongly reduces high fat diet-induced adiposity although the mechanism is still unclear. We now aimed to elucidate the mechanism by which caspase-1 deficiency reduces modulates resistance to high fat diet-feeding fat accumulation in adipose tissue by focusing on the role of caspase-1 in the regulation of triglyceride (TG)-rich lipoprotein metabolism. Methods: Caspase-1 deficient and wild-type mice (both C57Bl/6 background) were used to determine postprandial TG kinetics, intestinal TG absorption, VLDL-TG production as well as TG clearance, all of which strongly contribute to the supply of TG for storage in adipose tissue. Micro-array and qPCR analysis were used to unravel intestinal and hepatic metabolic pathways involved. Results: Caspase-1 deficiency reduced the postprandial response to an oral lipid load, while tissue specific clearance of TG-rich lipoproteins was not changed. Indeed, an oral olive oil gavage containing [3H]TG revealed that caspase-1 deficiency significantly decreased intestinal chylomicron-TG production and reduced the uptake of [3H]TG-derived FA by liver, muscle, and adipose tissue. Similarly, caspase-1 deficiency reduced the hepatic VLDL-TG production without reducing VLDL-apoB production, despite an elevated hepatic TG content. Pathway analysis revealed that caspase-1 deficiency reduces intestinal and hepatic expression of genes involved in lipogenesis. Conclusions: Absence of caspase-1 reduces assembly and secretion of TG-rich lipoproteins, thereby reducing the availability of TG-derived FA for uptake by peripheral organs including adipose tissue. We anticipate that caspase-1 represents a novel link between innate immunity and lipid metabolism. Keywords: Expression profiling by array Wild-type (WT) and Casp1-null mice were maintained at lab chow. Animals, aged between 14 and 16 weeks (n=3 per genotype), were killed and liver and intestinal segments were removed. Livers were isolated from mice that were fasted over night, whereas intesines were removed from mice 2 hrs after they received an oral lipid load.Total RNA was isolated and subjected to gene expression profiling.

Dataset Information

Triglyceride-rich lipoprotein binding and uptake by heparan sulfate proteoglycan receptors in a CRISPR/Cas9 library of Hep3B mutants.

Publications

Triglyceride-rich lipoprotein binding and uptake by heparan sulfate proteoglycan receptors in a CRISPR/Cas9 library of Hep3B mutants.

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