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.

Keywords: heparan sulfate 6-O-sulfotransferases; lipoprotein receptors; syndecan-1; triglyceride-rich lipoprotein remnants.

Fig. 1

Expression of HSPGs and heparan sulfate biosynthetic genes in primary human hepatocytes and Hep3B cells. mRNA was isolated from human primary hepatocytes (open bars; N = 3 technical replicates) and human hepatoma cell line, Hep3B (filled bars; n = 2 technical replicates). Amplification primers are listed in Supporting Table S1. This figure is available in black and white in print and in color at Glycobiology online.

Fig. 2

Composition of heparan sulfate in wild-type and mutant Hep3B cells. (A) Heparan sulfate was isolated and purified from wild-type and mutant cell lines, and the disaccharide composition was determined (n = 2 biological replicates). The relative content of individual disaccharides is shown. (B) The number of N-acetylated (N-acetyl), N-sulfated (N-SO₃), 6-O-sulfated (6-O-SO₃) glucosamine units and 2-O-sulfated uronic acids (2-O-SO3) was calculated. Statistics was calculated by two-way analysis of variance (ANOVA). n.d. means not determined. The disaccharide structure code is used: D0H0, ΔUA-GlcNH₂; D0A0, ΔUA-GlcNAc; D0H6, ΔUA-GlcNH₂6S; D2H0, ΔUA2S-GlcNH₂; D0S0, ΔUA-GlcNS; D2H6, ΔUA2S-GlcNH₂6S; D0A6, ΔUA-GlcNAc6S; D0S6, ΔUA-GlcNS6S; D2A0, ΔUA2S-GlcNAc; D2S0, ΔUA2S-GlcNS; D2A6, ΔUA2S-GlcNAc6S; D2S6, ΔUA2S-GlcNS6S; where ΔUA = 4,5-unsaturated uronic acid and I = iduronic acid (Lawrence, Lu, et al. 2008). This figure is available in black and white in print and in color at Glycobiology online.

Fig. 3

Silencing of HS6ST3 in HS6ST1^−/−;HS6ST2^−/− cell line. Wild-type Hep3B and HS6ST1^−/−;HS6ST2^−/− cells were transfected either scrambled or HS6ST3 siRNA (Sigma-Aldrich). (A) HS6ST3 gene expression was analyzed (n = 2 technical replicates). (B) Heparan sulfate was isolated and the disaccharide composition was determined (n = 2 biological replicates). (C) The number of N-acetylated (N-acetyl), N-sulfated (N-SO₃), 6-O-sulfated (6-O-SO₃) glucosamine units and 2-O-sulfated uronic acids (2-O-SO₃) was calculated. Statistics was calculated by two-way ANOVA. This figure is available in black and white in print and in color at Glycobiology online.

Fig. 4

Cell surface heparan sulfate determines TRL and FGF2 binding. (A) Wild-type and mutant Hep3B cells were incubated with [³H] TRLs (100 μg/mL) for 1 h on ice. Bound [³H] TRLs were determined by liquid scintillation counting (n = 3 technical replicates). (B) Cells were incubated with biotinylated FGF2 at 4°C for 1 h. Binding was determined by PE/Cy5-conjugated streptavidin and flow cytometry. Data were analyzed using FlowJo software. A set of Hep3B wild-type cells was treated with heparin lyases I, II and III for 30 min at 37°C prior to incubation with FGF2 (n = 2 technical replicates). Statistics was calculated by one-way ANOVA. This figure is available in black and white in print and in color at Glycobiology online.

Fig. 5

Heparan sulfate 6-O-sulfation is important for TRL uptake. (A) Uptake of TRLs in wild-type and SDC1^−/− cells was measured using DiD-VLDL particles. Cells were treated with 100 μg/mL of DiD-VLDL particles in the presence and absence of heparin lyases (HL), or heparin (100 μg/mL). Cell uptake was measured using fluorescence reader, and the values were normalized to total cell protein (n = 2 biological replicates each performed in triplicate). (B) Postprandial clearance was measured by retinol excursion as described (Ishibashi et al. 1996). Briefly, 20 μCi of [11,12-³H] retinol (44.4 Ci/mmol; PerkinElmer) in ethanol was mixed with 1 mL of corn oil. Each mouse received 200 μL of the mixture by oral gavage. Blood was sampled at the times indicated by tail bleed, and radioactivity was measured in triplicate (10 μL serum) by scintillation counting. This figure is available in black and white in print and in color at Glycobiology online.

Fig. 6

SDC1 null Hep3B cells. (A) Sanger sequencing of a region within exon 2 of SDC1 in wild-type Hep3B cells and in a cloned mutant cell line. The arrows indicate the start site of the altered DNA sequence in the mutant and the predicted amino acid sequence. Each allele resulted in a downstream frameshift mutation. (B) Extracts of wild-type and SDC1^−/− cells western blotted with an antibody to human SDC1 after heparin lyase treatment. Anti-actin was used as a loading control. (C) Cell surface SDC1 was detected by flow cytometry. SDC1 null cell line (black filled) does not exhibit cell surface SDC1 as compared to wild type (gray filled). The background is indicated (not filled). (D) Heparan sulfate from wild-type (white bars) and SDC1^−/− (black bars) cells was digested with heparin lyases, and the liberated disaccharides were analyzed by liquid chromatography/mass spectrometry (n = 2 biological replicates). (E) FGF2 binding in wild-type and SDC1^−/− Hep3B cells before and after treatment of heparin lyases (n = 2 technical replicates). Statistics was calculated by one-way ANOVA. This figure is available in black and white in print and in color at Glycobiology online.

Fig. 7

SDC1 regulates TRL uptake. Binding (A) and uptake (B) of TRLs in wild-type and SDC1^−/− cells were measured using DiD-VLDL particles. (A) Cells were treated with 100 μg/mL of DiD-VLDL particles in the presence and absence of heparin lyases, PIPLC or heparin for 1 h in the dark on ice. Cell associated particles were measured using fluorescence reader, and the values were normalized to total cell protein (n = 2 biological replicates each performed in triplicate). (B) Wild-type and SDC1^−/− cells were incubated at the indicated times at 37°C with 100 μg/mL of DiD-VLDL particles in the presence and absence of heparin lyases, PIPLC and heparin. The cells were treated with trypsin and the fluorescence of internalized particles was measured. The values represent the average fluorescence intensity values normalized to cell protein (n = 2 biological replicates each performed in triplicate). (C) WT and SDC1^−/− cell lines were treated with heparin lyase I, II and III (each at 5 mU/mL) and neo-epitopes were detected using 3G10 antibody. SDC1 is indicated by the black arrowhead. Statistics calculated by t-test and two-way ANOVA. This figure is available in black and white in print and in color at Glycobiology online.