Hypomorphic sialidase expression decreases serum cholesterol by downregulation of VLDL production in mice

Abstract

Lipoprotein metabolism is an important contributing factor in the development and progression of atherosclerosis. Plasma lipoproteins and their receptors are heavily glycosylated and sialylated, and levels of sialic acids modulate their biological functions. Sialylation is controlled by the activities of sialyltranferases and sialidases. To address the impact of sialidase (neu1) activity on lipoprotein metabolism, we have generated a mouse model with a hypomorphic neu1 allele (B6.SM) that displays reduced sialidase expression and sialidase activity. The objectives of this study are to determine the impact of sialidase on the rate of hepatic lipoprotein secretion and lipoprotein uptake. Our results indicate that hepatic levels of cholesterol and triglycerides are significantly higher in B6.SM mice compared with C57Bl/6 mice; however, VLDL-triglyceride production rate is lower. In addition, B6.SM mice show significantly lower levels of hepatic microsomal triglyceride transfer protein (MTP) and active sterol-regulatory element binding protein (SREBP)-2 but higher levels of diglyceride acyltransferase (DGAT)2; these are all indicative of increased hepatic lipid storage. Rescue of sialidase activity in hypomorphic sialidase mice using helper-dependent adenovirus resulted in increased VLDL production and an increase in MTP levels. Furthermore, hypomorphic sialidase expression results in stabilization of hepatic LDL receptor (LDLR) protein expression, which enhances LDL uptake. These findings provide novel evidence for a central role of sialidase in the cross talk between the uptake and production of lipoproteins.

Fig. 1.

Sialidase protein expression and activity in C57Bl/6 and B6.SM mice. (A) B6.SM males show a significant reduction in hepatic neu1 sialidase expression compared with C57Bl/6 controls (P = 0.03). Representative blots of n = 3 for each group. Liver lysates were subjected to SDS-PAGE (8%). Membranes were probed with anti-neu1 Sialidase antibody and anti-β-actin as a control. Intensities of bands were measured by ImageJ densitometry software. (B) B6.SM tissues have significantly lower levels of sialidase activity, and this is especially prominent in the liver where levels are reduced to approximately 20% of C57Bl/6. Brain, liver, spleen, and kidney lysates were assessed for sialidase activity using fluorescent 4-Mu-NANA. *P = 0.01, **P = 0.001, ***P < 0.0001. mU = μMol/hr.

Fig. 2.

FPLC cholesterol profiles of C57Bl/6 and B6.SM mice. Serum lipoproteins were fractionated by size using FPLC using a superose 6 gel filtration column. The FPLC cholesterol profiles of the unfasted male B6.SM animals (n = 3) have significantly less total cholesterol in LDL-sized particles than those of the corresponding C57Bl/6 controls (n = 3). In addition, there is a tendency of the HDL and VLDL (inset) peaks to be shifted slightly to the right. Each circle represents mean ± SE (*P < 0.05).

Fig. 3.

In vivo hepatic VLDL-TG production in C57Bl/6 and B6.SM mice. C57Bl/6 (n = 3) and B6.SM mice (n = 3) were fasted overnight and injected with the lipoprotein lipase inhibitor Triton WR1339 (500 mg/kg). Serum samples were drawn just before the injection (time 0 h) and at 1, 2, 3, and 4 h postinjection. There is a decrease in serum (A) VLDL-triglyceride, (B) VLDL-cholesterol, (C) VLDL-free cholesterol, and (D) VLDL-cholesteryl esters concentrations at different indicated time points after 0 h (*P < 0.05). Also note that there is a decrease in the steepness of the slope of the B6.SM mice compared with that of the C57Bl/6 mice. This indicates that hypomorphic sialidase expression causes decreased hepatic VLDL-TG production rates. Values represent means ± SE. (E) B6.SM mice have a significant decrease in the protein expression of MTP compared with C57Bl/6 (P = 0.002), which further supports that hypomorphic sialidase expression results in decreased VLDL-TG production. Mouse liver lysates were subjected to SDS-PAGE (8%), and membranes were probed with anti-MTP and anti-β-actin antibodies. Intensities of bands were measured by ImageJ densitometry software.

Fig. 4.

In vivo hepatic VLDL-TG production in B6.SM mice rescued with sialidase gene therapy. (A) Plasmid map of pC4HSUmsial helper-dependent adenovirus displaying fragment sizes corresponding to an EcoRI digestion. Restriction analysis of pC4HSUmsial using EcoRI reveals the appropriate fragments following digestion (lane 1, pC4HSUmsial; lane 2, 1 kb ladder). This virus was combined into a helper-dependent vector, yielding HD-AdSial (see Methods). (B) Sialidase activity of sialidosis fibroblast cells (WG544) that were infected with HD-AdSial at various particles/cell. Viral infection increases sialidase activity at a particle-dependent rate (n = 3, error bars represent SE). B6.SM male mice (n = 4 for each group) were infected with helper-dependent adenovirus containing mouse sialidase (HD-AdSial) or LacZ (HD-AdlacZ) and monitored for 14 days. (C) Neu1 sialidase protein is upregulated in liver lysates of the HD-AdSial group as measured by Western blotting, indicating expression of the viral vector. VLDL-TG production was analyzed by hourly serum triglyceride measurement following injection of LPL inhibitor Triton WR1339, as described above. (D) B6.SM mice infected with mouse sialidase helper-dependent adenovirus have significantly higher VLDL-TG production compared with B6.SM mice infected with HD-AdlacZ control adenovirus. (E) MTP protein expression is increased in B6.SM mice that were infected with HD-AdSial compared with HD-AdlacZ as measured by Western blotting. *P < 0.05.

Fig. 5.

Hepatic SREBP-2, SREBP-1, ACAT-2, and DGAT2 expression in C57Bl/6 and B6.SM mice. Liver lysates were subjected to SDS-PAGE (8%). Membranes were probed with (A) anti-SREBP-2, (B) anti-SREBP-1a/c, (C) anti-ACAT-2, and (D) anti-DGAT2 and anti-β-actin or anti-β-tubulin or anti-GAPDH antibodies as a control. Intensities of bands were measured by ImageJ densitometry software. B6.SM mice have a significant decrease in the protein expression of cleaved SREBP-2 (P = 0.004), a significant increase in DGAT2 protein expression (P = 0.01), and a trend of increase in the protein expression of ACAT-2 (P = 0.11) in liver compared with C57Bl/6 mice. Representative blots of n = 3 for each group.

Fig. 6.

Hypomorphic sialidase expression influences hepatic expression and glycosylation of LDLR. (A) B6.SM male mice have equal hepatic protein levels of LDLR compared with C57Bl/6 controls. Representative blots of n = 3 for each group. Liver lysates were subjected to SDS-PAGE followed by Western blotting for LDLR and β-actin (as a control). Intensities of bands were measured by ImageJ densitometry software. (B) B6.SM male mice have a trend of increase in hepatic protein levels of LRP-1 compared with C57Bl/6 mice, although this is not significant (P = 0.06). (C) B6.SM mice have lower levels of hepatic LDLR mRNA compared with C57Bl/6 controls while showing no difference in SREBP-2 mRNA (P = 0.03 and P = 0.3, n = 3 in each group). (D) PCSK9 TrueBlot immunoprecipitation of B6.SM and C57Bl/6 mouse serum. B6.SM mice have lower levels of secreted PCSK9 compared with C57Bl/6 controls (P = 0.026, n = 3 in each group). (E) LDLR from B6.SM mice has higher levels of terminal α-2,6 linked and α-2,3 linked sialic acids compared with C57Bl/6 mice, due to lower sialidase levels. Sialylated conjugates from membrane-enriched liver lysates (equal protein) were pulled down using biotinylated SNA and MALII lectins followed by streptavidin sepharose. Pulled down glycoproteins were immunoblotted for LDLR. Control LDLR blotting of LDLR IP samples show equal quantities of LDLR in starting lysates.

Fig. 7.

LDLR expression and function in human fibroblasts with a Neu1 sialidase-null mutation. Cells were serum starved in Optimem media to upregulate LDLR and analyzed by Western blotting, immunofluorescence, Oil Red O staining, and enzymatic cholesterol assay. (A) Sialidase-null fibroblasts do not show significantly different LDLR protein expression, although their receptors appear to be of a higher molecular weight (representative of four blots). (B) LDLR immunofluorescence shows localization and clustering of receptors in fibroblasts (arrows), but no gross expression changes between the two cell types (bar = 20 μm). (C) Oil Red O staining after LDL incubation shows lipoprotein uptake and lipid droplet formation (arrows) in the fibroblasts, although there was no significant difference in neutral lipid accumulation (bar = 20 μm). (D) Sialidase-null fibroblasts show significantly higher total cholesterol levels as measured by enzymatic cholesterol assay after LDL incubation compared with wild-type cells, indicating more LDL uptake (P = 0.048, n = 3 in each group).