Isolation of heparan sulfates with antithrombin III affinity and anticoagulant potency from BALB/c 3T3, B16.F10 melanoma, and cutaneous fibrosarcoma cell lines

The heparan sulfates synthesized in vitro by three cell lines were isolated by proteolysis and preparative anion exchange chromatography and purified free of other glycosaminoglycans by selective enzymatic degradation. The isolates from the medium of BALB/c 3T3 fibroblasts, B16.F10 melanoma cells, and a cutaneous fibrosarcoma line, along with that from the detergent-extracted cell layer of the fibroblasts, were affinity-fractionated on columns of matrix-immobilized human antithrombin III. Each heparan sulfate contained subfractions with high affinity for the proteinase inhibitor, ranging from 3-34% of the starting material. The high affinity species possessed measurable anticoagulant activities by a clotting assay (6 to 30 units/mg). Since none of the lines were derived from cell types having any known biological role in vascular homeostasis, we suggest that anticoagulant activity of the glycosaminoglycan is a random property of its primary structure.     

Proteoglycan and glycosaminoglycan free chain expression in keratinocytes, endothelium, and mesenchymal cells

Cultured fibroblasts, bovine aortic endothelial cells, and human keratinocytes synthesize both proteoglycans and glycosaminoglycan free chains, the proportions varying between cell types. The major metabolic labeling is in proteoglycans, except for keratinocytes with approximately 60% of product as free chains. The proteoglycans range from approximately 50- greater than 1000 kDa, and the glycosaminoglycan side chains derived by alkaline elimination are approximately 30- greater than 100 kDa. The glycosaminoglycan free chains, in contrast, are smaller, from approximately 7-40 kDa in mass. The proteoglycans are both medium and cell layer constituents, whereas the glycosaminoglycan free chains are essentially confined to cells. The cellular proteoglycans and a portion of the free chains are accessible to in situ digestion by Flavobacterial glycosaminoglycan lyases, presumably reflecting localization to the cell surface. Collectively, the data show the free chains to be a common feature of all cells studied and to be partly expressed on cell surfaces. We hypothesize that the processing that creates these free chains occurs on cell surfaces, in which location they could serve ligand receptor functions.     

Structural studies of heparitin sulfates.

Heparitin sulfate fractions with a large range in sulfate content were subjected to degradation by Flavobacterium heparinase and by nitrous acid. The products obtained were fractionated by chromatography, characterized, and used to arrive at tentative structures for these complex polysaccharides. The heparitin sulfate chains examined appear to be composed of: 1. uninterrupted blocks of N-acetylglucosamine containing disaccharides; 2. larger blocks with a molecular weight range of 5000 to 6000 which include the N-acetyl block but do not contain heparinase sensitive linkages; 3. segments containing mainly areas where N-acetyl, N-sulfate and some disulfated units alternate in the chain. The size and arrangement of these polymer segments seem to vary with the sulfate content of a particular heparitin sulfate. For instance, the polysaccharides with the highest degree of sulfation do not appear to contain N-acetyl blocks of significant size.     

Truncation mutations in ABCA1 suppress normal upregulation of full-length ABCA1 by 9-cis-retinoic acid and 22-R-hydroxycholesterol

Mutations in ABCA1 uniformly decrease plasma HDL-cholesterol (HDL-C) and reduce cholesterol efflux, yet different mutations in ABCA1 result in different phenotypic effects in heterozygotes. For example, truncation mutations result in significantly lower HDL-C and apoliprotein A-I (apoA-I) levels in heterozygotes compared with nontruncation mutations, suggesting that truncation mutations may negatively affect the wild-type allele. To specifically test this hypothesis, we examined ABCA1 protein expression in response to 9-cis-retinoic acid (9-cis-RA) and 22-R-hydroxycholesterol (22-R-OH-Chol) in a collection of human fibroblasts representing eight different mutations and observed that truncation mutations blunted the response to oxysterol stimulation and dominantly suppressed induction of the remaining full-length allele to 5-10% of wild-type levels. mRNA levels between truncation and nontruncation mutations were comparable, suggesting that ABCA1 expression was suppressed at the protein level. Dominant negative activity of truncated ABCA1 was recapitulated in an in vitro model using transfected Cos-7 cells. Our results suggest that the severe reduction of HDL-C in patients with truncation mutations may be at least partly explained by dominant negative suppression of expression and activity of the remaining full-length ABCA1 allele. These data suggest that ABCA1 requires a physical association with itself or other molecules for normal function and has important pharmacogenetic implications for individuals with truncation mutations.     

Cholesteryl ester transfer protein and hyperalphalipoproteinemia in Caucasians

It is unclear whether cholesteryl ester transfer protein (CETP) contributes to high density lipoprotein cholesterol (HDL-C) levels in hyperalphalipoproteinemia (HALP) in Caucasians. Moreover, even less is known about the effects of hereditary CETP deficiency in non-Japanese. We studied 95 unrelated Caucasian individuals with HALP. No correlations between CETP concentration or activity and HDL-C were identified. Screening for CETP gene defects led to the identification of heterozygosity for a novel splice site mutation in one individual. Twenty-five heterozygotes for this mutation showed reduced CETP concentration (-40%) and activity (-50%) and a 35% increase of HDL-C compared with family controls. The heterozygotes presented with an isolated high HDL-C, whereas the remaining subjects exhibited a typical high HDL-C/low-triglyceride phenotype. The increase of HDL-C in the CETP-deficient heterozygotes was primarily attributable to increased high density lipoprotein containing apolipoprotein A-I and A-II (LpAI:AII) levels, contrasting with an increase in both high density lipoprotein containing apolipoprotein A-I only and LpAI:AII in the other group. This study suggests the absence of a relationship between CETP and HDL-C levels in Caucasians with HALP. The data furthermore indicate that genetic CETP deficiency is rare among Caucasians and that this disorder presents with a phenotype that is different from that of subjects with HALP who have no mutation in the CETP gene.     

Heterozygosity for a loss-of-function mutation in GALNT2 improves plasma triglyceride clearance in man

Genome-wide association studies have identified GALNT2 as a candidate gene in lipid metabolism, but it is not known how the encoded enzyme ppGalNAc-T2, which contributes to the initiation of mucin-type O-linked glycosylation, mediates this effect. In two probands with elevated plasma high-density lipoprotein cholesterol and reduced triglycerides, we identified a mutation in GALNT2. It is shown that carriers have improved postprandial triglyceride clearance, which is likely attributable to attenuated glycosylation of apolipoprotein (apo) C-III, as observed in their plasma. This protein inhibits lipoprotein lipase (LPL), which hydrolyses plasma triglycerides. We show that an apoC-III-based peptide is a substrate for ppGalNAc-T2 while its glycosylation by the mutant enzyme is impaired. In addition, neuraminidase treatment of apoC-III which removes the sialic acids from its glycan chain decreases its potential to inhibit LPL. Combined, these data suggest that ppGalNAc-T2 can affect lipid metabolism through apoC-III glycosylation, thereby establishing GALNT2 as a lipid-modifying gene.     

Inherited disorders of HDL metabolism and atherosclerosis

PURPOSE OF REVIEW: Genetic disorders of HDL metabolism are rare and, as a result, the assessment of atherosclerosis risk in individuals suffering from these disorders has been difficult. Ultrasound imaging of carotid arteries has provided a tool to assess the risk in hereditary hypo and hyperalphalipoproteinemia. This review gives a comprehensive summary. RECENT FINDINGS: Epidemiological studies have unequivocally shown that HDL cholesterol levels are inversely related to coronary artery disease risk, but the literature concerning genetic disorders of HDL metabolism provides less convincing information. Fortuitously, we were able to directly compare carotid intima media thickness data of substantial numbers of individuals with mutations in either apolipoprotein A-I (apoA-I), ATP binding cassette AI (ABCA1), lecithin: cholesterol acyltransferase (LCAT) or cholesteryl ester transfer protein. These data show that carriers of an apoA-I mutation exhibit the most pronounced accelerated atherosclerosis compared with those carrying mutations in ABCA1 and LCAT. Heterozygosity for a non-sense mutation in cholesteryl ester transfer protein did, by contrast, not distinguish carriers from controls in terms of intima media thickness progression. We will discuss these results in the context of the current literature. SUMMARY: Intima media thickness studies have provided evidence that hypoalphalipoproteinemia due to mutations in apoA-I, ABCA1, and LCAT is associated with increased progression of atherosclerosis. In contrast, hyperalphalipoproteinemia as a result of loss of cholesteryl ester transfer protein function is associated with unaltered atherosclerosis progression compared with family controls. This insight is of interest, since it can assist in the prioritizing of antiatherogenic therapy by increasing HDL cholesterol levels.     

Increased risk of coronary artery disease in Caucasians with extremely low HDL cholesterol due to mutations in ABCA1, APOA1, and LCAT

Mutations in ABCA1, APOA1, and LCAT reduce HDL cholesterol (HDLc) in humans. However, the prevalence of these mutations and their relative effects on HDLc reduction and risk of coronary artery disease (CAD) are less clear. Here we searched for ABCA1, APOA1, and LCAT mutations in 178 unrelated probands with HDLc < 10th percentile but no other major lipid abnormalities, including 89 with ≥ 1 first-degree relative with low HDLc (familial probands) and 89 where familial status of low HDLc is uncertain (unknown probands). Mutations were most frequent in LCAT (15.7%), followed by ABCA1 (9.0%) and APOA1 (4.5%), and were found in 42.7% of familial but only 14.6% of unknown probands (p = 2.44 ∗ 10^− 5). Interestingly, only 16 of 24 (66.7%) mutations assessed in families conferred an average HDLc < 10th percentile. Furthermore, only mutation carriers with HDLc < 5th percentile had elevated risk of CAD (odds ratio (OR) = 2.26 for 34 ABCA1 mutation carriers vs. 149 total first-degree relative controls, p = 0.05; OR = 2.50 for 26 APOA1 mutation carriers, p = 0.04; OR = 3.44 for 38 LCAT mutation carriers, p = 1.1 ∗ 10^− 3). These observations show that mutations in ABCA1, APOA1, and LCAT are sufficient to explain > 40% of familial hypoalphalipoproteinemia in this cohort. Moreover, individuals with mutations and large reductions in HDLc have increased risk of CAD. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Segregation of LIPG, CETP, and GALNT2 Mutations in Caucasian Families with Extremely High HDL Cholesterol

To date, few mutations are described to underlie highly-elevated HDLc levels in families. Here we sequenced the coding regions and adjacent sequence of the LIPG, CETP, and GALNT2 genes in 171 unrelated Dutch Caucasian probands with HDLc≥90th percentile and analyzed segregation of mutations with lipid phenotypes in family members. In these probands, mutations were most frequent in LIPG (12.9%) followed by GALNT2 (2.3%) and CETP (0.6%). A total of 6 of 10 mutations in these three genes were novel (60.0%), and mutations segregated with elevated HDLc in families. Interestingly, the LIPG mutations N396S and R476W, which usually result in elevated HDLc, were unexpectedly found in 6 probands with low HDLc (i.e., ≤10th percentile). However, 5 of these probands also carried mutations in ABCA1, LCAT, or LPL. Finally, no CETP and GALNT2 mutations were found in 136 unrelated probands with low HDLc. Taken together, we show that rare coding and splicing mutations in LIPG, CETP, and GALNT2 are enriched in persons with hyperalphalipoproteinemia and segregate with elevated HDLc in families. Moreover, LIPG mutations do not overcome low HDLc in individuals with ABCA1 and possibly LCAT and LPL mutations, indicating that LIPG affects HDLc levels downstream of these proteins.