apoC-III Isoforms Research Articles

Distinct patterns of apolipoprotein C-I, C-II, and C-III isoforms are associated with markers of Alzheimer’s disease

Apolipoproteins C-I, C-II, and C-III interact with ApoE to regulate lipoprotein metabolism and contribute to Alzheimer's disease pathophysiology. In plasma, apoC-I and C-II exist as truncated isoforms, while apoC-III exhibits multiple glycoforms. This study aimed to 1) delineate apoC-I, C-II, and C-III isoform profiles in cerebrospinal fluid (CSF) and plasma in a cohort of nondemented older individuals (n = 61), and 2) examine the effect of APOE4 on these isoforms and their correlation with CSF Aβ42, a surrogate of brain amyloid accumulation. The isoforms of the apoCs were immunoaffinity enriched and measured with MALDI-TOF mass spectrometry, revealing a significantly higher percentage of truncated apoC-I and apoC-II in CSF compared with matched plasma, with positive correlation between CSF and plasma. A greater percentage of monosialylated and disialylated apoC-III isoforms was detected in CSF, accompanied by a lower percentage of the two nonsialylated apoC-III isoforms, with significant linear correlations between CSF and plasma. Furthermore, a greater percentage of truncated apoC-I in CSF and apoC-II in plasma and CSF was observed in individuals carrying at least one APOE Ɛ4 allele. Increased apoC-I and apoC-II truncations were associated with lower CSF Aβ42. Finally, monosialylated apoC-III was lower, and disialylated apoC-III greater in the CSF of Ɛ4 carriers. Together, these results reveal distinct patterns of the apoCs isoforms in CSF, implying CSF-specific apoCs processing. These patterns were accentuated in APOE Ɛ4 allele carriers, suggesting an association between APOE4 genotype and Alzheimer's disease pathology with apoCs processing and function in the brain.

Identification of New Apolipoprotein-CIII Glycoforms with Ultrahigh Resolution MALDI-FTICR Mass Spectrometry of Human Sera

Apolipoprotein-CIII (apoCIII) is an abundant blood glycoprotein associated with lipoprotein particles. Three different glycoforms have been described, all containing a mucin-type core-1 O-glycosylation with either zero, one or two sialic acids. Changes in the relative abundance of these glycoforms have been observed in a variety of different pathologies. In this study, ultrahigh resolution 15T MALDI Fourier transform ion cyclotron resonance (FTICR) MS was used to analyze apoCIII isoforms in serum protein profiles. For this purpose, serum proteins were purified using both a fully automated RPC18-based magnetic bead method and an RPC4 cartridge-based solid phase extraction method. Six new apoCIII isoforms were identified with low-ppm mass measurement errors and ultrahigh precision. These were characterized by more complex glycan moieties that are fucosylated instead of sialylated. To confirm the glycan moiety and localize the glycosylation site, top-down ESI-FTICR-MS/MS and bottom-up LC-ion trap MS/MS were used. A large variation in the presence and abundance of the fucosylated isoforms was found in a set of 96 serum samples. These findings of fucosylated apolipoprotein-CIII isoforms warrant further research to elucidate the implications these glycoforms may have for the plethora of studies where alterations in apoCIII have been linked to the development of many different pathologies.

Plasma apolipoprotein C-III metabolism in patients with chronic kidney disease

Moderate chronic kidney disease (CKD) (defined by an estimated glomerular filtration rate of 30-60 ml/min) is associated with mild hypertriglyceridemia related to delayed catabolism of triglyceride-rich lipoprotein particles. Altered apolipoprotein C-III (apoC-III) metabolism may contribute to dyslipidemia in CKD. To further characterize the dyslipidemia of CKD, we investigated the kinetics of plasma apoC-III in 7 nonobese, nondiabetic, non-nephrotic CKD subjects and 7 age- and sex-matched healthy controls, using deuterated leucine ([5, 5, 5, ²H₃]leucine), gas chromatography-mass spectrometry, and multicompartmental modeling. Compared with controls, CKD subjects had higher concentrations of plasma and VLDL triglycerides and plasma and VLDL apoC-III (P < 0.05). The increased plasma apoC-III concentration was associated with a decreased apoC-III fractional catabolic rate (FCR) (1.21 ± 0.15 vs. 0.74 ± 0.12 pools/day, P = 0.03). There were no differences between apoC-III production rates of controls and those of CKD subjects. In CKD subjects, plasma apoC-III concentration was significantly and negatively correlated with apoC-III FCR (r = -0.749, P = 0.05) but not with apoC-III production rate. Plasma apoC-III concentration was positively correlated with plasma and VLDL triglycerides and VLDL apoB concentrations and negatively correlated with VLDL apoB FCR (P < 0.05 for all). ApoC-III FCR was negatively correlated with plasma and VLDL triglycerides and VLDL apoB concentration and positively correlated with VLDL apoB FCR (P < 0.05 for all). Altered plasma apoC-III metabolism is a feature of dyslipidemia in moderate CKD. Modification of apoC-III catabolism may be an important therapeutic target for reducing cardiovascular disease risk in moderate CKD.

Analysis of N‐ and O‐linked protein glycosylation in children with Prader‐Willi syndrome

Current genotype-phenotype correlations in Prader-Willi syndrome (PWS) are struggling to give an explanation of the diversity in phenotype and there is a need to move towards a molecular understanding of PWS. A range of functions related to glycoproteins are involved in the pathophysiology of PWS and it may be that abnormal glycosylation is contributing to the biological phenotype. The objective of this study was to investigate the state of N- and O-linked glycosylation in children with Prader-Willi syndrome. Twenty-three children with PWS and 20 non-PWS controls were included in the study. Protein N-linked glycosylation was assessed by analysing serum transferrin through mass spectrometry and protein O-linked through isoelectric focusing (IEF) of serum apolipoprotein C-III (apoC-III), confirmed by mass spectrometry. The results of this analysis indicated that the N-linked glycosylation pathway in PWS is normal. A subgroup of PWS individuals was found to have a hyposialylated pattern of apoC-III isoforms. This was independent of the underlying genetic mechanism and is the first report of an apoC-III IEF abnormality in PWS. This is the first report of apoC-III hyposialylation in PWS. As this field is in its infancy, additional study is required before these findings may be used in clinical settings.

ApoCIII-Enriched LDL in Type 2 Diabetes Displays Altered Lipid Composition, Increased Susceptibility for Sphingomyelinase, and Increased Binding to Biglycan

OBJECTIVEApolipoprotein CIII (apoCIII) is an independent risk factor for cardiovascular disease, but the molecular mechanisms involved are poorly understood. We investigated potential proatherogenic properties of apoCIII-containing LDL from hypertriglyceridemic patients with type 2 diabetes.RESEARCH DESIGN AND METHODSLDL was isolated from control subjects, subjects with type 2 diabetes, and apoB transgenic mice. LDL-biglycan binding was analyzed with a solid-phase assay using immunoplates coated with biglycan. Lipid composition was analyzed with mass spectrometry. Hydrolysis of LDL by sphingomyelinase was analyzed after labeling plasma LDL with [3H]sphingomyelin. ApoCIII isoforms were quantified after isoelectric focusing. Human aortic endothelial cells were incubated with desialylated apoCIII or with LDL enriched with specific apoCIII isoforms.RESULTSWe showed that enriching LDL with apoCIII only induced a small increase in LDL-proteoglycan binding, and this effect was dependent on a functional site A in apoB100. Our findings indicated that intrinsic characteristics of the diabetic LDL other than apoCIII are responsible for further increased proteoglycan binding of diabetic LDL with high-endogenous apoCIII, and we showed alterations in the lipid composition of diabetic LDL with high apoCIII. We also demonstrated that high apoCIII increased susceptibility of LDL to hydrolysis and aggregation by sphingomyelinases. In addition, we demonstrated that sialylation of apoCIII increased with increasing apoCIII content and that sialylation of apoCIII was essential for its proinflammatory properties.CONCLUSIONSWe have demonstrated a number of features of apoCIII-containing LDL from hypertriglyceridemic patients with type 2 diabetes that could explain the proatherogenic role of apoCIII.

Apolipoprotein C-III isoforms: kinetics and relative implication in lipid metabolism

Apolipoprotein C-III (apoC-III) production rate (PR) is strongly correlated with plasma triglyceride (TG) levels. ApoC-III exists in three different isoforms, according to the sialylation degree of the protein. We investigated the kinetics and respective role of each apoC-III isoform in modulating intravascular lipid/lipoprotein metabolism. ApoC-III kinetics were measured in a sample of 18 healthy men [mean age (+/-SD) 42.1 +/- 9.5 years, body mass index 29.8 +/- 4.6 kg/m2] using a primed-constant infusion of l-(5,5,5-D3) leucine for 12 h. Mono-sialylated and di-sialylated apoC-III (apo-CIII1 and apoC-III2) exhibited similar PRs (means +/- SD, 1.22 +/- 0.49 mg/kg/day vs. 1.15 +/- 0.59 mg/kg/day, respectively) and similar fractional catabolic rates (FCRs) (0.51 +/- 0.13 pool/day vs. 0.61 +/- 0.24 pool/day, respectively). Nonsialylated apoC-III (apoC-III0) had an 80% lower PR (0.25 +/- 0.12 mg/kg/day) and a 60% lower FCR (0.21 +/- 0.07 pool/day) (P < 0.0001 for comparison with CIII1 and CIII2 isoforms). The PRs of apoC-III1 and apoC-III2 were more strongly correlated with plasma TG levels (r > 0.8, P < 0.0001) than was apoC-III0 PR (r = 0.54, P < 0.05). Finally, the PR of apoC-III2 was strongly correlated with the proportion of LDL <255 A (r = 0.72, P = 0.002). These results suggest that all apoC-III isoforms, especially the predominant CIII1 and CIII2 isoforms, contribute to hypertriglyceridemia and that apoC-III2 may play a significant role in the expression of the small, dense LDL phenotype.

Mass spectral study of polymorphism of the apolipoproteins of very low density lipoprotein

New isoforms of apolipoprotein (apo)C-I and apoC-III have been detected in delipidated fractions from very low density lipoprotein (VLDL) using matrix-assisted laser desorption (MALDI) and electrospray ionization (ESI) mass spectrometry (MS). The cleavage sites of truncated apoC-III isoforms have also been identified. The VLDL fractions were isolated by fixed-angle single-spin ultracentrifugation using a self-generating sucrose density gradient and delipidated using a newly developed C18 solid phase extraction protocol. Fifteen apoC isoforms and apoE were identified in the MALDI spectra and the existence of the more abundant species was verified by ESI-MS. The relative intensities of the apoCs are closely correlated in three normolipidemic subjects. A fourth subject with type V hyperlipidemia exhibited an elevated apoC-III level and a suppressed level of the newly discovered truncated apoC-I isoform. ApoC-II was found to be particularly sensitive to in vitro oxidation. The dynamic range and specificity of the MALDI assay shows that the complete apoC isoform profile and apoE phenotype can be obtained in a single measurement from the delipidated VLDL fraction. —Bondarenko, P. V., S. L. Cockrill, L. K. Watkins, I. D. Cruzado, and R. D. Macfarlane. Mass spectral study of polymorphism of the apolipoproteins of very low density lipoprotein. J. Lipid Res. 1999. 40: 543–555.

Isoforms of rat apolipoproteins and changes induced by insulin deficiency and fasting

Reasons for the disordered lipoprotein metabolism in insulin deficiency are not completely understood. In this study the apolipoproteins from plasma of fed and fasted streptozotocin-induced insulin-deficient rats were compared with normal control rats. Analysis of the apolipoprotein isoforms by two-dimensional electrophoresis revealed increased proportions of sialylated apo E and of sialylated apo C-III in diabetic rats compared with control rats. Fasting increased the proportion of sialylated apo E but not the proportion of sialylated apo C-III. 3H-labeled leucine was injected into normal and insulin-deficient rats, followed by a chase of unlabeled leucine after 30 min. Blood samples were collected at intervals over 24 h and the apolipoprotein components were separated by two-dimensional electrophoresis. The relative specific activities of sialylated isoforms of apo E were less than the relative specific activities of non-sialylated apo E isoforms. In contrast, sialylated isoforms of apo C-III had higher relative specific activities than non-sialylated apo C-III. No interconversions of apo E or apo C-III isoforms were found within the lipoprotein fractions. In insulin-deficient diabetic rats the relative specific activities of sialylated apo E and apo C-III isoforms were both increased relative to non-sialylated isoforms when compared with control rats. The results of this study suggest that the isoforms of apo E and apo C-III associated with the plasma lipoproteins of diabetic rats are changed in parallel with changes in synthesis of the isoforms. The changes in association with the isoforms of the apolipoproteins possibly contribute to abnormal metabolism of plasma lipoproteins in insulin deficiency.

Site-specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity of apolipoproteins A-I, C-II, and C-III.

ApoA-I and apoC-II are eluted in two isoforms and apoC-III2 is eluted in three isoforms by reversed phase high performance liquid chromatography (HPLC). The structural basis of these nongenetic heterogeneities was unravelled using HPLC of proteolytic peptides and time-of-flight secondary ion mass spectrometry (TOF-SIMS). In apoA-I, the chromatographic microheterogeneity was caused by the formation of methionine sulfoxides (MetSO). However, only residues Met112 and Met148 were found oxidized, whereas Met86 was unaffected and also resistant towards artificial oxidation. To assess whether and to what extent amino acid substitutions in apoA-I might affect methionine sulfoxidation, the tryptic peptides of 13 different mutant apoA-I proteins from 24 heterozygous apoA-I variant carriers were analyzed by HPLC. In normal apoA-I, the ratios MetSO112/Met112 and MetSO148/Met148 were highly variable. By contrast, the relative ratio of oxidation of methionine residues 112 and 148 was constant. The amino acid changes Lys107----Met, Lys107----O, Glu139----Gly, Glu147----Val, and Pro165----Arg resulted in the preferential oxidation of Met112, and Asp103----Asn resulted in a preferential oxidation of Met148; whereas Pro3----Arg, Pro3----His, Pro4----Arg, Asp89----Glu, Ala158----Asp, Glu198----Lys, and Asp213----Gly had no impact. ApoC-II and apoC-III isoforms differed by the oxidation of the two methionine residues in these proteins. Whereas in apoC-II both methionine residues were oxidized in parallel, in apoC-III the two methionine residues differed in their susceptibility towards oxidation. We conclude that the formation of MetSO depends on the molecular microenvironment within a protein.

Characterization of an apolipoprotein C-III mutant by high-performance liquid chromatography and time-of-flight secondary ion mass spectrometry

Apolipoprotein (apo) C-III isoforms from a patient with a mutant apo C-III and from controls were isolated to homogeneity byisoelectric focusing and subjected to proteolytic digestion. The peptides obtained were separated by reversed-phase high-perforance liquid chromatography, and their molecular masses were determined by time-of-flight secondary ion mass spectrometry. Molecular masses of peptides derived from apo C-III 0, C-III 1 and C-III 2 were indistinguishable from control preparations, whereas the mutant apo C-III contained a COOH-terminal, carbohydrate-containing peptide with an abnormal retention time in high-performance liquid chromatography and a molecular mass higher by 291 daltons owing to oversialation at position 74 of the amino acid sequence (apo C-III 3).

Effects of severe caloric restriction on the degree of sialylation of apoprotein C-III in obese women.

Alterations in the relative proportions of very low density lipoprotein (VLDL) apoprotein C-III (apoC-III) isoforms have been previously observed under various dietary and metabolic states. This study was designed to evaluate the effects of a very high caloric restriction on plasma triglycerides and on the relative proportions of VLDL apoprotein C-II (apoC-II) and apoC-III isoforms in obese subjects. VLDL were isolated by preparative ultra-centrifugation from 12 obese women. ApoC-II and apoC-III subspecies were separated by analytical ultrathin-layer isoelectrofocusing. The mean body weight decreased significantly from 96.2 +/- 8.7 to 90.6 +/- 7.6 kg (p less than 0.01). Mean total and VLDL triglycerides did not vary significantly. Apoprotein C-III2 (apoC-III2) as percentage of total apoprotein C increased (p less than 0.01) and apoprotein CIII0 (apoC-III0) decreased (p less than 0.01). Apoprotein C-III1 (apoC-III1) did not vary significantly. An inverse correlation was found between the percentage variation of apoC-III2 and that of apoC-III1 (r = -0.94; p less than 0.01). The variations of apoC-III2 correlated positively (r = 0.83; p less than 0.01), while those of apoC-III1 correlated inversely (r = -0.65, p less than 0.025) with the changes of VLDL triglycerides. The apoC-III1 to apoC-III2 ratio as well as the apoC-III0 to apoC-III2 and apo C-III1 ratios decreased after diet (p from less than 0.01 to less than 0.001). Total apoC-III as well as apoC-III2 and apoC-III1 to apoC-II ratios did not vary.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of guar gum on plasma lipoproteins and apolipoproteins C-II and C-III in patients affected by familial combined hyperlipoproteinemia

In recent years, guar gum has been shown to be a potent hypocholesterolemic agent. The effects of this fiber on triglycerides are less clear. In order to evaluate the influence of guar supplementation on plasma lipoproteins and apolipoprotein C-II (apoC-II) and apolipoprotein C-III (apoC-III) isoforms (apoC-III2, apoC-III1, apoC-III0), 16 g/day of guar gum were administered to 12 outpatients affected by familial combined hyperlipoproteinemia for a period of 60 days. Mean total cholesterol and triglyceride levels significantly decreased after 15 days of treatment and persisted reduced at the 30th and 60th day of guar supplementation. While low-density lipoprotein cholesterol paralleled the reduction of total plasma cholesterol, we did not observe any change in the cholesterol content of high-density lipoprotein (HDL) subfractions (HDL2 and HDL3) during the study. A redistribution of the relative content of very low-density lipoprotein apoC-III isoforms with a significant increase of apoC-III1 and a decrease of apoC-III0 was observed after 15, 30, and 60 days of guar gum administration. The results show that guar gum reduces not only cholesterol but also triglyceride levels in patients affected by familial combined hyperlipoproteinemia. Further studies are needed to confirm the suggestion that the different distribution of very low-density lipoprotein apoC-III isoforms induced by guar supplementation may influence the behavior of plasma triglycerides.

The Degree of Sialylation of ApoC-III Is Altered by Diet

ApoC-III, an apolipoprotein that contains from 0 to 3 or more moles of sialic acid per mole of protein, plays important roles in lipoprotein metabolism. A high carbohydrate diet alters the relative proportions of the variously sialylated isoforms of ApoC-III in the plasma VLDL of man and of rat. Hepatic perfusion experiments in the rat indicate that the diet produces its affects on plasma VLDL by altering the hepatic output of ApoC-III. Diet is a relatively long-term stimulus to VLDL secretion. Our aim was to ascertain whether the changes in ApoC-III sialylation patterns could be reproduced by an acute stimulus to increased hepatic VLDL secretion, i.e., the perfusion of rat livers with media containing high levels of free fatty acid (FFA). Male Sprague-Dawley rats (∼250 g) were fed either Purina rat meal or a high carbohydrate formula for 3 wk. Livers were perfused with Krebs-Ringers bicarbonate, 3% bovine serum albumin containing either no added FFA, or 200 mg of added oleate. ApoB and ApoC-III3 were measured by radioimmunoassay and lipids by enzymatic assays. Lipoproteins were isolated by preparative or zonal ultracentrifugation. The relative proportions of the various isoforms of ApoC-III were quantified by isoelectric focusing. The carbohydrate diet increased the plasma levels of triglycerides and ApoC-III3 ∼ twofold. The diet also increased the proportions of ApoC-III0 relative to total ApoC-III from 33 to 42%, in plasma VLDL (P &amp;lt; 0.02). Perfusates of livers of carbohydrate-fed rats accumulated VLDL-TG and ApoC-III3 at increased rates (93 versus 211, P &amp;lt; 0.001, and 12.7 versus 23.5, P &amp;lt; 0.05, μg/g/h, respectively) and the relative proportions of perfusate VLDL ApoC-lll were altered: ApoC-III0 rose from 37% to 59% and ApoC-III3 fell from 60% to 38% (P &amp;lt; 0.02 for each). Perfusion of livers of control (meal-fed) animals with 200 mg oleate yielded twofold increases in the rates of accumulation of VLDL-TG over meal fed non-FFA perfused controls and changes in gross VLDL composition that resembled those produced by carbohydrate feeding, but ApoC-III3 levels were not increased and the relative proportions of ApoC-III0, ApoC-III, 2 and ApoC-III3 were not altered. Perfusion of livers of carbohydrate-fed rats with oleate produced fivefold and 2.5-fold increases in the rates of accumulation of VLDL-TG and ApoC-III3, respectively. The proportion of ApoC-III isoforms were identical with those seen in the livers of carbohydrate-fed rats perfused without added oleate. Thus, the changes in ApoC-lll sialylation were related more to diet than to the presence of added FFA, i.e., an acute stimulus did not reproduce the changes seen with diet. These data indicate that the sialylation of ApoC-III is under metabolic control.