Apolipoprotein E ε4 (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), yet whether its pathogenic effects are driven by HDL lipidation state or by elevated APOE4 levels in the CNS remains unclear. Using ADNI data, we analyzed cerebrospinal fluid (CSF) and plasma small and large HDL particle concentrations, total APOE levels, and isoform-specific APOE3 and APOE4 protein levels in 144 participants with APOE ε3/ε3, ε3/ε4, or ε4/ε4 genotypes, grouped by cognitive state and memory progression over 4 years. Memory loss was defined as ≥ 10% decline on the Rey Auditory Verbal Learning Test (RAVLT) delayed recall between baseline and 48-month follow-up. Cross-sectional associations with CSF Aβ1-42, total tau, and p-tau181 were evaluated using covariate-adjusted linear regression, and longitudinal trajectories were examined using linear mixed-effects models over 6 years. CSF small and large HDL levels were higher in cognitively normal individuals and non-progressors, while APOE4 carriers exhibited reduced CSF small HDL relative to ε3 homozygotes. Importantly, APOE4 status moderated HDL-biomarker associations in opposing directions: in carriers, higher CSF small HDL was associated with lower tau and Aβ1-42, whereas in non-carriers, higher CSF small HDL was associated with higher CSF tau levels. Higher CSF APOE4 protein levels were associated with elevated tau and p-tau181, while plasma APOE measures showed minimal and often opposing associations with CSF biomarkers. Longitudinally, higher baseline CSF APOE4 proportion was associated with greater memory decline over 6 years but did not predict biomarker change. These findings argue against a protective role of APOE4 lipidation in AD and instead support a CNS-compartment model in which APOE4 protein burden aligns more closely with tau pathology and cognitive decline than peripheral APOE or HDL measures.

The apolipoprotein E (ApoE) Ɛ4 allele is associated with a significant risk for both late-onset Alzheimer’s Disease (AD) development and cerebral amyloidosis, but the degree to which cerebrospinal fluid (CSF) apoE glycosylation affects disease progression is unclear. The objective of this study was to examine the relationship of CSF apoE glycosylation with t-tau, p-tau181, and Aβ1-42 CSF levels, and to delineate the effect of the APOE4+ genotype (vs E4-) on glycosylation. Total glycosylation and apoE isoform-specific glycosylation were analyzed in baseline plasma and CSF samples from a longitudinal cohort of older individuals (n=188, ages 55 - 89) from the Alzheimer’s Disease Neuroimaging Initiative (ADNI). ApoE glycosylation was assayed using Mass Spectrometric Immunoassay (MSIA) that simultaneously detects the apoE isoforms and their glycosylation percentage. We performed pairwise comparisons between each apoE isoform and utilized linear mixed-effects modeling to analyze longitudinal associations of apoE glycosylation with AD biomarkers, adjusting for age, sex, race, education, and clinical status. We examined glycosylation as a function of individual apoE isoforms and found that both total and secondary CSF glycosylation had the same trend: the E4 isoform had an overall lower % glycosylation compared to E2 and E3 isoforms (p<0.0001). Similarly, the E3 isoform had lower total and secondary CSF % glycosylation compared to the E2 isoform (p<0.0001). We further assessed whether the % apoE glycosylation in the baseline samples were associated with CSF t-tau, p-tau181, and Aβ1-42 CSF levels in the longitudinal samples. Total CSF and secondary CSF glycosylation demonstrated highly significant associations with decreased levels of t-tau (p < 0.002; p < 0.02 respectively) and p-Tau181 (p< 0.006; p<0.02 respectively) over a 10-year period. Secondary CSF glycosylation was significantly positively correlated with increased CSF Aβ1-42 levels over a 10-year period (p < 0.04). The degree of CSF ApoE glycosylation is strongly associated with concentrations of CSF t-tau, p-Tau181, and Aβ1-42, and is predictive of biomarker changes over time. These novel discoveries underscore the importance of understanding the mechanisms involved in apoE glycosylation in the brain, which can lead to new targets and therapies for AD.

Apolipoprotein A-I (apoA-I) plays beneficial roles as the major structural and functional protein on plasma high-density lipoproteins (HDL). However, APOA1 gene mutations can cause protein misfolding and pathologic amyloid deposition in various organs in human hereditary AApoAI amyloidosis, a potentially lethal systemic disease. We report esophageal and duodenal AApoAI amyloidosis in a 56-year-old patient with Barrett's esophagus, a condition involving chronic acid reflux. Amyloid deposits contained full-length apoA-I featuring a novel D20Y mutation identified by gene sequencing and protein mass spectrometry. Genetic analysis of asymptomatic family members revealed autosomal dominant inheritance. Fibril formation by the full-length variant apoA-I rather than its fragments and the location of the mutation in a conserved amyloid-prone N-terminal segment were highly unusual for hereditary AApoA-I amyloidosis. Structural and stability studies of the recombinant D20Y and wild-type apoA-I showed small but significant mutation-induced structural perturbations in the native lipid-free protein at pH 7.4. Major destabilization and aggregation of the variant protein were observed at pH 4.0. We propose that acidic conditions in Barrett's esophagus promoted protein misfolding and amyloid formation by the D20Y variant. These findings expand our understanding of the clinical features and molecular basis of AApoAI amyloidosis and suggest clinical strategies.

The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD). ApoE is glycosylated with an O-linked Core-1 sialylated glycan at several sites; however, the impact and function of this glycosylation on AD biomarkers remain unclear. We examined apoE glycosylation (total and secondary) in a cohort of cerebrospinal fluid (CSF, n = 181) and plasma (n = 178) samples from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) stratified into 4 groups: cognitively normal (CN), Mild Cognitive Impairment (MCI), progressors, and non-progressors based on delayed word recall performance over 4 years. We observed decreasing glycosylation (reduced

Apolipoprotein C-I (apoC-I) is a key regulator of triglyceride and HDL metabolism. Preclinical data also indicate a role of apoC-I in insulin resistance (IR) and hyperglycemia. In circulation, apoC-I appears as native (C-I) and truncated (C-I’) proteoform. Higher ratio of C-I’ to C-I (C-I’/C-I) is associated with more favorable cardiometabolic risk profiles. We now tested if C-I’/C-I is associated with longitudinal changes in fasting glucose (FG) and IR, and incident diabetes. ApoC-I proteoforms were measured by mass spectrometry immunoassay in plasma from 4,747 participants of the Multi-Ethnic Study of Atherosclerosis that were without diabetes at baseline. IR estimated by HOMA-IR was measured at baseline and at ~10-year follow-up. FG and incident diabetes (FG > 6.99 mmol/l or on diabetes medications) were evaluated for up to 16 years. Lower baseline C-I’/C-I was associated with greater longitudinal increases in FG and IR (by 2.7% [95%CI: 1.5, 3.9] and 4.3% [1.1, 7.3] per 1 SD decrease in C-I’/C-I), and increased risk of diabetes (n=768 events) after adjusting for baseline age, sex, race & ethnicity, income, education, BMI, systolic BP, FG, fasting insulin, antihypertensives and statins use, eGFR and plasma lipids (Hazard ratio: 1.19 [95% CI: 1.10, 1.28]). The association with diabetes was stronger in those with normal than impaired FG (1.20 [1.08, 1.33] vs. 1.09 [0.96, 1.23], p-interaction 0.023). Total apoC-I concentrations were not associated with changes in FG and IR in all models, and with incident diabetes upon adjusting for risk factors. In conclusion, lower posttranslational apoC-I truncation was associated, independently of typical diabetes risk factors, with greater worsening of IR, greater increase in FG and higher risk of diabetes. The relationship of lower C-I’/C-I with diabetes risk appeared present prior to development of impaired FG. In contrast, total apoC-I concentrations were not related to worsening of glucose metabolism. Disclosure J. Koska: None. Y. Hu: None. D. Billheimer: None. D. Nedelkov: None. M. Budoff: Speaker's Bureau; Boehringer-Ingelheim, Lilly Diabetes, AstraZeneca. A. Bertoni: None. R. McClelland: None. P. Reaven: Research Support; Dexcom, Inc. Funding National Heart, Lung, and Blood Institute (R01-HL-138969)

Objective: Higher truncated-to-native apolipoprotein (apo) C-I proteoform ratios (C-I’/C-I) are associated with favorable cardiometabolic risk profiles, but its relationship with longitudinal changes in insulin resistance and incident diabetes is unknown. Research Design and Methods: Plasma apoC-I proteoforms were measured by mass spectrometry immunoassay at baseline in 4,742 non-diabetic MESA and 524 prediabetes ACT NOW participants. The primary outcome was incident diabetes (fasting glucose [FG] ≥7.0 mmol/l or hypoglycemic medications use in MESA; FG ≥7.0 mmol/l or 2-hour OGTT glucose ≥ 11.1 mmol/l in ACT NOW). Secondary outcomes were changes in FG and HOMA-IR in MESA, and OGTT-glucose area under the curve (AUCglucose) and Matsuda insulin sensitivity index (ISI) in ACT NOW. Results: In MESA, higher C-I’/C-I was associated with lower risk of diabetes (n=564 events, HR: 0.87 [95%CI: 0.79, 0.95] per SD, p=0.0036, median follow-up 9 years), and smaller increases (follow-up adjusted for baseline) in FG (-0.5%, p<0.0001) and HOMA-IR (-2.9%, p=0.011) after adjusting for baseline clinical and demographic covariates, including plasma triglycerides and HDL-C. Total apoC-I concentrations were not associated with changes in FG, HOMA-IR and incident diabetes. In ACT NOW, higher C-I’/C-I was associated with smaller increases in AUCglucose (-1.8%, p=0.0052), greater increases in ISI (7.2%, p=0.0095), and lower risk of diabetes (n=59 events, 0.66 [0.48, 0.91], p=0.004, median follow-up 2.5 years) after adjusting for treatment group and diabetes risk factors, including plasma lipids. Conclusion: Our results indicate that apoC-I truncation may contribute to changes in glucose levels and insulin resistance, and risk of diabetes.

Background and aims: Apolipoprotein C-III (apoC-III) proteoform composition shows distinct relationships with plasma lipids and cardiovascular risk. The present study tested whether apoC-III proteoforms are associated with risk of peripheral artery disease (PAD). Methods: ApoC-III proteoforms, i.e., native (C-III0a), and glycosylated with zero (C-III0b), one (C-III1) or two (C-III2) sialic acids, were measured by mass spectrometry immunoassay on 5,734 Multi-Ethnic Study of Atherosclerosis participants who were subsequently followed for clinical PAD over 17 years. Ankle-brachial index (ABI) was also assessed at baseline and then 3 and 10 years later in 4,830 participants. Results: Higher baseline C-III0b/C-III1 and lower baseline C-III2/C-III1 were associated with slower decline in ABI (follow-up adjusted for baseline) over time, independently of cardiometabolic risk factors, and plasma triglycerides and HDL cholesterol levels (estimated difference per 1 SD was 0.31 % for both, p < 0.01). The associations between C-III2/C-III1 and changes in ABI were stronger in men (-1.21 % vs. -0.27 % in women), and in Black and Chinese participants (-0.83 % and -0.86 % vs. 0.12 % in White). Higher C-III0b/C-III1 was associated with a trend for lower risk of PAD (HR = 0.84 [95%CI: 0.67-1.04]) that became stronger after excluding participants on lipid-lowering medications (0.73 [95%CI: 0.57-0.94]). Neither change in ABI nor clinical PAD was related to total apoC-III levels. Conclusions: We found associations of apoC-III proteoform composition with changes in ABI that were independent of other risk factors, including plasma lipids. Our data further support unique properties of apoC-III proteoforms in modulating vascular health that go beyond total apoC-III levels.

Apolipoprotein C-I (apoC-I) is key regulator of triglyceride and HDL metabolism. It appears as native and truncated posttranslational proteoforms. Higher truncated-to-native apoC-I proteoforms ratio (C-I’/C-I) is associated with favorable cardiometabolic risk profile. Whether apoC-I proteoform composition is associated with declines in insulin sensitivity and secretion, and incident diabetes is unknown. Plasma apoC-I proteoforms (by mass spectrometry immunoassay) were measured at baseline in 524 participants of the ACT NOW, a placebo-controlled study of pioglitazone in impaired glucose tolerance. Baseline and follow-up indices of insulin sensitivity (ISI, by Matsuda’s equation) and secretion (∆I/∆G calculated as AUCinsulin/AUCglucose) were derived from glucose and insulin concentrations during an OGTT. At baseline, C-I’/C-I correlated with both ISI and ∆I/∆G (r=0.26; r=-0.17, both p<0.0001). Prospectively, higher baseline C-I’/C-I was associated with higher ISI (by 9.4% [95% CI: 4.1, 13.9] per 1 SD; follow-up adjusted for baseline, time of follow-up and treatment group), lower ∆I/∆G (-4.9% [-7.7, -1.0]) and reduced diabetes risk (n=59 events, HR 0.75 [95%CI: 0.57-0.98]). The C-I’/C-I associations with insulin sensitivity and diabetes were independent of diabetes risk factors (e.g., BMI) and plasma lipids (HR 0.72 [0.54-0.96]). The C-I’/C-I association with diabetes was attenuated after adjusting for change (follow-up adjusting for baseline) in ISI (HR 0.82 [0.61-1.11]) but not in ∆I/∆G (HR 0.67 [0.49-0.93]). In conclusion, lower apoC-I truncation was associated, independently of typical diabetes risk factors, with decline in insulin sensitivity and increased risk of diabetes in individuals with impaired glucose tolerance. The association with incident diabetes appears to reflect favorable effects of apoC-I truncation on insulin sensitivity but not on insulin secretion. Disclosure J. Koska: None. D. Nedelkov: None. D. Billheimer: None. D. Schwenke: None. P. Reaven: Research Support; Dexcom, Inc. Funding National Institutes of Health (R24-DK090958); Takeda Pharmaceuticals

Higher truncated-to-native proteoform ratios of apolipoproteins (apo) C-I (C-I’/C-I) and C-II (C-II’/C-II) are associated with less atherogenic lipid profiles, including lower triglycerides (TG) and higher HDL levels. Here we examined the relationships of C-I’/C-II and C-II’/C-II, and total apoC-I and apoC-II concentrations with coronary artery calcium (CAC) progression. Apo C-I and C-II proteoforms were measured by mass spectrometry immunoassay in 5,791 baseline MESA plasma samples. Total apo C-I and C-II concentrations were assayed in 3,851 samples. CAC was measured 1 to 4 times (mean 2.5) over 10 years. Repeated measures regression models were adjusted for age, gender, race/ethnicity, and follow-up time (Model 1), then for BMI, diabetes, systolic BP, use of tobacco, statins and antihypertensive medications (Model 2), and then for plasma HDL and TG (Model 3). Among 5,069 participants with at least one follow-up CAC CT scan, 2,513 had CAC progression defined as events of CAC changing from 0 to > 0, or ≥ 10 units/yr from CAC > 0 but ≤ 100, or ≥ 10%/yr from CAC > 100. Progression was inversely associated with C-I’/C-I (Risk Ratio per 1 SD: 0.93 [95% CI 0.87, 0.97], Model 3). In those with baseline CAC = 0 (n = 2,581), risk of CAC > 0 (897 events) was inversely associated with C-I’/C-I in Model 1 only (0.85 [0.79, 0.93]). In those with baseline CAC > 0, C-II’/C-II was positively associated with follow-up change in density score (β-estimate per 1 SD 0.02 [95% CI: 0.001, 0.03] units) in Model 3, indicating a potentially more stable plaque. Baseline-adjusted follow-up density score was also positively related to total apoC-I in Model 2 (0.02 [0.004, 0.04]). None of the CAC measures were related to total apoC-II. In conclusion, increased truncation of apo C-I and C-II is associated with reduced CAC progression and/or improved stability of coronary atherosclerotic plaque that is not explained by total concentrations of both apolipoproteins. Disclosure J.Koska: None. J.Furtado: Employee; Biogen. Y.Hu: None. D.Billheimer: Advisory Panel; AstraZeneca. M.Allison: None. M.Budoff: Research Support; Novo Nordisk, Novartis, Boehringer Ingelheim Inc., Speaker's Bureau; Boehringer Ingelheim and Eli Lilly Alliance. D.Nedelkov: None. R.Mcclelland: None. P.Reaven: Research Support; Dexcom, Inc. Funding National Heart, Lung, and Blood Institute (R01HL138969, 75N92020D00001, HHSN268201500003I, N01HC95159, 75N92020D00005, N01HC95160, 75N92020D00002, N01HC95161, 75N92020D00003, N01HC95162, 75N92020D00006, N01HC95163, 75N92020D00004, N01HC95164, 75N92020D000

Carrying the apolipoprotein E (ApoE) Ɛ4 allele is associated with an increased risk of cerebral amyloidosis and late-onset Alzheimer’s disease, but the degree to which apoE glycosylation affects its development is not clear. In a previous pilot study, we identified distinct total and secondary isoform-specific cerebral spinal fluid (CSF) apoE glycosylation profiles, with the E4 isoform having the lowest glycosylation percentage (E2 > E3 > E4). In this work, we extend the analysis to a larger cohort of individuals ( n = 106), utilizing matched plasma and CSF samples with clinical measures of AD biomarkers. The results confirm the isoform-specific glycosylation of apoE in CSF, resulting from secondary CSF apoE glycosylation patterns. CSF apoE glycosylation percentages positively correlated with CSF Aβ 42 levels ( r = 0.53, p < 0.0001). These correlations were not observed for plasma apoE glycosylation. CSF total and secondary apoE glycosylation percentages also correlated with the concentration of CSF small high-density lipoprotein particles (s-HDL-P), which we have previously shown to be correlated with CSF Aβ 42 levels and measures of cognitive function. Desialylation of apoE purified from CSF showed reduced Aβ 42 degradation in microglia with E4 > E3 and increased binding affinity to heparin. These results indicate that apoE glycosylation has a new and important role in influencing brain Aβ metabolism and can be a potential target of treatment.

Background: Apo CIII (apolipoprotein CIII) is an important regulator of triglyceride metabolism and was associated with cardiovascular risk in several cohorts. It is present in 4 major proteoforms, a native peptide (CIII 0a ), and glycosylated proteoforms with zero (CIII 0b ), 1 (CIII 1 , most abundant), or 2 (CIII 2 ) sialic acids, which may differentially modify lipoprotein metabolism. We studied the relationships of these proteoforms with plasma lipids and cardiovascular risk. Methods: Apo CIII proteoforms were measured by mass spectrometry immunoassay in baseline plasma samples of 5791 participants of Multi-Ethnic Study of Atherosclerosis, an observational community-based cohort. Standard plasma lipids were collected for up to 16 years and cardiovascular events (myocardial infarction, resuscitated cardiac arrest, or stroke) were adjudicated for up to 17 years. Results: Apo CIII proteoform composition differed by age, sex, race and ethnicity, body mass index, and fasting glucose. Notably, CIII 1 was lower in older participants, men and Black and Chinese (versus White) participants, and higher in obesity and diabetes. In contrast, CIII 2 was higher in older participants, men, Black, and Chinese persons, and lower in Hispanic individuals and obesity. Higher CIII 2 to CIII 1 ratio (CIII 2 /III 1 ) was associated with lower triglycerides and higher HDL (high-density lipoprotein) in cross-sectional and longitudinal models, independently of clinical and demographic risk factors and total apo CIII. The associations of CIII 0a /III 1 and CIII 0b /III 1 with plasma lipids were weaker and varied through cross-sectional and longitudinal analyses. Total apo CIII and CIII 2 /III 1 were positively associated with cardiovascular disease risk (n=669 events, hazard ratios, 1.14 [95% CI, 1.04–1.25] and 1.21 [1.11–1.31], respectively); however, the associations were attenuated after adjustment for clinical and demographic characteristics (1.07 [0.98–1.16]; 1.07 [0.97–1.17]). In contrast, CIII 0b /III 1 was inversely associated with cardiovascular disease risk even after full adjustment including plasma lipids (0.86 [0.79–0.93]). Conclusions: Our data indicate differences in clinical and demographic relationships of apo CIII proteoforms, and highlight the importance of apo CIII proteoform composition in predicting future lipid patterns and cardiovascular disease risk.

Higher truncated-to-native proteoform ratios of apolipoproteins (apo) C-I (C-I’/C-I) and C-II (C-II’/C-II) are associated with lower triglycerides (TG) and higher HDL levels. Here we examined the relationships of C-I’/C-II and C-II’/C-II, and total apo C-I and C-II concentrations with atherosclerotic cardiovascular disease (ASCVD) and coronary heart disease (CHD). Apo C-I and C-II proteoforms were measured by mass spectrometry immunoassay at baseline in 5,765 MESA participants followed for CHD (definite myocardial infarction, resuscitated cardiac arrest or fatal CHD) and ASCVD (CHD or stroke) for up to 17 years. Baseline total apo C-I and C-II levels were measured in a subgroup of 3,848 participants. After adjusting for age, gender, and race/ethnicity, lower C-II’/C-II was associated with both ASCVD and CHD (Figure). The association of C-II’/C-II with CHD remained significant after further adjusting for BMI, diabetes, systolic BP, eGFR, use of tobacco, statins, antihypertensive medications, total apoC-II, and plasma TG, but was abolished after adjusting for HDL. Neither ASCVD nor CHD were related to total apo C-I and C-II or CI’/CI. In conclusion, increased posttranslational apoC-II truncation (but not total apo C-I or C-II) is associated with reduced cardiovascular risk, possibly explained by differences in HDL cholesterol metabolism. Disclosure J.Koska: None. J.Furtado: Employee; Biogen. Y.Hu: None. D.Nedelkov: None. D.Billheimer: Advisory Panel; AstraZeneca. M.Budoff: Research Support; Novo Nordisk, Novartis, Boehringer Ingelheim Inc., Speaker's Bureau; Boehringer Ingelheim and Eli Lilly Alliance. M.Allison: None. R.Mcclelland: None. P.Reaven: Research Support; Dexcom, Inc. Funding National Heart, Lung, and Blood Institute (R01HL138969, 75N92020D00001, HHSN268201500003I, N01HC95159, 75N92020D00005, N01HC95160, 75N92020D00002, N01HC95161, 75N92020D00003, N01HC95162, 75N92020D00006, N01HC95163, 75N92020D00004, N01HC95164, 75N92020D00007, N01HC95165, N01HC95166, N01HC95167, N01HC95168, N01HC95169); National Center for Advancing Translational Sciences (UL1TR000040, UL1TR001079, UL1TR001420)

Apolipoproteins (apo) C-I and C-II are key regulators of triglyceride and HDL metabolism. Both exist as full-size native and truncated (apoC-I'; apoC-II') posttranslational proteoforms. However, the determinants and the role of these proteoforms in lipid metabolism are unknown. Here, we measured apoC-I and apoC-II proteoforms by mass spectrometry immunoassay in baseline and 10-year follow-up plasma samples from the Multi-Ethnic Study of Atherosclerosis. We found that baseline total apoC-I (mean = 9.2 mg/dl) was lower in African Americans (AA), Chinese Americans (CA), and Hispanics (by 1.8; 1.0; 1.0 mg/dl vs. whites), higher in women (by 1.2 mg/dl), and positively associated with plasma triglycerides and HDL. Furthermore, we observed that the truncated-to-native apoC-I ratio (apoC-I'/C-I) was lower in CA, negatively associated with triglycerides, and positively associated with HDL. We determined that total apoC-II (8.8 mg/dl) was lower in AA (by 0.8 mg/dl) and higher in CA and Hispanics (by 0.5 and 0.4 mg/dl), positively associated with triglycerides, and negatively associated with HDL. In addition, apoC-II'/C-II was higher in AA and women, negatively associated with triglycerides, and positively associated with HDL. We showed that the change in triglycerides was positively associated with changes in total apoC-I and apoC-II and negatively associated with changes in apoC-I'/C-I and apoC-II'/C-II, whereas the change in HDL was positively associated with changes in total apoC-I and apoC-II'/C-II and negatively associated with change in total apoC-II. This study documents racial/ethnic variation in apoC-I and apoC-II plasma levels and highlights apolipoprotein posttranslational modification as a potential regulator of plasma lipids.

The mechanisms of how APOE4 allele (APOE4) increases the risk of Alzheimer’s disease (AD) pathology have not been fully elucidated. In cerebrospinal fluid (CSF), apoE is heavily glycosylated but its contribution to AD pathology is not known. The objective of this study was to determine the impact of APOE genotype and cognitive status on the relative abundance of apoE protein isoforms and their specific glycosylation patterns in CSF and plasma.

Apolipoprotein C-III (apoC-III) exists in four major proteoforms - unglycosylated form (C-III0a) , and glycosylated forms with zero (C-III0b) , one (C-III1, most abundant) or two (C-III2) sialic acids - which modify lipoprotein metabolism. The relation of these proteoforms with plasma lipids and cardiovascular disease (CVD) risk in a general population is unknown. Total apoC-III concentrations and apoC-III proteoforms were measured in baseline plasma of 5,787 MESA participants. Plasma lipids and CVD risk were assessed over 15 years. Proteoform percentages correlated modestly with total apoC-III (r2<0.10) . ApoC-III0b decreased and C-III2 increased with age (by 11% per 5 yrs) . Compared to non-Hispanic whites, African Americans had lower (35%) total apoC-III and C-III0b and higher C-III2 (88%) , and Chinese Americans and Hispanics had higher C-III0a (88%; 40%) . Females had higher total apoC-III, C-III1 and C-III0b (23%; 31%; 30%) , and lower C-III0a and C-III2 (17%; 28%) . Total apoC-III, C-III0b, C-III1 and C-III2 were higher in diabetes. Prospectively, in models that included baseline characteristics, lipid-lowering medications and total apoC-III, higher C-III2 to C-III1 ratio (C-III2/III1) was associated with lower triglycerides (22%) and higher HDL (19%) . After adjustment for baseline characteristics (including lipids) and total apoC-III, higher C-III0b to C-III1 ratio was associated with reduced risk of CVD (n=629 events, HR= 0.89) and coronary heart disease (n=426, HR=0.86) . In contrast, despite links with better lipids, higher C-III2/III1 was associated with increased risk of CVD (HR=1.09) . No CVD events were related to total apoC-III. Our data indicate differences in clinical and demographic determinants of total apoC-III concentrations and apoC-III proteoforms and highlight the importance of apoC-III proteoform distribution in predicting future lipid concentrations and CVD risk. Disclosure J.Koska: None. S.Sinari: None. Y.Hu: None. J.Furtado: Research Support; Eli Lilly and Company, Pfizer Inc. D.Billheimer: None. D.Nedelkov: None. R.Mcclelland: None. P.Reaven: Research Support; AstraZeneca, Dexcom, Inc. Funding National Institutes of Health (R01-HL138969)

Background: beta 2-microglobulin amyloidosis was first described in the 1980s as a protein deposition disease associated with long-term haemodialysis. More recently, two inherited forms resulting from separate point mutations in the beta 2-microglobulin gene have been identified. In this report, we detail a novel beta 2M variant, P32L, caused by a unique dinucleotide mutation that is linked to systemic hereditary beta 2-microglobulin amyloidosis. Methods: Three family members from a Portuguese kinship featured cardiomyopathy, requiring organ transplantation in one case, along with soft tissue involvement; other involvements included gastrointestinal, neuropathic and sicca syndrome. In vitro studies with recombinant P32L, P32G, D76N and wild-type beta 2-microglobulin were undertaken to compare the biophysical properties of the proteins. Results: The P32L variant was caused by the unique heterozygous dinucleotide mutation c.154_155delinsTT. Amyloid disease featured lowered serum beta 2-microglobulin levels with near equal amounts of circulating P32L and wild-type proteins; amyloid deposits were composed exclusively of P32L variant protein. In vitro studies of P32L demonstrated thermodynamic and chemical instability and enhanced susceptibility to proteolysis with rapid formation of pre-fibrillar oligomeric structures by N- and C-terminally truncated species under physiological conditions. Conclusions: This work provides both clinical and experimental evidence supporting the critical role of P32 residue replacement in beta 2M amyloid fibrillogenesis.

Apolipoproteins (apo) C-I, C-II and C-III in circulation are in several post-translational forms with distinct effects on lipoprotein metabolism. ApoC-I and apoC-II exist as dominant native and minor truncated (C-I’; C-II’) forms; apoC-III appears as unglycosylated (C-III0a) , or glycosylated with zero (C-III0b) , one (C-III1, most abundant) or two (C-III2) sialic acids. The association of these proteoforms with peripheral artery disease (PAD) in a general population is unstudied. Total apoC-I, C-II and C-III concentrations and relative amounts of their proteoforms were measured in baseline plasma of 5,681 MESA participants. The ankle-brachial index (ABI) was assessed at baseline, 3 and years. Prevalent PAD was defined as baseline ABI ≤ 0.9. Incident PAD was follow-up ABI ≤ 0.9 or a hospital-based diagnosis of PAD to include revascularization. After adjustment for baseline characteristics, lipid lowering therapy and plasma lipids, low baseline ABI was associated with low C-III0b to C-III1 ratio (C-III0b/III1, p=0.01) and high C-III2 to C-III1 ratio (C-III2/III1, p<0.01) . Prevalent PAD (n=226) was only associated with C-III2/III1 (OR 1.16, 95% CI: 1.01-1.33, p=0.03) . Lower ABI at follow-up was associated with lower C-III0b/III1 and higher C-III2/III1 at baseline (p<0.both) . Incident PAD (n=244) was associated with C-III2/III1 (HR 1.22, 1.06-1.41, p=0.01) . Total apoC-I, C-II and C-III concentrations, or apoC-I and C-II truncations were not associated with ABI at baseline and follow-up, or with cross-sectional and prospective PAD determinations. ApoC-I and apoC-II and their truncations do not appear linked with PAD. In contrast, we find different associations of apoC-III0b and apoC-III2 with prevalent and incident PAD that are independent of other risk factors, including plasma lipids. Our data provide further support that proteoforms may have unique properties not captured by “total” apolipoprotein measures. Disclosure P.Reaven: Research Support; AstraZeneca, Dexcom, Inc. J.Koska: None. S.Hansen: None. Y.Hu: None. J.Furtado: Research Support; Eli Lilly and Company, Pfizer Inc. S.Sinari: None. D.Billheimer: None. D.Nedelkov: None. M.Budoff: Research Support; Amarin Corporation, Novo Nordisk, Speaker's Bureau; Boehringer Ingelheim International GmbH, Lilly. R.Mcclelland: None. Funding National Institutes of Health (R01-HL138969)

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.

Background: The mechanisms of how APOE epsilon 4 allele (APOE4) increases the risk of Alzheimer's disease (AD) pathology have not been fully elucidated. In cerebrospinal fluid (CSF), apoE is heavily glycosylated. Objective: To determine the impact of APOE genotype on the relative abundance of apoE protein isoforms and their specific glycosylation patterns in CSF and plasma via a newly developed mass spectrometric immunoassay (MSIA) assay. Methods: Total glycosylation and isoform-specific glycosylation were analyzed in plasma and CSF from a group of nondemented older individuals (n = 22), consisting of homozygous epsilon 3 and epsilon 4 or heterozygous epsilon 3/epsilon 4, epsilon 2/epsilon 3, or epsilon 2/epsilon 4 carriers. The glycan structures were further confirmed after treatment with sialidase. Results: In heterozygous individuals, the apoE3/E2, E4/E2, and E4/E3 isoform ratios were all significantly lower in plasma compared to CSF. For all individuals, a single O-linked glycan was observed in plasma, while two glycans (of the same type) per apoE were observed in CSF. The ratio of glycosylated to total apoE was greater in CSF compared to plasma for all apoE isoforms. In plasma and CSF, a trend of decreasing glycosylation was observed from apoE2 > apoE3 > apoE4. The difference in the percentage of secondary glycosylation in CSF was significantly greater in apoE4 compared to the other isoforms. Conclusion: The new MSIA apoE assay robustly distinguishes among apoE isoforms and glycoforms in plasma and CSF. ApoE4 is the predominant isoform and least glycosylated in CSF. Assessing apoE isoform-specific glycosylation by MSIA may help clarify brain apoE metabolism and AD risk.