Site-Specific Glycoprofiles of HDL-Associated ApoE are Correlated with HDL Functional Capacity and Unaffected by Short-Term Diet

Abstract

Since HDL glycoprofiles are associated with HDL functional capacity we set out to determine whether diet can alter the glycoprofiles of key HDL-associated proteins, including ApoE, a potent driver of chronic disease risk. Ten healthy subjects consumed a fast food (FF) and a Mediterranean (Med) diet for 4 days in randomized order, with a 4-day wash-out between treatments. A multiple reaction monitoring (MRM) method was used to characterize the site-specific glycoprofiles of HDL proteins, and HDL functional capacity was analyzed. We describe for the first time that ApoE has 7 mucin-type Oglycosylation sites, which were not affected by short-term diet. The glycoprofiles of other HDLassociated proteins were also unaffected, except a di-sialylated ApoC-III glycan was enriched after Med diet, while a non-sialylated ApoC-III glycan was enriched after FF diet. Twenty-five individual glycopeptides were significantly correlated with cholesterol efflux capacity and 21 glycopeptides were correlated with immunomodulatory capacity. Results from this study indicate that the glycoprofiles of HDL-associated proteins including ApoE are correlated with HDL functional capacity but generally unaffected by diet in the short-term, except ApoC-III sialylation. These results suggest that HDL protein glycoprofiles are affected by both acute and long-term factors, and may be useful for biomarker discovery.

Simple and Fast Assay for Apolipoprotein E Phenotyping and Glycotyping: Discovering Isoform-Specific Glycosylation in Plasma and Cerebrospinal Fluid

“…Subsequently, Ser 290 was also identified as a glycan attachment site [22]. Additional possible glycan attachment sites have since been identified through the use of mass spectrometry (MS)-based approaches, and they include Thr 8 , Thr 18 , Ser 197 , Ser 263 , Thr 289 , and Ser 296 [23][24][25][26]. A larger proportion of apoE is glycosylated and sialylated in cerebrospinal fluid (CSF) than in plasma [22,[27][28][29].…”

“…Consequently, only 4 plasma and 4 CSF unmatched samples from healthy individuals were analyzed. Similarly, Zhu et al employed a lengthy apoE glycolprofiling method with multiple centrifugations for HDL isolation, 18 hours of trypsin digestion, and LC-MS analysis of the glycopeptides on plasma samples from 10 healthy individuals [26]. Not only are these LC-MS-based approaches complex, but they also delivered limited information: only the glycosylation sites and the carbohydrate glycan composition can be revealed from the tryptic glycopeptides, but not the total number of glycans attached per apoE molecule.…”

“…Analysis of homozygous individuals (2/2, 3/3, 4/4) could yield such isoform-specific glycosylation information, but such studies have not been performed thus far. In the first two apoE glycosylation sites-identification studies, only 3/3 individuals were analyzed [21,22]; in the latest glycosylation studies, the apoE phenotypes could not be determined [25,26], because they were undifferentiable after tryptic digestion. Hence, there is a knowledge gap in the apoE isoform-specific glycosylation and its role in AD.…”

“…Interestingly, the glycosylated apoE peaks in CSF indicated, for the first time, the presence of two glycans per one molecule of apoE in CSF (Table 1). It is quite possible that apoE can be glycosylated at two sites at the same time since several glycosylation sites for apoE have been identified so far [23][24][25][26]. Indeed, Lee et al have detected two glycans per one molecule of cellular and secreted apoE from primary human macrophages using two-dimensional gel electrophoresis (2DE) and MS analysis of in-gel generated tryptic glycopeptides [22].…”

“…It has been demonstrated that the C-terminus mediates apoE's stability, wherein it is rich in Ser and Thr glycosylation sites that may play a key role in protection against selfassociation and spontaneous aggregation [22]. The NETOGlyc 4.0 Server predicts several sites for Oglycosylation in the C-terminus, including Thr 289 and Ser 290 , and there is experimental evidence for glycosylation at these sites, plus Ser 296 [22,25,26]. Thus, the second glycan may be attached to the C-terminus of apoE in CSF.…”