O-glycosylation on cerebrospinal fluid and plasma apolipoprotein E differs in the lipid-binding domain

Abstract

<jats:title>Abstract</jats:title> <jats:p>The O-glycoprotein apolipoprotein E (APOE), the strongest genetic risk factor for Alzheimer’s disease, associates with lipoproteins. Cerebrospinal fluid (CSF) APOE binds only high-density lipoproteins (HDLs), while plasma APOE attaches to lipoproteins of diverse sizes with binding fine-tuned by the C-terminal loop. To better understand the O-glycosylation on this critical molecule and differences across tissues, we analyzed the O-glycosylation on APOE isolated from the plasma and CSF of aged individuals. Detailed LC-MS/MS analyses allowed the identification of the glycosite and the attached glycan and site occupancy for all detectable glycosites on APOE and further three-dimensional modeling of physiological glycoforms of APOE. APOE is O-glycosylated at several sites: Thr8, Thr18, Thr194, Ser197, Thr289, Ser290 and Ser296. Plasma APOE held more abundant (20.5%) N-terminal (Thr8) sialylated core 1 (Neu5Acα2–3Galβ1–3GalNAcα1-) glycosylation compared to CSF APOE (0.1%). APOE was hinge domain glycosylated (Thr194 and Ser197) in both CSF (27.3%) and plasma (10.3%). CSF APOE held almost 10-fold more abundant C-terminal (Thr289, Ser290 and Ser296) glycosylation (36.8% of CSF peptide283–299 was glycosylated, 3.8% of plasma peptide283–299), with sialylated and disialylated (Neu5Acα2–3Galβ1–3(Neu5Acα2–6) GalNAcα1-) core 1 structures. Modeling suggested that C-terminal glycosylation, particularly the branched disialylated structure, could interact across domains including the receptor-binding domain. These data, although limited by sample size, suggest that there are tissue-specific APOE glycoforms. Sialylated glycans, previously shown to improve HDL binding, are more abundant on the lipid-binding domain of CSF APOE and reduced in plasma APOE. This indicates that APOE glycosylation may be implicated in lipoprotein-binding flexibility.</jats:p>

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].…”

“…This is followed by LC-MS analysis of the resulting glycans or glycopeptides, respectively. As an example, in a recent study of apoE glycoforms by Flowers et al [25], the approach included 16-hours of immunocapture, gel separation, 16-hours of digestion with trypsin, and LC-MS separation and detection of glycancontaining peptides. Consequently, only 4 plasma and 4 CSF unmatched samples from healthy individuals were analyzed.…”

“…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.…”