Jincui Huang
Jincui Huang; Hyeyoung Lee; Angela Zivkovic; Jennifer Smilowitz; Bruce German; Carlito Lebrilla
UC Davis, Davis, CA
NOVEL ASPECT: Extensive site-specific glycosylation of HDL proteins and glycolipid analysis indicate that HDL is a highly glycosylated and sialylated particle.
INTRODUCTION:
High-density lipoprotein (HDL) particles are composed of lipids and lipoproteins and are considered as a strong inverse predictor of risk of cardiovascular disease. The function of HDL in health and diseases is related to its role in reverse cholesterol transport and in its interactions with cell membrane receptors, enzymes, and lipid-transport proteins. The proteins and lipids of HDL are potentially glycosylated. Because of the role of glycosylation in recognition, protein folding, and protein half-life in blood, it may contribute significantly to the overall function of HDL However, very little is known concerning the glycosylation of  HDL. Using glycomics, glycolipidomics and glycoproteomics approaches, we have determined for the first time the site-specific glycosylation in proteins and lipid glycosylation of HDL.
METHODS:
Isolation of HDL was performed by ultracentrifugation. N-Linked glycans were released from the HDL solution using PNGase F. Pronase E, covalently coupled to CNBr activated sepharose beads, was reacted to the HDL by incubating at 37 ºC overnight. The digestion mixtures were desalted and enriched via solid phase extraction (SPE) prior to the analyses. Extraction of gangliosides was performed by mixing water, methanol and chloroform with a HDL solution after centrifugation. The supernatant was collected and cleaned by SPE. MS and MS/MS analyses of the glycopeptides and gangliosides were achieved via nano-LC/Q-TOF MS with a nano-LC column integrated in a micro-chip packed with porous graphitized carbon for glycan/glycopeptides and C18 for gangliosides.
ABSTRACT:
Proteomics analysis of HDL particles revealed a list of apolipoproteins as well as the HDL associated plasma proteins, approximately 60% of which are glycosylated.Glycans from HDL were separated with a PGC stationary phase and corresponded to over 60 N-linked glycan compounds with over 20 distinct compositions. Each composition produced two or more chromatographic peaks corresponding to structural isomers. Sialylated glycans including complex type with bi- or tri- antenna were found to be the dominant glycan types. Interestingly, a biantennary glycan with two sialic acids was the most abundant glycan species as is found in blood. The structures of N-glycans from HDL associated glycoproteins were further elucidated by tandem MS.Comprehensive glycan-microheterogeneity of HDL associated glycoproteins was determined using non-specific proteolysis and analysis with nano-LC/Q-TOF MS.  An in-house software, Glycopeptide Finder, was used to interpret the tandem MS. This method lead to the characterization of both N- or O-glycans whose site was determined based on the associated peptide tags. In all, over 50 glycopeptides corresponding to 20 glycosites from more than 10 glycoproteins were observed and determined in the HDL protein mixture. These glycopeptides were a mixture of N-linked glycopeptides (mainly biantennary complex type sialylated glycans) and O-linked glycopeptides (mainly sialylated). The results yielded comprehensive glycoprotein analysis with detailed glycan microheterogeneity.Ganglioside analysis showed that GM3 (monosialoganglioside, NeuAc2–3Gal1–4Glc–Cer) and GD3 (disialoganglioside, NeuAc2–8NeuAc2–3Gal1–4Glc–Cer) are the most abundant glycolipids in human HDL. A proportion of 75% GM3 and 25% GD3 were obtained. Both GM3 and GD3 are composed of heterogeneous ceramide lipids, including d34:1, d39:1, d40:1 and d41:1.