Structural Elucidation of the N- andO-Glycans of Human Apolipoprotein(a)

Abstract

Apolipoprotein(a) (apo(a)) is a multikringle domain glycoprotein that exists covalently linked to apolipoprotein B100 of low density lipoprotein, to form the lipoprotein(a) (Lp(a)) particle, or as proteolytic fragments. Elevated plasma concentrations of apo(a) and its fragments may promote atherosclerosis, but the underlying mechanisms are incompletely understood. The factors influencing apo(a) proteolysis are also uncertain. Here we have used exoglycosidase digestion and mass spectrometry to sequence the Asn (N)-linked and Ser/Thr (O)-linked oligosaccharides of human apo(a). We also assessed the potential role of apo(a) O-glycans in protecting thermolysin-sensitive regions of the polypeptide. Apo(a) contained two major N-glycans that accounted for 17% of the total oligosaccharide structures. The N-glycans were complex biantennary structures present in either a mono-or disialylated state. The O-glycans were mostly (80%) represented by the monosialylated core type 1 structure, NeuNAc␣2-3Gal␤1-3GalNAc, with smaller amounts of disialylated and non-sialylated O-glycans also detected. Removal of apo(a) O-glycans by sialidase and O-glycosidase treatment dramatically increased the sensitivity of the polypeptide to thermolysin digestion. These studies provide the first direct sequencing data for apo(a) glycans and indicate a novel function for apo(a) O-glycans that is potentially related to the atherogenicity of Lp(a).Lipoprotein(a) (Lp(a)) 1 consists of a low density lipoprotein (LDL) particle covalently linked through a single disulfide bond to apolipoprotein(a) (apo(a)) (1). Apo(a) contains multikringle domains that are homologous to plasminogen kringles IV and V (2). This homology is thought to underlie the atherogenicity of Lp(a) because apo(a) and its naturally occurring proteolytic fragments compete with plasminogen for fibrin(ogen) binding (3, 4). The predicted apo(a)-mediated decrease in fibrinolysis may contribute to the accumulation of fibrin at sites of atherosclerotic lesion development. Apo(a) also exhibits additional proatherogenic properties including binding to fibronectin (5) and decorin (6), direct inhibition of tissue-type plasminogen activator 1 action (7), and modulation of fibrinolysis after cellsurface binding (8,9).The concentration of plasma and urinary apo(a) proteolytic fragments is increased concomitantly with elevated plasma Lp(a) levels (10, 11). In some cases, the apo(a) fragments have a higher atherogenic potential than the intact Lp(a). For example, when the apo(a) moiety of Lp(a) is cleaved by protease to generate "mini-Lp(a)" (12), the resulting particle binds fibrinogen (12) and fibronectin (13) more avidly than undigested Lp(a). Because apo(a) fragments have potentially atherogenic properties and because they are also known to accumulate in atherosclerotic lesions (14), it is important to understand the factors that regulate apo(a) proteolysis. Previous studies have focused on the role of serine proteases and metalloproteinase as relevant enzymes (5, 12, 15, 16); however, the factor...

O-linked glycosylation of von Willebrand factor modulates the interaction with platelet receptor glycoprotein Ib under static and shear stress conditions

“…First, they can block the accessibility of the peptide backbone to proteases and thus alter the plasma half-life of proteins. [8][9][10][11] Second, they mediate intracellular trafficking and are ligands for cell surface receptors; for example, they modulate leukocyte adhesion and rolling at sites of inflammation and on the endothelial surface. 12,13 Finally, O-linked oligosaccharides influence the structure of the protein by introducing steric hindrance to folding and confer a more rigid, extended conformation to the peptide backbone.…”

Rapid Discrimination of Fatty Acid Composition in Fats and Oils by Electrospray Ionization Mass Spectrometry

“…Some fats and oils, particularly castor oil and fish oils, take longer to analyze than others because they contain more types of fatty acids. Recently, electrospray ionization mass spectrometry (ESI-MS) has been applied to the analysis of the fatty acid in both the positive [15][16][17][18] and negative 19,20 ion modes. The saponifiables of the fats and oils (fatty acids) transform relatively easily into negative ions due to the compounds' carboxyl group.…”

Report of the National Heart, Lung, and Blood Institute Workshop on Lipoprotein(a) and Cardiovascular Disease: Recent Advances and Future Directions

“…The hydrophilic O-linked glycans are primarily (ϳ80%) composed of monosialylated core structures (13 ), which may have implications for Lp(a) catabolism. apo(a), and thus Lp(a), is heterogeneous in its glycosylation, although the significance of the glycosylation modification with respect to the in vivo properties of Lp(a) is still unclear.…”