Site-specific N-glycosylation identification of recombinant human lectin-like oxidized low density lipoprotein receptor-1 (LOX-1)

Abstract

Human LOX-1/OLR 1 plays a key role in atherogenesis and endothelial dysfunction. The N-glycosylation of LOX-1 has been shown to affect its biological functions in vivo and modulate the pathogenesis of atherosclerosis. However, the N-glycosylation pattern of LOX-1 has not been described yet. The present study was aimed at elucidating the N-glycosylation of recombinant human LOX-1 with regard to N-glycan profile and N-glycosylation sites. Here, an approach using nonspecific protease (Pronase E) digestion followed by MALDI-QIT-TOF MS and multistage MS (MS(3)) analysis is explored to obtain site-specific N-glycosylation information of recombinant human LOX-1, in combination with glycan structure confirmation through characterizing released glycans using tandem MS. The results reveal that N-glycans structures as well as their corresponding attached site of LOX-1 can be identified simultaneously by direct MS analysis of glycopeptides from non-specific protease digestion. With this approach, one potential glycosylation site of recombinant human LOX-1 on Asn(139) is readily identified and found to carry heterogeneous complex type N-glycans. In addition, manual annotation of multistage MS data utilizing diagnostic ions, which were found to be particularly useful in defining the structure of glycopeptides and glycans was addressed for proper spectra interpretation. The findings described herein will shed new light on further research of the structure-function relationships of LOX-1 N-glycan.

Plasma High-Mannose and Complex/Hybrid N-Glycans Are Associated with Hypercholesterolemia in Humans and Rabbits

“…Changes in the glycosylation of Alpha-2-macroglobulin and Alpha-2-HS-glycoprotein have been associated with cancer and autoimmune diseases[ 34 ]. The N-glycosylation of lectin-like oxidized low density lipoprotein receptor-1 has been shown to modulate the pathogenesis of atherosclerosis[ 29 , 31 ]. N-glycosylation defect on scavenger receptor expressed by endothelial cells(SREC-I) could impair the uptake of modified LDL into macrophages and endothelial cells[ 32 ].…”

Discovery of Specific Metastasis-Related N-Glycan Alterations in Epithelial Ovarian Cancer Based on Quantitative Glycomics

“…The released glycans were further purified and desalted using a Porous Graphic Carbon Solid-Phase Extraction (PGC-SPE). To this end, the PGC microcolumns were packed with porous graphic carbon powder and prepared using GELoader tips as described before [44]. The microcolumns were preconditioned with 6 column volumes of 0.1% (v/v) trifluoroacetic acid (TFA) in 80% acetonitrile (ACN)/H 2 O (v/v) and equilibrated with equal volume of water.…”

Targeting LOX‐1 in atherosclerosis and vasculopathy: current knowledge and future perspectives

“…LOX‐1 is initially synthesized as a 40‐kDa precursor with N‐linked mannose‐type carbohydrate modifications, followed by further glycosylation, and finally processed into the 50‐kDa mature LOX‐1 protein . Importantly, the N‐linked glycosylation of asparagine residues regulates the transport of LOX‐1 to the cell membrane, where it binds to ox‐LDL . Structurally, there are two cleavage sites (Arg86‐Ser87 and Lys89‐Ser90) in LOX‐1 .…”