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

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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 (MS3) 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 Asn139 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.

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Abbreviations

ACN:

Acetonitrile

Asn:

Asparagine

Asp:

Aspartate

DHB:

2 5-dihydroxybenzoic acid

DTT:

Dithiothreitol

ESI:

Electrospray ionization

Fuc:

L-fucose

Gal:

D-galactose

GlcNAc:

N-acetyl-D-glucosamine

Hex:

Hexose

HexNAc:

N-acetylhexosamine

IAA:

Iodacetamide

LOX-1:

Lectin-like oxidized low density lipoprotein receptor-1

MALDI:

Matrix assisted laser desorption/ionization

Man:

D-mannose

MS:

Mass spectrometry

PGC:

Porous graphic carbon

PNGase F:

Peptide N-Glycosidase F

QIT:

Quadropole ion trap

RT:

Room temperature

SDS-PAGE:

Dodecyl sulfate sodium salt-polyacrylamide gel electrophoresis

SPE:

Solid-phase extraction

TFA:

Trifluoroacetic acid

TOF:

Time of flight

v/v :

Volume/volume

w/w :

Weight/weight

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Acknowledgments

We gratefully acknowledge the financial support from National natural science fund (31100586, 31010103906, 30930025), National Basic Research Program of China (973 Program) (2012CB8221004, 2011CB910604) and National High-tech R&D Program (863 Program) (2012AA020203).

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Authors and Affiliations

  1. Key Laboratory of Glycoconjugate Research Ministry of Public Health, Shanghai Medical College, Fudan University, Shanghai, 200032, People’s Republic of China

    Yifan Qian, Xingwang Zhang, Lei Zhou, Xiaojing Yun, Jianhui Xie, Jiejie Xu, Yuanyuan Ruan & Shifang Ren

  2. Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai, 200032, People’s Republic of China

    Yifan Qian, Xingwang Zhang, Lei Zhou, Xiaojing Yun, Jianhui Xie, Jiejie Xu, Yuanyuan Ruan & Shifang Ren

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  1. Yifan Qian
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Qian, Y., Zhang, X., Zhou, L. et al. Site-specific N-glycosylation identification of recombinant human lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). Glycoconj J 29, 399–409 (2012). https://doi.org/10.1007/s10719-012-9408-z

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