Site-specific N-glycosylation identification of recombinant human lectin-like oxidized low density lipoprotein receptor-1 (LOX-1).
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.
Key Laboratory of Glycoconjugate Research Ministry of Public Health, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China.
Yifan
Y
Qian
Xingwang
X
Zhang
Lei
L
Zhou
Xiaojing
X
Yun
Jianhui
J
Xie
Jiejie
J
Xu
Yuanyuan
Y
Ruan
Shifang
S
Ren
D002240:Carbohydrate Sequence; D045744:Cell Line, Tumor; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D011402:Pronase / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051127:Scavenger Receptors, Class E / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
C495137:OLR1 protein, human; D011994:Recombinant Proteins; D051127:Scavenger Receptors, Class E; D011402:Pronase