title: Elucidation of differences in N-glycosylation between different molecular weight forms of recombinant CLEC-2 by LC MALDI tandem MS.
abstract: C-type lectin-like receptor 2 (CLEC-2) is a newly identified receptor expressed on the platelet surface. It has been reported that CLEC-2 exists as a higher molecular weight (HMW) and a lower molecular weight (LMW) form, which share the same protein core but differ in glycans. The two forms appear to have different ligand-binding abilities, indicating that the differential glycosylation of CLEC-2 possibly produces functionally distinct glycoforms. This study aimed to explore an easy method to directly elucidate the N-glycosylation difference by employing a glycoproteomics approach. The off-line coupling of nano-LC with a MALDI-QIT-TOF mass spectrometer was demonstrated to be capable of sensitive and direct elucidation of the glycosylation difference between HMW and LMW CLEC-2, simultaneously providing information about their oligosaccharide structures and the glycosylation sites. The results reveal that a specific glycosylation site, Asn 134, is differently glycosylated in the two forms, with complex types of bi-antennary, tri-antennary and tetra-antennary, N-linked, fucosylated glycans identified at this site in the HMW form but not in the LMW form. The observed difference in glycosylation might provide new insights into the underlying mechanisms of biological functions of CLEC-2. Because of its simplicity and sensitivity, the method explored in this work suggests that it holds promise as a method of elucidating differences in direct N-glycosylation of target glycoprotein, even in small amount of samples.
authors.affiliation: Key Laboratory of Glycoconjugate Research Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai 200032, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Glycoconjugate Research Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai 200032, PR China.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Qian
authors.affiliation: Key Laboratory of Glycoconjugate Research Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai 200032, PR China.
authors.forename: Xingwang
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Glycoconjugate Research Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai 200032, PR China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Ruan
authors.affiliation: Key Laboratory of Glycoconjugate Research Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai 200032, PR China. Electronic address: renshifang@fudan.edu.cn.
authors.forename: Shifang
authors.initials: S
authors.lastname: Ren
authors.affiliation: Key Laboratory of Glycoconjugate Research Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai 200032, PR China. Electronic address: jxgu@shmu.edu.cn.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Gu
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D037181:Lectins, C-Type / Q000737:chemistry* / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D008970:Molecular Weight; D011134:Polysaccharides / Q000378:metabolism; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
chemical_list: C104778:CLEC2B protein, human; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D014357:Trypsin
keywords: Intact glycopeptides; Liquid chromatography; Mass spectrometry; N-glycosylation difference; Recombinant CLEC-2