Limited Addition of the 6-Arm β1,2-linked N-Acetylglucosamine (GlcNAc) Residue Facilitates the Formation of the Largest N-Glycan in Plants

Abstract

Background:The largest N-glycan in plants is the paucimannosidic N-glycan with Man 3 XylFuc(GlcNAc) 2 structure. Results: A sophisticated mechanism producing the largest N-glycan in plants is proposed. Conclusion: Limited addition of the 6-arm GlcNAc to the common N-glycan acceptor (GlcNAcMan 3 (GlcNAc) 2 ) facilitates formation of the largest N-glycan in plants.Significance: This sophisticated mechanism expands our knowledge of the energy-efficient N-glycan processing in plants.

Metabolic Isotope Labeling of Polysaccharides with Isotopic Glucose for Quantitative Glycomics in Cell Culture

“…6,19 The analysis was performed in reflector positive ion mode using 2,5-dihydroxybenzoic acid (20 mg/mL solution in 50% methanol) as a matrix. 6,19 The analysis was performed in reflector positive ion mode using 2,5-dihydroxybenzoic acid (20 mg/mL solution in 50% methanol) as a matrix.…”

“…6,19 Equivalent amounts (200 μg) of protein samples were mixed and digested with 20 μg of trypsin in reaction buffer (40 mM NH 4 HCO 3 ) at 37 C overnight. Plant tissues (3 g) were ground in liquid nitrogen, resuspended in 10 mL of a 50 mM Hepes (pH 7.5) buffer containing 20 mM sodium metabisulfite, 5 mM ethylenediaminetetraacetic acid (EDTA), 0.1% (w/v) sodium dodecyl sulfate (SDS), and 1.7% polyvinylpolypyrrolidone, and then insoluble material was eliminated by centrifugation (15 min at 13 000 × g) at 4 C. Protein samples were processed by acetone precipitation three times at 4 C for 3 h to remove contaminants.…”

“…[4][5][6] In order to understand the biological relevance of glycans, many mass spectrometry (MS)-based methods have been developed for the relative quantitation of glycan. 1,2 The majority of extracellular matrix and the membranous proteins are highly glycosylated by N-linked glycans as well as other types of glycans.…”

“…8,9 Another version of relative isotopic labeling of glycans utilizes heavy vs. light iodomethane ( 13 CH 3 vs. 12 CDH 2 ) in a standard permethylation workflow for the incorporation of isobaric structures. 6,19 The analysis was performed in reflector positive ion mode using 2,5-dihydroxybenzoic acid (20 mg/mL solution in 50% methanol) as a matrix. 13,14 In contrast to the aforementioned in vivo labeling methods, a metabolic labeling method, isotopic detection of aminosugars with glutamine (IDAWG), has been reported by Wells and coworkers for comparative glycomic studies using the murine embryonic stem cells.…”

“…1,2 The majority of extracellular matrix and the membranous proteins are highly glycosylated by N-linked glycans as well as other types of glycans. [4][5][6] In order to understand the biological relevance of glycans, many mass spectrometry (MS)-based methods have been developed for the relative quantitation of glycan. [4][5][6] In order to understand the biological relevance of glycans, many mass spectrometry (MS)-based methods have been developed for the relative quantitation of glycan.…”