Profiling the Bisecting N-acetylglucosamine Modification in Amniotic Membrane via Mass Spectrometry

Abstract

Bisecting N-acetylglucosamine (GlcNAc), a GlcNAc linked to the core β-mannose residue via a β1,4 linkage, is a special type of N-glycosylation that has been reported to be involved in various biological processes, such as cell adhesion and fetal development. This N-glycan structure is abundant in human trophoblasts, which is postulated to be resistant to natural killer cell-mediated cytotoxicity, enabling a mother to nourish a fetus without rejection. In this study, we hypothesized that the human amniotic membrane, which serves as the last barrier for the fetus, may also express bisected-type glycans. To test this hypothesis, glycomic analysis of the human amniotic membrane was performed, and bisected N-glycans were detected. Furthermore, our proteomic data, which have been previously employed to explore human missing proteins, were analyzed and the presence of bisecting GlcNAc-modified peptides was confirmed. A total of 41 glycoproteins with 43 glycopeptides were found to possess a bisecting GlcNAc, and 25 of these glycoproteins were reported to exhibit this type of modification for the first time. These results provide insights into the potential roles of bisecting GlcNAc modification in the human amniotic membrane, and can be beneficial to functional studies on glycoproteins with bisecting GlcNAc modifications and functional studies on immune suppression in human placenta.

Keywords: Amniotic membrane; Bisecting N-acetylglucosamine; Glycan; Glycoprotein; Mass spectrometry.

Figure 1

Annotated MALDI–TOF MS spectra of permethylated N-glycans from the human amniotic membrane The spectra have been smoothened and the baselines have been subtracted with flexAnalysis default setting. A. The glycans in the mass range from m/z 1500 to m/z 3000. B. The glycans in the mass range from m/z 3000 to m/z 4500. All ions are [M+Na]⁺. Peaks are labeled with their m/z values, and putative structures are described based on the molecular weight and N-glycan biosynthetic pathway. Annotations are simplified to biantennary structures, with additional LacNAc, Fuc, and GlcNAc listed outside the bracket. MALDI–TOF MS, matrix-assisted laser desorption ionization time of flight mass spectrometry; LacNAc, N-acetyllactosamine; Fuc, fucose; GlcNAc, N-acetylglucosamine; Man, mannose; Gal, galactose; NeuAc, N-acetylneuraminic acid.

Figure 2

The MS8 approach confirms the presence of bisecting GlcNAc The fragment ion at m/z 444.18 (theoretical value) in the red frame is the characteristic ion of the bisected-type glycan. MS, mass spectrometry.

Figure 3

Annotated ESI ITMS MS7 spectrum of thepermethylated N-glycanion at m/z2489.25from the human amniotic membrane Assignments of the possible fragment ions are indicated on the cartoons and on the spectrum. The number indicated above the peak is the m/z value of the fragment ion (resulting ion) that has been detected by the mass spectrometer. Data were acquired in the form of [M+Na]⁺. The energy under CID and HCD modes first breaks glycosidic bonds to form ‘b’ ions or ‘y’ ions, and further fragmentation will produce ‘by’ ions or ‘yy’ ions or ‘bb’ ions, and so on. ESI, electrospray ionization; ITMS, ion trap mass spectroscopy; CID, collision-induced dissociation; HCD, high-energy collision dissociation.

Figure 4

Annotated ESI MS2 spectrum showing N-glycosylation on the N8 in the peptide KLHINHNNLTESVGPLPK This Pep is from lumican (UniProt: P51884). All ions are [P + H]⁺ or [P + 2H]²⁺ or [P + 3H]³⁺. Double charged ions are annotated as z = 2, triple charged ions are annotated as z = 3, and others are monocharged. The number indicated above the peak in the spectrum is the m/z value of the ion that has been detected by the mass spectrometer. To make the annotation clearer, ions are labeled in different colors. Pep, peptide.

Supplementary figure S1

Human placenta processing. A. Human placenta and umbilical cord. B. chorion and amniotic membrane section. C. amniotic membrane after washing with PBS. D. SDS–PAGE image of the extracted proteins from human amniotic membrane. PBS, phosphate buffered saline; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Supplementary figure S4

Distribution of identified bisecting GlcNAc containing glycoproteins in terms of molecular function, cellular component, and biological process. One protein may be involved in multiple categories.

Supplementary figure S5

KEGG analysis results of the identified bisecting GlcNAc containing glycoproteins. KEGG, Kyoto Encyclopedia of Genes and Genomes.