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Identification of Poly-N-Acetyllactosamine-Carrying Glycoproteins from HL-60 Human Promyelocytic Leukemia Cells Using a Site-Specific Glycome Analysis Method, Glyco-RIDGE.

ABSTRACT: To elucidate the relationship between the protein function and the diversity and heterogeneity of glycans conjugated to the protein, glycosylation sites, glycan variation, and glycan proportions at each site of the glycoprotein must be analyzed. Glycopeptide-based structural analysis technology using mass spectrometry has been developed; however, complicated analyses of complex spectra obtained by multistage fragmentation are necessary, and sensitivity and throughput of the analyses are low. Therefore, we developed a liquid chromatography/mass spectrometry (MS)-based glycopeptide analysis method to reveal the site-specific glycome (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile, Glyco-RIDGE). This method used accurate masses and retention times of glycopeptides, without requiring MS2, and could be applied to complex mixtures. To increase the number of identified peptide, fractionation of sample glycopeptides for reduction of sample complexity is required. Therefore, in this study, glycopeptides were fractionated into four fractions by hydrophilic interaction chromatography, and each fraction was analyzed using the Glyco-RIDGE method. As a result, many glycopeptides having long glycans were enriched in the highest hydrophilic fraction. Based on the monosaccharide composition, these glycans were thought to be poly-N-acetyllactosamine (polylactosamine [pLN]), and 31 pLN-carrier proteins were identified in HL-60 cells. Gene ontology enrichment analysis revealed that pLN carriers included many molecules related to signal transduction, receptors, and cell adhesion. Thus, these findings provided important insights into the analysis of the glycoproteome using our novel Glyco-RIDGE method. Graphical Abstract ?.

SUBMITTER: Togayachi A

PROVIDER: S-EPMC6004004 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Identification of Poly-N-Acetyllactosamine-Carrying Glycoproteins from HL-60 Human Promyelocytic Leukemia Cells Using a Site-Specific Glycome Analysis Method, Glyco-RIDGE.

Togayachi Akira A Tomioka Azusa A Fujita Mika M Sukegawa Masako M Noro Erika E Takakura Daisuke D Miyazaki Michiyo M Shikanai Toshihide T Narimatsu Hisashi H Kaji Hiroyuki H

Journal of the American Society for Mass Spectrometry 20180419 6

To elucidate the relationship between the protein function and the diversity and heterogeneity of glycans conjugated to the protein, glycosylation sites, glycan variation, and glycan proportions at each site of the glycoprotein must be analyzed. Glycopeptide-based structural analysis technology using mass spectrometry has been developed; however, complicated analyses of complex spectra obtained by multistage fragmentation are necessary, and sensitivity and throughput of the analyses are low. The ...[more]

PMID: 29675740

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Project description:Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which has been shown to play a role in the differentiation of haematopoietic cells. We report here that neutrophils are the first nucleated mammalian cell type demonstrated to be devoid of immunoreactive PARP. Both NB4 acute promyelocytic leukaemia and HL-60 (acute myelocytic leukaemia) cells were differentiated into non-malignant neutrophils with all-trans-retinoic acid (ATRA). Western blot analysis demonstrated that ATRA had no effect on PARP expression in HL-60 cells. However, PARP was completely down-regulated in NB4 cells within 36 h of treatment initiation. This decrease in PARP polypeptide coincided with growth arrest and preceded the appearance of neutrophilic differentiation features. NB4 cells require a combination of 1,25-dihydroxyvitamin D3 (1,25-D3) and phorbol 12-myristate 13-acetate (PMA) to differentiate completely into monocyte/macrophages, whereas HL-60 cells can be made to differentiate by combined or single agents. PARP expression was up-regulated 90-fold when NB4 cells were treated with PMA and 1,25-D3 together, and this increase accompanied expression of the monocyte/macrophage phenotype. Only modest changes in PARP expression were observed when each agent was used alone in NB4 cells or when HL-60 cells were differentiated along the monocyte/macrophage pathway. In addition, PARP activity was modulated in a pattern similar to protein levels when NB4 cells were induced to differentiate along the neutrophilic and monocyte/macrophage pathways. This suggests that the activity of PARP may be controlled through regulation of protein levels during NB4 cell differentiation. We conclude that PARP levels are dramatically modulated during monocyte/macrophage and neutrophilic differentiation. On the basis of the tremendous changes in PARP polypeptide and total activity during myeloid differentiation, we propose that modulation of PARP gene expression is required for cellular maturation in both lineages.

Dataset Information

Identification of Poly-N-Acetyllactosamine-Carrying Glycoproteins from HL-60 Human Promyelocytic Leukemia Cells Using a Site-Specific Glycome Analysis Method, Glyco-RIDGE.

Publications

Identification of Poly-N-Acetyllactosamine-Carrying Glycoproteins from HL-60 Human Promyelocytic Leukemia Cells Using a Site-Specific Glycome Analysis Method, Glyco-RIDGE.

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