Large‐scale identification of secretome glycoproteins recognized by <i>Wisteria floribunda</i> agglutinin: A glycoproteomic approach to biomarker discovery

Sugahara, Daisuke; Tomioka, Azusa; Sato, Takashi; Narimatsu, Hisashi; Kaji, Hiroyuki

doi:10.1002/pmic.201400443

Cited by 19 publications

(26 citation statements)

References 57 publications

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“…2A, lanes 1-4), whereas the expression of WGA-binding glycoproteins did not change ( Fig. 2A, lanes [5][6][7][8]. In line with the WFA blotting data, the number and amounts of glycoproteins captured by WFA beads increased as evidenced by silver staining (Fig.…”

Section: Capturing Type-i Ldn Glycan-carrying Proteins From Lec8 Cellssupporting

confidence: 80%

“…In other studies, type-II LDN glycans have been detected in the secreted glycoproteins found in conditioned media and serum samples (5). Nonetheless, the glycoproteins identified here as type-I carriers were mainly intracellular glycoproteins (Table S1 and Fig.…”

Section: Preferences Of B3galnt2 During the Synthesis Of Type-i Ldn Isupporting

confidence: 50%

“…Ohkura et al (4) have demonstrated that most of the LDNcarrying glycoproteins are in the culture medium rather than on the cell membrane of Madin-Darby canine kidney cells and HEK293T cells, which contain high B4GALNT activity. Recently, Sugahara et al (5) also identified many LDN-carrying glycoproteins in the cultured medium of HEK293T cells, indicating the general prevalence of LDN on glycoproteins in addition to secreted hormones. Because only LDN (GalNAc␤1-4GlcNAc) structures had been observed in mammalian cells until recently, the glycoproteins identified have been assumed to carry LDN (GalNAc␤1-4GlcNAc).…”

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confidence: 99%

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Identification of mammalian glycoproteins with type-I LacdiNAc structures synthesized by the glycosyltransferase B3GALNT2

Nakane

Angata

Sato

et al. 2019

Journal of Biological Chemistry

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The type-I LacdiNAc (LDN; GalNAc␤1-3GlcNAc) has rarely been observed in mammalian cells except in the O-glycan of ␣-dystroglycan, in contrast to type-II LDN structures (GalNAc␤1-4GlcNAc) in N-and O-glycans that are present in many mammalian glycoproteins, such as pituitary and hypothalamic hormones. Although a ␤1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2; type-I LDN synthase) has been cloned, the function of type-I LDN in mammalian cells is still unclear, as its carrier protein(s) has not been identified. In this study, using HeLa cells, we demonstrate that inhibition of Golgi-resident glycosyltransferase increases the abundance of B3GALNT2-synthesized type-I LDN structures, recognized by Wisteria floribunda agglutinin (WFA). Using isotope-coded glycosylation site-specific tagging (IGOT)-LC/MS analysis of Lec8 Chinese hamster cells lacking galactosylation and of cells transfected with the B3GALNT2 gene, we identified the glycoproteins that carry B3GALNT2-generated type-I LDN in their N-glycans. Our results further revealed that LDN presence on low-density lipoprotein receptor-related protein 1 and nicastrin depends on B3GALNT2, indicating the occurrence of type-I LDN in vivo in mammalian cells. Our analysis also uncovered that most of the identified glycoproteins localize to intracellular organelles, particularly to the endoplasmic reticulum. Whereas B4GALNT3 and B4GALNT4 synthesized LDN on extracellular glycoproteins, B3GALNT2 primarily transferred LDN to intracellular glycoproteins, thereby clearly delineating proteins that carry type-I or type-II LDNs. Taken together, our results indicate the presence of mammalian glycoproteins carrying type-I LDN on N-glycans and suggest that type-I and type-II LDNs have different roles in vivo. cro ARTICLE

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Section: Capturing Type-i Ldn Glycan-carrying Proteins From Lec8 Cellssupporting

confidence: 80%

Section: Preferences Of B3galnt2 During the Synthesis Of Type-i Ldn Isupporting

confidence: 50%

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confidence: 99%

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Identification of mammalian glycoproteins with type-I LacdiNAc structures synthesized by the glycosyltransferase B3GALNT2

Nakane

Angata

Sato

et al. 2019

Journal of Biological Chemistry

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“…We therefore developed a method for large-scale identification of glycopeptides (peptide sequences and glycosylated sites) by glycopeptide capture, PNGase-mediated removal of glycans, and liquid chromatography (LC)/mass spectrometry (MS) analysis [ 7 , 8 ]. This approach has facilitated the discovery of glycobiomarker candidates in hepatocellular carcinoma, lung adenocarcinoma, small cell lung carcinoma, and ovarian clear cell carcinoma [ 9 – 14 ] and has enabled the identification of glycosylated proteins from various organisms, including Caenorhabditis elegans , mice, and humans [ 15 – 19 ]. This knowledge was then used to construct a glycoprotein database, GlycoProtDB [ 16 ].…”

Section: Introductionmentioning

confidence: 99%

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

Togayachi

Tomioka

Fujita

et al. 2018

J. Am. Soc. Mass Spectrom.

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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 Abstractᅟ Electronic supplementary materialThe online version of this article (10.1007/s13361-018-1938-6) contains supplementary material, which is available to authorized users.

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“…The IGOT-LC/MS method was developed in 2003 as an early step technology for large-scale identification of N-glycosylated proteins [40,44,45]. The advantage of the IGOT-LC/MS is that the biological samples such as cell or tissue homogenates, serum or other body fluids can be applied directly.…”

Section: As Listed Inmentioning

confidence: 99%

Development of M2BPGi: a novel fibrosis serum glyco-biomarker for chronic hepatitis/cirrhosis diagnostics

Narimatsu

2015

Expert Review of Proteomics

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Many proteins in the living body are glycoproteins, which present glycans linked on their surface. Glycan structures reflect the degree of cell differentiation or canceration and are cell specific. These characteristics are advantageous in the development of various disease biomarkers. Glycoprotein-based biomarkers (glyco-biomarkers) are developed by utilizing the specific changes in the glycan structure on a glycoprotein secreted from the diseased cells of interest. Therefore, quantification of the altered glycan structures is the key to developing a new glyco-biomarker. Glycoscience is a relatively new area of molecular science, and recent advancement of glycotechnologies is remarkable. In the author's institute, new glycoscience technologies have been designed to be efficiently utilized for the development of new diagnostic agents. This paper introduces a strategy for glyco-biomarker development, which was successfully applied in the development of Wisteria floribunda agglutinin-positive Mac-2 binding protein M2BPGi, a liver fibrosis marker now commercially available for clinical use.

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Large‐scale identification of secretome glycoproteins recognized by Wisteria floribunda agglutinin: A glycoproteomic approach to biomarker discovery

Cited by 19 publications

References 57 publications

Identification of mammalian glycoproteins with type-I LacdiNAc structures synthesized by the glycosyltransferase B3GALNT2

Identification of mammalian glycoproteins with type-I LacdiNAc structures synthesized by the glycosyltransferase B3GALNT2

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

Development of M2BPGi: a novel fibrosis serum glyco-biomarker for chronic hepatitis/cirrhosis diagnostics

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