Glycomic Profiling of Cells and Tissues by Mass Spectrometry: Fingerprinting and Sequencing Methodologies

Jang-Lee, Jihye; North, Simon J.; Sutton-Smith, Mark; Goldberg, David; Panico, Maria; Morris, Howard R.; Haslam, Stuart M.; Dell, Anne

doi:10.1016/s0076-6879(06)15005-3

Cited by 148 publications

(144 citation statements)

References 31 publications

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“…Glycomic Analysis. Myocytes isolated from neonatal and adult atria and ventricles were processed and analyzed by Core C of the Consortium for Functional Glycomics (http://www.functionalglycomics.org), as described previously (47). N-glycans were released from extracted glycoproteins of tissue preparations by PNGase F digestion, permethylated using the sodium hydroxide procedure, and purified by Sep-Pak C18 cartridges, as previously described (47).…”

Section: Methodsmentioning

confidence: 99%

Regulated and aberrant glycosylation modulate cardiac electrical signaling

Montpetit

Stocker

Schwetz

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Millions afflicted with Chagas disease and other disorders of aberrant glycosylation suffer symptoms consistent with altered electrical signaling such as arrhythmias, decreased neuronal conduction velocity, and hyporeflexia. Cardiac, neuronal, and muscle electrical signaling is controlled and modulated by changes in voltage-gated ion channel activity that occur through physiological and pathological processes such as development, epilepsy, and cardiomyopathy. Glycans attached to ion channels alter channel activity through isoform-specific mechanisms. Here we show that regulated and aberrant glycosylation modulate cardiac ion channel activity and electrical signaling through a cell-specific mechanism. Data show that nearly half of 239 glycosylation-associated genes (glycogenes) were significantly differentially expressed among neonatal and adult atrial and ventricular myocytes. The N-glycan structures produced among cardiomyocyte types were markedly variable. Thus, the cardiac glycome, defined as the complete set of glycan structures produced in the heart, is remodeled. One glycogene, ST8sia2, a polysialyltransferase, is expressed only in the neonatal atrium. Cardiomyocyte electrical signaling was compared in control and ST8sia2 (؊/؊) neonatal atrial and ventricular myocytes. Action potential waveforms and gating of less sialylated voltage-gated Na ؉ channels were altered consistently in ST8sia2 (؊/؊) atrial myocytes. ST8sia2 expression had no effect on ventricular myocyte excitability. Thus, the regulated (between atrium and ventricle) and aberrant (knockout in the neonatal atrium) expression of a single glycogene was sufficient to modulate cardiomyocyte excitability. A mechanism is described by which cardiac function is controlled and modulated through physiological and pathological processes that involve regulated and aberrant glycosylation. action potentials ͉ cardiomyocyte ͉ glycomics ͉ ion channels ͉ sialic acids

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Section: Methodsmentioning

confidence: 99%

Regulated and aberrant glycosylation modulate cardiac electrical signaling

Montpetit

Stocker

Schwetz

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…N-linked, O-linked, and GSL-derived glycans were extracted from ;70 to 100 3 10 6 WT and variant HL-60 cells as described previously (Jang-Lee et al 32 ; supplemental Methods). Glycans were permethylated prior to matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MALDI-TOF-TOF MS/MS analysis.…”

Section: Mass Spectrometrymentioning

confidence: 99%

ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes

Mondal

Buffone

Stolfa

et al. 2015

Blood

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• A single a(2,3) sialyltransferase, ST3Gal-4, controls sLe X biosynthesis on N-and O-glycans in cells of human myeloid lineage.• Blocking this enzyme activity prevents human neutrophil adhesion to E-, P-, and L-selectin.The precise glycosyltransferase enzymes that mediate selectin-ligand biosynthesis in human leukocytes are unknown. This knowledge is important because selectin-mediated cell tethering and rolling is a critical component of both normal immune response and various vascular disorders. We evaluated the role of 3 a(2,3)sialyltransferases, ST3Gal-3, -4, and -6, which act on the type II N-Acetyllactosamine structure (Galb1,4GlcNAc) to create sialyl Lewis-X (sLe X ) and related sialofucosylated glycans on human leukocytes of myeloid lineage. These genes were either silenced using lentiviral short hairpin RNA (shRNA) or functionally ablated using the clustered regularly interspaced short palindromic repeat/ Cas9 technology. The results show that ST3Gal-4, but not ST3Gal-3 or -6, is the major sialyltransferase regulating the biosynthesis of E-, P-, and L-selectin ligands in humans. Reduction in ST3Gal-4 activity lowered cell-surface HECA-452 epitope expression by 75% to 95%. Glycomics profiling of knockouts demonstrate an almost complete loss of the sLe X epitope on both leukocyte N-and O-glycans. In cell-adhesion studies, ST3Gal-4 knockdown/knockout cells displayed 90% to 100% reduction in tethering and rolling density on all selectins. ST3Gal-4 silencing in neutrophils derived from human CD34 1 hematopoietic stem cells also resulted in 80% to 90% reduction in cell adhesion to all selectins. Overall, a single sialyltransferase regulates selectin-ligand biosynthesis in human leukocytes, unlike mice where multiple enzymes contribute to this function. (Blood. 2015;125(4):687-696)

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“…The supernatant was lyophilized before being dissolved in 200 l of ammonium bicarbonate (50 mM (pH 8.4)) and incubated with 5 units of PNGase F (EC 3.5.1.52; Roche Applied Science) at 37°C for 20 h. The reaction was terminated by lyophilization. N-glycans were separated from the peptides by C 18 Sep-Pak (Waters) purification, permethylated (15), and analyzed by MALDI-TOF MS as previously described (16).…”

Section: Glycomic Profiling Of N-glycans Of Surface ␤2 Subunits With mentioning

confidence: 99%

Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating

Lagrange

Hernández

et al. 2010

Journal of Biological Chemistry

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N-Linked glycosylation plays a pivotal role in biogenesis and functions of secreted and membrane proteins, including ligand-gated ion channels. Mutations of enzymes involved in the N-linked glycosylation pathway have been associated with congenital disorders of glycosylation, many of which cause psychomotor retardation and seizures (1). The Cys-loop receptor superfamily of ligand-gated ion channels, which contains ␥-aminobutyric acid type A (GABA A ), 2 nicotinic acetylcholine (nACh), glycine, and 5-hydroxytryptamine type 3 (5-HT 3 ) receptors, mediates fast synaptic transmission in the nervous system and is involved in nearly every aspect of brain activity. It has been shown that blocking N-linked glycosylation of Cysloop receptors using tunicamycin substantially decreased receptors surface expression (2-4).GABA A receptor ␤1-3 subunits are essential components of virtually all GABA A receptors expressed in brain (5, 6). Based on the glycosylation sequons, Asn-Xaa-Ser/Thr, where Xaa is any amino acid except proline (7), each subunit contains three potential glycosylation sites in equivalent positions (Asn-32, Asn-104, and Asn-173 in the ␤2 subunit). Furthermore, multiple sequence alignment using Multalin software (8) and homology modeling suggested that about three quarters of all human Cys-loop receptor subunits contain potential glycosylation sites in the region containing the Asn-32 site, and about onehalf of the subunits contain potential glycosylation sites in the region containing the Asn-104 or Asn-173 sites (Table 1). The clustering of the glycosylation sites in Cys-loop receptor subunits further suggests that N-linked glycosylation might regulate Cys-loop receptor biogenesis and functions similarly in a spatially related manner. In support of this prediction, an intact Asn-32 equivalent site of the GABA A receptor ␣1 subunit or the nACh receptor ␣7 subunit (9, 10), an intact Asn-104 equivalent site of the nACh receptor ␣7 subunit or the mouse 5-HT 3 A subunit (4, 10), and an intact Asn-173 equivalent site of the mouse nACh receptor ␣1 subunit or the 5-HT 3 A subunit are required for functional receptor surface expression (4,11).In this study we determined the importance of glycosylation of ␤2 subunits for surface targeting and function of ␣1␤2 receptors in transfected human embryonic kidney (HEK293T) cells. We demonstrated that glycosylation efficiency of Asn-32 was lower than Asn-104 or Asn-173. Although glycosylation of Asn-173 was important for stability of singly expressed sub-

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Glycomic Profiling of Cells and Tissues by Mass Spectrometry: Fingerprinting and Sequencing Methodologies

Cited by 148 publications

References 31 publications

Regulated and aberrant glycosylation modulate cardiac electrical signaling

Regulated and aberrant glycosylation modulate cardiac electrical signaling

ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes

Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating

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