Establishment of a novel monoclonal antibody against truncated glycoforms of α-dystroglycan lacking matriglycans

Yamasaki, Fumiya; Umezawa, Fumiko; Sensui, Tomohiro; Anzo, Masashi; Abo, Hirohito; Kuo, Chu-Wei; Khoo, Kay‐Hooi; Kato, Koichi; Yagi, Hirokazu; Kawashima, Hiroto

doi:10.1016/j.bbrc.2021.09.043

Cited by 4 publications

(12 citation statements)

References 21 publications

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“…In this plasmid vector, the coding sequence of human α-DG (1-373) with amino acid substitution of threonine with arginine at position 322 was subcloned into pEF-Fc. For Flag-tagged expression vector of TagD was constructed as previously described (24). In the expression plasmid for Flag-tagged PCYT2 (Uniprot KB: Q99447-1) recombinant protein, the coding sequence of human PCYT2 with flag peptide (DYKDDDDK) purchased from addgene (#81074) was cloned into the pCAG-Neo vector (WAKO).…”

Section: Methodsmentioning

confidence: 99%

“…Digested peptides purified using a ZipTip C18 (Merck Millipore) were analyzed by LC-MS/MS on an Orbitrap Fusion Tribrid (Thermo Fisher Scientific) coupled to an Easy-nLC 1200 (Thermo Fisher Scientific). The identities of glycopeptides were then manually verified based on positive detection of the expected peptide pyr QIHATPTPVR b and y ions, and/or the peptide core ion at m/z 551.804 (z=2) or 1102.600 (z=1) in their respective HCD MS/MS spectra, as described previously (20, 24).…”

Section: Methodsmentioning

confidence: 99%

“…The dual action of FKTN catalyzes this novel modification and, therefore, competes for the first Rbo5P conjugation (20, 21). Furthermore, the GroP modification is enhanced by elevation of intracellular CDP-Gro level, which can be artificially realized by overexpression of TagD, a CDP-Gro synthase from Gram-positive bacteria (22, 23), resulting in a concomitant decrease of the laminin-binding glycans (24). These findings raise the possibility that GroP serves as a terminator of the laminin-binding glycan elongation.…”

Section: Introductionmentioning

confidence: 99%

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Cancer malignancy is correlated with up-regulation of PCYT2-mediated glycerol phosphate modification of α-dystroglycan

Umezawa

Natsume

Fukusada

et al. 2021

Preprint

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The dystrophin-glycoprotein complex connects the cytoskeleton with base membrane components such as laminin through unique O-glycans displayed on α-dystroglycan (α-DG). Genetic impairment of elongation of these glycans causes congenital muscular dystrophies. We previously identified that glycerol phosphate (GroP) can cap the core part of the α-DG O-glycans and terminate their further elongation. This study examined the possible roles of the GroP modification in cancer malignancy, focusing on colorectal cancer. We found that the GroP modification critically depends on PCYT2, which serves as CDP-Gro synthase. Furthermore, we identified a significant positive correlation between cancer progression and GroP modification, which also correlated positively with PCYT2 expression. Moreover, we demonstrate that GroP modification promotes the migration of cancer cells. Based on these findings, we propose that the GroP modification by PCYT2 disrupts the glycan-mediated cell adhesion to the extracellular matrix and thereby enhances cancer metastasis. Thus, the present study suggests the possibility of novel approaches for cancer treatment by targeting the PCYT2-mediated GroP modification.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cancer malignancy is correlated with up-regulation of PCYT2-mediated glycerol phosphate modification of α-dystroglycan

Umezawa

Natsume

Fukusada

et al. 2021

Preprint

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“…If there is a critical amino acid of PCYT2 that affects specifically either CDP-Etn or CDP-Gro synthetic activity, it may be useful to distinguish between the CDP-Etn-and CDP-Gro-related functions of PCYT2. Very recently, Yamasaki et al [59] showed that overexpression of bacterial GCT in HCT116 cells significantly increased cellular CDP-Gro, and GroP modification of α-DG increased and laminin-binding glycan largely decreased. These results suggest the inhibitory role of GroP modification in the functional glycan synthesis of α-DG at the cellular level.…”

Section: Biological Roles Of Grop-containing Glycan On α-Dgmentioning

confidence: 99%

“…Because GroP modification of α-DG is considered as a stop signal of functional glycan synthesis, it may be involved in the pathogenesis of α-dystroglycanopathies or cancer progression. Yamasaki et al [59] established an antibody that is highly reactive with GroPterminated glycan. This antibody may be useful to elucidate the tissue distribution and the role of GroP-modification under the physiological and pathological conditions.…”

Section: Prospectsmentioning

confidence: 99%

Biosynthetic Mechanisms and Biological Significance of Glycerol Phosphate-Containing Glycan in Mammals

2021

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Bacteria contain glycerol phosphate (GroP)-containing glycans, which are important constituents of cell-surface glycopolymers such as the teichoic acids of Gram-positive bacterial cell walls. These glycopolymers comprising GroP play crucial roles in bacterial physiology and virulence. Recently, the first identification of a GroP-containing glycan in mammals was reported as a variant form of O-mannosyl glycan on α-dystroglycan (α-DG). However, the biological significance of such GroP modification remains largely unknown. In this review, we provide an overview of this new discovery of GroP-containing glycan in mammals and then outline the recent progress in elucidating the biosynthetic mechanisms of GroP-containing glycans on α-DG. In addition, we discuss the potential biological role of GroP modification along with the challenges and prospects for further research. The progress in this newly identified glycan modification will provide insights into the phylogenetic implications of glycan.

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Inhibitory machinery for the functional dystroglycan glycosylation

Okajima

2023

The Journal of Biochemistry

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Dystroglycan (DG), a muscular transmembrane protein, plays a critical role in transducing extracellular matrix-derived signals to the cytoskeleton and provides physical strength to skeletal muscle cell membranes. The extracellular domain of DG, α-DG, displays unique glycosylation patterns. Fully functional glycosylation is required for this domain to interact with components of extracellular matrices, including laminin. One of the unique sugar compositions found in such functional glycans on DG is two ribitol phosphates (RboP) that are transferred by the sequential actions of fukutin (FKTN) and fukutin-related protein (FKRP), which use CDP-ribitol as a donor substrate. These are then further primed for matriglycan biosynthesis. A recent in vitro study reported that glycerol phosphate (GroP) could be similarly added to α-DG by FKTN and FKRP if they used CDP-glycerol (CDP-Gro) as a donor substrate. However, the physiological relevance of these findings remains elusive. Imae et al. addressed the knowledge gap regarding whether CDP-Gro is present in mammals and how CDP-Gro is synthesized and functions in mammals.

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Establishment of a novel monoclonal antibody against truncated glycoforms of α-dystroglycan lacking matriglycans

Cited by 4 publications

References 21 publications

Cancer malignancy is correlated with up-regulation of PCYT2-mediated glycerol phosphate modification of α-dystroglycan

Cancer malignancy is correlated with up-regulation of PCYT2-mediated glycerol phosphate modification of α-dystroglycan

Biosynthetic Mechanisms and Biological Significance of Glycerol Phosphate-Containing Glycan in Mammals

Inhibitory machinery for the functional dystroglycan glycosylation

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