Biology subjects

Stanley, P.

Publications and source records attributed to Stanley, P..

6 recordsLinked to original sources

bioRxiv2026

Absence of EOGT Precludes Defective Development in Fringe-null Mouse Intestine

Identifying biological roles for glycosyltransferases is a continuing challenge and important for defining morbidities associated with congenital disorders of glycosylation. Here we investigate the consequences to intestinal development of conditionally deleting Lfng alone or Lfng, Mfng and Rfng together in a mixed or Eogt-null genetic background. Each Fringe transfers N-acetylglucosamine (GlcNAc) to fucose (Fuc) attached to Ser or Thr by POFUT1 in a consensus sequence found in certain epithelial growth factor-like (EGF) repeats. EOGT transfers GlcNAc directly to Ser/Thr in a separate consensus sequence of the EGF repeat. Notch receptors and Notch ligands contain the largest number of EGF repeats with consensus sites for these O-glycans. Conditional deletion of Pofut1 in mouse intestine causes similar developmental defects to deletion of Notch1 and Notch2 or Dll1 and Dll4. LFNG also contributes to optimal Notch signaling in mouse intestine. In this work, we generated Lfng[F/F]:Villin-Cre and Lfng[F/F]Mfng[-/-]Rfng[-/-]:Villin-Cre mice in which extension of O-Fuc on EGF repeats was inhibited or prevented in intestinal epithelium. Conditional deletion of either Lfng alone or all three Fringe activities together led to defective intestinal development with a marked increase in goblet and Paneth cells, increased crypt width and reduced villus length. Unexpectedly, in mice globally lacking EOGT, conditional inactivation of the three Fringe genes did not lead to defective intestinal development. Thus, the absence of EOGT prevented disruption of development in Fringe-null intestine, identifying a novel role for EOGT in regulating intestinal development.

cell biology↗

bioRxiv2024

The N-glycosylation defect in Lec5 and Lec9 CHO cells is caused by absence of the DHRSX gene

Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.

biochemistry↗

bioRxiv2023

EOGT Enables Residual Notch Signaling in Mouse Intestinal Cells Lacking POFUT1

Notch signaling determines cell fates in mouse intestine. Notch receptors contain multiple epidermal growth factor-like (EGF) repeats modified by O-glycans that regulate Notch signaling. Conditional deletion of protein O-fucosyltransferase 1 (Pofut1) substantially reduces Notch signaling and markedly perturbs lineage development in mouse intestine. However, mice with inactivated Pofut1 are viable, whereas complete elimination of Notch signaling in intestine is lethal. Here we investigate whether residual Notch signaling enabled by EOGT permits mice lacking Pofut1 in intestine to survive. Mice globally lacking Eogt alone were grossly unaffected in intestinal development. In contrast, mice lacking both Eogt and Pofut1 died at [~]28 days after birth with greater loss of body weight, a greater increase in the numbers of goblet and Paneth cells, and greater downregulation of Notch target genes, compared to Pofut1 deletion alone. These data establish that both O-fucose and O-GlcNAc glycans are fundamental to Notch signaling in the intestine and provide new insights into roles for O-glycans in regulating Notch ligand binding. Finally, EOGT and O-GlcNAc glycans provide residual Notch signaling and support viability in mice lacking Pofut1 in the intestine.

developmental biology↗

bioRxiv2022

Synergistic Regulation of Notch Signaling by Different O-Glycans Promotes Hematopoiesis

Glycosylation of Notch receptors by O-fucose glycans regulates Notch ligand binding and Notch signaling during hematopoiesis. However, roles in hematopoiesis for other O-glycans that modify Notch receptors have not been determined. Here we show that the EGF domain-specific GlcNAc transferase EOGT is required in mice for the optimal production of lymphoid and myeloid cells. The phenotype of Eogt null mice was largely cell-autonomous, and Notch target gene expression was reduced in T cell progenitors. Moreover, EOGT supported residual Notch signaling following conditional deletion of Pofut1 in hematopoietic stem cells (HSC). Eogt:Pofut1 double mutant HSC had more severe defects in bone marrow, and in T and B cell development in thymus and spleen, compared to deletion of Pofut1 alone. The combined results show that EOGT and O-GlcNAc glycans are required for optimal hematopoiesis and T and B cell development, and that they act synergistically with POFUT1 and O-fucose glycans to promote Notch signaling in lymphoid and myeloid differentiation. Key pointsO_LIO-GlcNAc glycans and EOGT promote lymphopoiesis and myelopoiesis C_LIO_LIEOGT supports Notch signaling in the absence of POFUT1 and O-fucose glycans C_LI

immunology↗

bioRxiv2019

The Golgi Glycoprotein MGAT4D Protects Testicular Germ Cells From Mild Heat Stress

Male germ cells are sensitive to heat stress and testes must be maintained outside the body for optimal fertility. However, no germ cell intrinsic mechanism that protects from heat has been reported. Here, we identify the germ cell specific Golgi glycoprotein MGAT4D as a protector of male germ cells from heat stress. Mgat4d is highly expressed in spermatocytes and spermatids. Unexpectedly, when the Mgat4d gene was inactivated globally or conditionally in spermatogonia, or mis-expressed in spermatogonia, spermatocytes or spermatids, neither spermatogenesis nor fertility were affected. On the other hand, when males were subjected to mild heat stress of the testis (43{degrees}C for 25 min), germ cells with inactivated Mgat4d were markedly more sensitive to the effects of heat stress, and transgenic mice expressing Mgat4d were partially protected from heat stress. Germ cells lacking Mgat4d generally mounted a similar heat shock response to control germ cells, but could not maintain that response. Several pathways activated by heat stress in wild type were induced to a lesser extent in Mgat4d[-/-] heat-stressed germ cells (NF{kappa}B response, TNF and TGF{beta} signaling, Hif1 and Myc genes). Thus, the Golgi glycoprotein MGAT4D is a novel, intrinsic protector of male germ cells from heat stress.

developmental biology↗

bioRxiv2019

In situ Fucosylation for Modulating Wnt Signaling in Live Cells

Wnt/{beta}-catenin signaling, also known as canonical Wnt signaling, regulates critical, context-dependent transcription in numerous (patho) physiological events. Amongst the well-documented mechanisms of canonical Wnt signaling, modification of N-glycans by L-fucose (Fuc) is the newest and the least understood. Using a combination of Chinese Hamster Ovary (CHO) cell mutants with different fucosylation levels and in situ cell-surface Fuc editing (ISF), we report that (1-3)-fucosylation of N-acetylglucosamine in the LacNAc (Gal{beta}(1-4)-GlcNAc) sequences of complex N-glycans modulates Wnt signaling by regulating the endocytosis of low density lipoprotein receptor-related protein 6 (LRP6). Pulse-chase experiments reveal that increasing N-glycan LacNAc fucosylation elevates endocytosis of lipid-raft-localized LRP6, leading to the suppression of Wnt-{beta}-catenin signaling. Inhibiting endocytosis by inhibiting dynamin 1, a GTPase responsible for endocytosis in eukaryotic cells, partially rescues Wnt signaling. Remarkably, inhibition of Wnt signaling by N-glycan LacNAc fucosylation is fully rescued by the addition of free Fuc to the medium, suggesting that endocytosis of N-glycan fucosylated LRP6 may be mediated by a receptor that recognizes the bound (1-3)-Fuc. This work provides the first evidence that in situ cell-surface fucosylation can be exploited to regulate a specific signaling pathway via endocytosis, revealing a novel regulatory mechanism linking glycosylation of a cell surface receptor with its intracellular signaling.

biochemistry↗