Protein O-fucosyltransferase 1 (Pofut1) regulates lymphoid and myeloid homeostasis through modulation of Notch receptor ligand interactions

Abstract

Notch signaling is essential for lymphocyte development and is also implicated in myelopoiesis. Notch receptors are modified by O-fucosylation catalyzed by protein O-fucosyltransferase 1 (Pofut1). Fringe enzymes add N-acetylglucosamine to O-fucose and modify Notch signaling by altering the sensitivity of Notch receptors to Notch ligands. To address physiologic functions in hematopoiesis of Notch modified by O-fucose glycans, we examined mice with inducible inactivation of Pofut1 using Mx-Cre. These mice exhibited a reduction in T lymphopoiesis and in the production of marginal-zone B cells, in addition to myeloid hyperplasia. Restoration of Notch1 signaling rescued T lymphopoiesis and the marrow myeloid hyperplasia. After marrow transfer, both cell-autonomous and environmental cues were found to contribute to lymphoid developmental defects and myeloid hyperplasia in Pofut1-deleted mice. Although Pofut1 deficiency slightly decreased cell surface expression of Notch1 and Notch2, it completely abrogated the binding of Notch receptors with Delta-like Notch ligands and suppressed downstream Notch target activation, indicating that O-fucose glycans are critical for efficient Notch-ligand binding that transduce Notch signals. The combined data support a key role for the O-fucose glycans generated by Pofut1 in Notch regulation of hematopoietic homeostasis through modulation of Notch-ligand interactions.

Endocytosis in Notch Signaling Activation

“…The requirement of fucosylation for Notch activation is even more evident in mammals, where constitutive inactivation of the Pofut1 gene produces developmental defects that are undistinguishable from the most aggressive Notch mutants [118]; notably, defects due to Pofut1 absence can be fully rescued by expressing a constitutively active form of Notch1, at least in the hematopoietic compartment [119]. Taken together, these data show that fucosylation is required for proper Notch signaling activation in all species.…”

NOTCH signaling in Sertoli cells regulates gonocyte fate

“…49,50 Results from both studies demonstrated that the NOTCH modifications brought about by POFUT1 are dispensable for testis development and spermatogenesis. 49,50 However, previous studies on POFUT1 demonstrate that the protein is not required for stable cell surface expression of NOTCH receptor in mammals; 51 that an unrelated ER glucosidases can substitute for POFUT1 in promoting NOTCH folding and function; 52 and that mammalian NOTCH receptors are capable of signaling in the absence of POFUT1 and O-fucose. 52 Therefore, the finding that POFUT1 in Sertoli cells is dispensable for testis development and spermatogenesis does not necessarily preclude the possibility that NOTCH signaling can still function, either in entirety or to a limited or different extent, in these cells in modulating or helping to maintain proper regulation of spermatogenesis.…”

Hematopoietic stem cells: to be or Notch to be

“…101 On the other hand, most of the phenotypes associated with Notch pathway manipulation suggest an almost exclusive cellautonomous requirement for Notch signaling in the maintenance of the hematopoietic system. However, mice deficient for Pofut1, which is required upstream of Fringe glycosyltransferases to permit Notch signaling through Delta, displayed both cell-autonomous and non-cell-autonomous defects that resulted in myeloproliferative disease, 100 suggestive of undetermined Notch functions in the regulation of the stromal HSC niche. In addition, defective Notch signaling in the murine skin results in an epidermal-barrierassociated inflammatory phenotype, which leads to massive thymic stroma lymphopoietin (TSLP) expression and produces lethal B-cell 102 or myeloid proliferative disorders.…”

“…Indeed, overexpression of Lunatic fringe that reduces the affinity between Notch and Delta ligands forces lymphoid progenitors to become B cells in a high Delta-presenting environment, 39 whereas modulating the density of Delta1 influences the generation of B or T cells in vitro. 96 Recent works describing the phenotype associated with loss-of-function mutations of positive Notch regulators, such as Mindbomb, 97 Nicastrin, 98 Presenilin, 99 or Pofut, 100 suggested that reduction in the levels of Notch activity is sufficient to induce a myeloproliferative syndrome that phenocopies the effects observed in one of the Mx-Cre-mediated Notch1 and Notch2 deletion, 98 despite that the same experimental model was previously generated showing an exclusive lymphoid phenotype. 88 Consistent with the possibility that myeloid differentiation/proliferation is favored by reduced Notch activity (see model in Figure 2), loss-of-function mutations of the pathway are present in approximately 12% of patients with chronic myelomonocytic leukemia.…”