Fc galactosylation follows consecutive reaction kinetics and enhances immunoglobulin G hexamerization for complement activation

Fc galactosylation is a critical quality attribute for anti-tumor recombinant immunoglobulin G (IgG)-based monoclonal antibody (mAb) therapeutics with complement-dependent cytotoxicity (CDC) as the mechanism of action. Although the correlation between galactosylation and CDC has been known, the underlying structure–function relationship is unclear. Heterogeneity of the Fc N-glycosylation produced by Chinese hamster ovary (CHO) cell culture biomanufacturing process leads to variable CDC potency. Here, we derived a kinetic model of galactose transfer reaction in the Golgi apparatus and used this model to determine the correlation between differently galactosylated species from CHO cell culture process. The model was validated by a retrospective data analysis of more than 800 historical samples from small-scale and large-scale CHO cell cultures. Furthermore, using various analytical technologies, we discovered the molecular basis for Fc glycan terminal galactosylation changing the three-dimensional conformation of the Fc, which facilitates the IgG1 hexamerization, thus enhancing C1q avidity and subsequent complement activation. Our study offers insight into the formation of galactosylated species, as well as a novel three-dimensional understanding of the structure–function relationship of terminal galactose to complement activation in mAb therapeutics

Biophysical Determinants of Notch Signaling.

Notch signaling is involved in many biological contexts such as cancer, stem cell development, and neural cell development. Because of the importance of Notch function in health and disease, the Notch signaling pathway has emerged as a potential therapeutic target. Mammalian Notch receptors are single-pass transmembrane glycoprotein receptors, which contain 29-36 EGF like repeats. The fucosyltransferase termed Pofut1 transfers fucose to the serine or threonine residue of the O-fucosylation consensus sequence on some EGF domains of Notch receptors. The glycosyltransferases termed Fringe can elongate O-fucose moieties by adding N-acetylglucosamine, which may be subsequently modified by galactose and sialic acid. These O-fucosylated glycans play key roles in modulating Notch-mediated signal transduction events. Here, we have observed how O-fucosylated glycan modifications modulate Notch receptor-ligand interactions using surface plasmon resonance techniques. A biphasic binding and dissociation pattern was observed, suggesting a two-state receptor-ligand interaction model characterized by initial formation of a transient receptor-ligand complex followed by a conformational change that leads to a more stable receptor-ligand complex. Primary and secondary on and off-rates for the four binding-competent Notch1-Notch ligand pairs were observed to be distinct and characteristic for each Notch ligand. The overall association constants observed when Dll-1 or Dll-4 interacted with Fringe-modified Notch1 were significantly greater than when these ligands interacted with unmodified Notch1, with enhancement likely due to Fringe modifications of fucose moieties within EGF domains 16-36. By contrast, Fringe modification of Notch1 did not significantly modulate interactions with Jag-1 or Jag-2. Mutational analyses confirm prior observations that the O-fucosylation site within EGF repeat 12 dictates much, if not all of the binding between Notch1 and its ligands. Finally, we observe that Fringe modification of Dll-4 enhances its ability to bind to Notch1. Our data reveal that the molecular basis of glycan-dependent Notch-Notch ligand binding. We propose a two-state binding model with triple stranded structure for Notch-Notch ligand complex arrangement. Here, O-fucosylation and Fringe modification of Notch receptors play a key role in both the binding and the conformational change.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/63631/1/jsshim_1.pd

O-Fucose Modulates Notch-Controlled Blood Lineage Commitment

Notch receptors are cell surface molecules essential for cell fate determination. Notch signaling is subject to tight regulation at multiple levels, including the posttranslational modification of Notch receptors by O-linked fucosylation, a reaction that is catalyzed by protein O-fucosyltransferase-1 (Pofut1). Our previous studies identified a myeloproliferative phenotype in mice conditionally deficient in cellular fucosylation that is attributable to a loss of Notch-dependent suppression of myelopoiesis. Here, we report that hematopoietic stem cells deficient in cellular fucosylation display decreased frequency and defective repopulating ability as well as decreased lymphoid but increased myeloid developmental potential. This phenotype may be attributed to suppressed Notch ligand binding and reduced downstream signaling of Notch activity in hematopoietic stem cells. Consistent with this finding, we further demonstrate that mouse embryonic stem cells deficient in Notch1 (Notch1−/−) or Pofut1 (Pofut1−/−) fail to generate T lymphocytes but differentiate into myeloid cells while coculturing with Notch ligand–expressing bone marrow stromal cells in vitro. Moreover, in vivo hematopoietic reconstitution of CD34+ progenitor cells derived from either Notch1−/− or Pofut1−/− embryonic stem cells show enhanced granulopoiesis with depressed lymphoid lineage development. Together, these results indicate that Notch signaling maintains hematopoietic lineage homeostasis by promoting lymphoid development and suppressing overt myelopoiesis, in part through processes controlled by O-linked fucosylation of Notch receptors

Notch-dependent control of myelopoiesis is regulated by fucosylation

Cell-cell contact–dependent mechanisms that modulate proliferation and/or differentiation in the context of hematopoiesis include mechanisms characteristic of the interactions between members of the Notch family of signal transduction molecules and their ligands. Whereas Notch family members and their ligands clearly modulate T lymphopoietic decisions, evidence for their participation in modulating myelopoiesis is much less clear, and roles for posttranslational control of Notch-dependent signal transduction in myelopoiesis are unexplored. We report here that a myeloproliferative phenotype in FX−/− mice, which are conditionally deficient in cellular fucosylation, is consequent to loss of Notch-dependent signal transduction on myeloid progenitor cells. In the context of a wild-type fucosylation phenotype, we find that the Notch ligands suppress myeloid differentiation of progenitor cells and enhance expression of Notch target genes. By contrast, fucosylation-deficient myeloid progenitors are insensitive to the suppressive effects of Notch ligands on myelopoiesis, do not transcribe Notch1 target genes when cocultured with Notch ligands, and have lost the wild-type Notch ligand-binding phenotype. Considered together, these observations indicate that Notch-dependent signaling controls myelopoiesis in vivo and in vitro and identifies a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in myeloid progenitors