Search

Notch is a cell-surface receptor that controls cell-fate decisions and is regulated by O-glycans attached to epidermal growth factor-like (EGF) repeats in its extracellular domain. Protein O-fucosyltransferase 1 (Pofut1) modifies EGF repeats with O-fucose and is essential for Notch signaling. Constitutive activation of Notch signaling has been associated with a variety of human malignancies. Therefore, tools that inhibit Notch activity are being developed as cancer therapeutics. To this end, we screened L-fucose analogs for their effects on Notch signaling. Two analogs, 6-alkynyl and 6-alkenyl fucose, were substrates of Pofut1 and were incorporated directly into Notch EGF repeats in cells. Both analogs were potent inhibitors of binding to and activation of Notch1 by Notch ligands Dll1 and Dll4, but not by Jag1. Mutagenesis and modeling studies suggest that incorporation of the analogs into EGF8 of Notch1 markedly reduces the ability of Delta ligands to bind and activate Notch1.

The bisecting GlcNAc is transferred to the core mannose residue of complex or hybrid N-glycans on glycoproteins by the β1,4-N-acetylglucosaminyltransferase III (GlcNAcT-III) or MGAT3. The addition of the bisecting GlcNAc confers unique lectin recognition properties to N-glycans. Thus, LEC10 gain-of-function Chinese hamster ovary (CHO) cells selected for the acquisition of ricin resistance, carry N-glycans with a bisecting GlcNAc, which enhances the binding of the erythroagglutinin E-PHA, but reduces the binding of ricin and galectins-1, -3 and -8. The altered interaction with galactose-binding lectins suggests that the bisecting GlcNAc affects N-glycan conformation. LEC10 mutants expressing polyoma middle T antigen (PyMT) exhibit reduced growth factor signaling. Furthermore, PyMT-induced mammary tumors lacking MGAT3, progress more rapidly than tumors with the bisecting GlcNAc on N-glycans of cell surface glycoproteins. In recent years, evidence for a new paradigm of cell growth control has emerged involving regulation of cell surface residency of growth factor and cytokine receptors via interactions and cross-linking of their branched N-glycans with a lattice of galectin(s). Specific cross-linking of glycoprotein receptors in the lattice regulates their endocytosis, leading to effects on growth factor-induced signaling. This review will describe evidence that the bisecting GlcNAc of N-glycans regulates cellular signaling and tumor progression, apparently through modulating N-glycan/galectin interactions.

The bisecting GlcNAc is transferred to complex or hybrid N-glycans by the action of N-acetylglucosaminyltransferase III (GlcNAc-TIII) encoded by the Mgat3 gene. CHO cells expressing mouse GlcNAc-TIII were shown by matrix-assisted laser desorption ionization (MALDI) mass spectrometry to produce mainly complex N-glycans with the predicted extra (bisecting) GlcNAc. In order to probe biological functions of the bisecting GlcNAc, antibodies that recognize this residue in the context of complex cell surface glycoconjugates were sought. The LEC10 gain-of-function Chinese hamster ovary (CHO) cell mutant that expresses GlcNAc-TIII and complex N-glycans with the bisecting GlcNAc was used to immunize Mgat3+/+ and Mgat3−/− mice. ELISA of whole sera showed that polyclonal antibodies that bound specifically to LEC10 cells were obtained solely from Mgat3−/− mice. Fluorescence-activated cell cytometry of different CHO glycosylation mutants and western blotting after glycosidase treatments were used to show that anti-LEC10 cell antisera from Mgat3−/− mice recognize cellular glycoproteins with complex N-glycans containing both a bisecting GlcNAc and Gal residues. The polyclonal antibody specificity was similar to that of the lectin E-PHA. IgM-depleted serum containing IgG and IgA antibodies retained full binding activity. Therefore Mgat3−/− mice but not wild type mice can be used effectively to produce polyclonal antibodies that specifically recognize glycoproteins bearing complex N-glycans with a bisecting GlcNAc. Published in 2003.

Cytotoxic plant lectins have been used for the single-step selection of mouse embryonal carcinoma cell mutants with altered expression of surface glycoconjugates. Following mutagenesis, several F9 and OTF9-63 cell lines resistant to the lectins from Triticum vulgaris or Ricinus communis were obtained. At least five distinct lectin-resistant (LecR) phenotypes have been identified on the basis of their relative sensitivities to four different plant lectins and their altered lectin-binding properties. None of the mutant types exhibits a significant change in the ability to bind a monoclonal antibody against the stage-specific embryonic antigen, SSEA-1. All of the mutants form aggregates when cultured in bacteriological petri dishes and appear to differentiate into endoderm-like cells following exposure to retinoic acid. However, two of the LecR cell lines exhibit an altered morphology when grown on a plastic substratum.

High-frequency transfection of CHO cells has been achieved for several plasmids, a cosmid library, and genomic DNA using Polybrene and dimethyl sulfoxide. All plasmid transfectants examined were stable and exhibited plasmid sequences in genomic DNA. The method is simple, reproducible, and succeeded with several independent CHO clones in the presence or the absence of carrier DNA, even at very low concentrations of plasmid DNA.

The cytotoxic plant lectins from P. sativum have been used to select new lectin-resistant mutants from Chinese hamster ovary (CHO) cells. Two novel phenotypes that behave recessively but fall into the same complementation group have been termed Lec13 and Lec13A. Both of these mutant types are phenotypically reverted to pea lectin sensitivity following growth in L-fucose. In contrast, two other unique phenotypes behave dominantly in somatic cell hybrids and maintain their pea lectin-resistance in the presence of L-fucose. They have been termed LEC14 and LEC18, respectively. The lectin-resistance and complementation properties of the four new mutant types suggest that they define three different glycosylation genes of the CHO genome.

Chinese hamster cell mutants resistant to the lectins PHA, WGA, RIC, LCA, and CON A were previously grouped into 8--10 distinct phenotypes on the basis of their unique patterns of lectin resistance and lectin-binding properties. All but one of these classes of lectin-resistant (LecR) mutants behave recessively in somatic cell hybrids. One ricin-resistant class (RicRII) behaves dominantly. Tests for complementation, by measuring the lectin-resistant properties of appropriate hybrids, show that seven distinct complimentation groups can be delineated among the phenotypically recessive mutants.

The cytotoxicity of 10 plant lectins with different carbohydrate recognition properties towards a number of mouse embryonal carcinoma (EC) cell lines (F9, OTF9-63, PCC4, PCC3/A/1, P19, and P19S1801A1) has been examined. Six of the lectins are toxic for the majority of the cell types at concentrations of less than or equal to 100 micrograms/ml and should be useful as direct selective agents for the isolation of EC glycosylation mutants (see accompanying manuscript). However, the concentration of the various lectins required to kill 90% of the cell population differs markedly between EC cell lines, the greatest variation being observed with the lectins from T. vulgaris (wheat germ agglutinin; WGA) and G. simplicifolia (GS-I). The lectin-binding abilities of different EC cell lines also vary and do not necessarily correlate with their relative lectin sensitivities. Certain lectins which are not toxic even at concentrations of 200 micrograms/ml, nevertheless exhibit significant binding at the cell surface. The extensive variation in lectin sensitivities and lectin-binding abilities between the EC cell lines is diagnostic of the expression of different carbohydrate structures at their respective cell surfaces. The results suggest that the EC lines examined will give rise to different families of glycosylation mutants.

The identification of causal variants in sequencing studies remains a considerable challenge that can be partially addressed by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and essentiality screens carried out on human cell lines. We propose a cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing biological properties. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented among genes non-essential for cell survival but required for organism development. After screening developmental disorder cases from three independent disease sequencing consortia, we identify potentially pathogenic variants in genes not previously associated with rare diseases. We therefore propose FUSIL as an efficient approach for disease gene discovery., Discovery of causal variants for monogenic disorders has been facilitated by whole exome and genome sequencing, but does not provide a diagnosis for all patients. Here, the authors propose a Full Spectrum of Intolerance to Loss-of-Function (FUSIL) categorization that integrates gene essentiality information to aid disease gene discovery.