The bisecting GlcNAc in cell growth control and tumor progression

Abstract

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.

Fig. 1

The bisecting GlcNAc and lectin binding. a A proposed complex N-glycan containing the bisecting GlcNAc added by MGAT3 expressed in LEC10 cells, and the β1,6GlcNAc branch initiated by MGAT5 and absent from Lec4 mutant cells. b Lectin resistance test of CHO wild type and LEC10B cells expressing MGAT3 using the lectins ricin and E-PHA (adapted from [14]). c Flow cytometry of FITC-labeled galectin-3 binding to CHO, LEC10 or LEC11 cells in the presence or absence of lactose (courtesy of Santosh Patnaik [36]). LEC11 cells express Fut6 and add Fuc to Lac-NAc to generate the LeX and SLeX epitopes (Fig. 1a [76])

Fig. 2

Galectin-1 binding to CHO and glycosylation mutants. CHO cell lines (CHO, Lec1, LEC10, and Lec4) grown in monolayer or suspension culture were washed, biotinylated, harvested and fixed. The biotinylated cells (50,000 cells/well) were arrayed on neutravidin-coated black ELISA plates. A subset of biotinylated glycans from the Consortium for Functional Glycomics (CFG) Glycan Array version 2.3, including known binders and non-binders to galectin-1, were also arrayed on the same plate at a concentration of 60 pmol/well. Alexa-488 labeled human galectin-1 (30 μg/ml) was applied to each well in binding buffer (20 mm Tris–HCl, pH 7.4, 150 mm NaCl, 2 mm CaCl₂, 2 mm MgCl₂, and 0.05% Tween 20 with 1% bovine serum albumin (BSA)) and incubated for 1 h at room temperature. After galectin-1 removal, plates were washed three times with binding buffer lacking BSA and relative fluorescence units measured. Structures of complex N-glycans typical of CHO mutant cells and a subset of glycans on the array are shown. CHO cells contain β1,6GlcNAc branched complex N-glycans that lack the bisecting GlcNAc; Lec1 cells have no complex N-glycans; LEC10/LEC10B cells contain complex N-glycans modified with the bisecting GlcNAc; Lec4 cells lack both β1,6GlcNAc branched N-glycans and the bisecting GlcNAc. A typical Lec8 mutant complex N-glycan is shown as it is included in the model in Fig. 3. See Fig. 1a for glycan symbols. NC, no cells or compound; PBS, phosphate buffered saline (no galectin-1)

Fig. 3

Model of galectin-dependent PDGFR signaling in CHO cells. Higher order clustering of PDGFRs on the CHO cell surface may be achieved through galectin-N-glycan interactions, which are thought to form a lattice that restrains endocytosis and promotes optimal ERK1/2 activation. Complex N-glycans on wild type CHO cells have the most binding sites for galectins and the greatest response to PDGF-AB. ERK1/2 activation is reduced in LEC10B cells that add the bisecting GlcNAc to complex N-glycans, and in Lec4 cells that lack a branch of complex N-glycans, and occurs at background levels in Lec8 cells that have few if any LacNAc units on complex N-glycans. Signaling strength correlates with the degree of interaction with the galectin lattice. Binding of N-glycans to galectin-3 pentamers is shown as an example, although other galectins are likely to participate in CHO galectin lattice(s). See Fig. 2 for complex N-glycan structures typical of CHO mutant cells