A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins

Abstract

Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO) cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal α2,3- or α2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b), CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1), core 3 (B3GNT6), core 4 (GCNT1 and B3GNT6), or extended core 1 (B3GNT3) chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galb3GlcNAc) or type 2 (Galb4GlcNAc) outer chains with or without α2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be important tools in studies aiming at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins.

Keywords: CHO; O-glycans; core saccharide; glycosyltransferase; mucin; sialic acid.

Figure 1

Stable CHO transfection scheme with designations of cell clones or bulk-selected cell populations and their respective target structures. CHO-K1 cells with their endogenous glycosylation machinery (green, oval box) were stably transfected with P-selectin glycoprotein ligand-1/mouse IgG2b Fc and O-glycan core chain glycosyltransferase cDNAs (blue) to generate stable CHO cell lines (blue boxes). In the name designation, C stands for CHO-K1, P for PSGL-1/mIgG2b, followed by the expected O-glycan core structures and (B) denotes bulk-selected cell populations. Based on the known specificity of the glycosyltransferases expressed, each cell line was expected to generate, among others, the indicated target structures (dotted line) based on different O-glycan core saccharide chains (dotted box). The O-glycan core structures were elongated by expressing B3GALT5 or by action of the endogenous B4GALTs to form type 1 or 2 outer chains, respectively. Terminal α2,3- or α2,6-sialylation was seen following the activity of a CHO-endogenous ST3GAL(s) or expression of β-galactoside α2,6-sialyltransferase 1 (ST6GAL1).

Figure 2

SDS-PAGE and Western blot analysis of purified PSGL-1/mIgG2b carrying α2,3-sialylated O-glycan core structures. For Western blot analyses, 0.2 μg of protein were loaded and analyzed on SDS-PAGE under reducing (A) or non-reducing (B) conditions. After blotting, membranes were probed with anti-mIgG Fc (A) and anti-PSGL-1 (B). The cell clone designation and how they were generated is shown in Figure 1.

Figure 3

LC-MS chromatograms of O-glycans of recombinant PSGL-1/mIgG2b expressed in CHO-K1 cells stably transfected with O-glycan core glycosyltransferases. CHO-K1 cells stably expressing B3GNT3 and PSGL-1/mIgG2b (CP-ext C1) produced a fusion protein carrying sialylated core 1 and extended core 1 O-glycans (A); those expressing GCNT1 (CP-C2) had mostly core 2 O-glycans on the fusion protein (B); cells expressing B3GNT6 (CP-C3(B)) secreted PSGL-1/mIgG2b decorated with predominantly core 3 O-glycans (C); and cells expressing both GCNT1 and B3GNT6 (CP-C4(B)) carried mostly core 4 O-glycans on PSGL-1/mIgG2b (D). Proposed structures are depicted using the Consortium for Functional Glycomics symbol nomenclature.

Figure 4

SDS-PAGE and Western blot analysis of purified PSGL-1/mIgG2b carrying α2,3-sialylated O-glycan core structures. For Western blot analyses, 0.2 μg of recombinant protein were loaded and analyzed on SDS-PAGE under non-reducing conditions. After blotting, membranes were probed with Maackia amurensis lectin-1 (MAL-1) (A), Maackia amurensis lectin-2 (MAL-2) (B), and Sambucus nigra agglutinin (SNA) (C). MAL-1 recognizes the type 2 chain (Galβ4GlcNAc) with or without α2,3-linked sialic acid; MAL-2 detects terminal α2,3-linked sialic acids; SNA detects terminal α2,6-linked sialic acids.

Figure 5

LC-MS/MS spectra of core 3 O-glycans containing type 1 and 2 outer core chains. MS/MS spectra of type 1 (A,B) and type 2 (C,D) chain-containing O-glycans on core 3 released from PSGL-1/mIgG2b produced in CP-C3-T1. Proposed structures are depicted using the Consortium for Functional Glycomics symbol nomenclature. A schematic characteristic glycosidic or cross-ring cleavage is shown according to the Domon and Costello nomenclature [37].

Figure 6

SDS-PAGE and lectin blot analysis of purified PSGL-1/mIgG2b carrying α2,6-linked sialic acid on different O-glycan core structures. For Western blot analyses, 0.2 μg of recombinant protein were loaded and analyzed on SDS-PAGE under non-reducing conditions. After blotting, membranes were probed with anti-PSGL-1 (A), MAL-1 (B), MAL-2 (C), and SNA (D).