Centralized modularity of N-linked glycosylation pathways in mammalian cells

Abstract

Glycosylation is a highly complex process to produce a diverse repertoire of cellular glycans that are attached to proteins and lipids. Glycans are involved in fundamental biological processes, including protein folding and clearance, cell proliferation and apoptosis, development, immune responses, and pathogenesis. One of the major types of glycans, N-linked glycans, is formed by sequential attachments of monosaccharides to proteins by a limited number of enzymes. Many of these enzymes can accept multiple N-linked glycans as substrates, thereby generating a large number of glycan intermediates and their intermingled pathways. Motivated by the quantitative methods developed in complex network research, we investigated the large-scale organization of such N-linked glycosylation pathways in mammalian cells. The N-linked glycosylation pathways are extremely modular, and are composed of cohesive topological modules that directly branch from a common upstream pathway of glycan synthesis. This unique structural property allows the glycan production between modules to be controlled by the upstream region. Although the enzymes act on multiple glycan substrates, indicating cross-talk between modules, the impact of the cross-talk on the module-specific enhancement of glycan synthesis may be confined within a moderate range by transcription-level control. The findings of the present study provide experimentally-testable predictions for glycosylation processes, and may be applicable to therapeutic glycoprotein engineering.

Figure 1. Glycan structures considered in network construction.

9-Mannose glycan enters the N-linked glycan biosynthetic pathways as the starting substrate (left), and can be processed into a fully sialylated complex-type glycan (right). Symbols indicate the sugar residues according to the CFG nomenclature . GlcNAc: N-acetylglucosamine; Man: mannose; Fuc: fucose; Gal: galactose; NeuAc: sialic acid.

Figure 2. Modular organization of N-linked glycosylation pathways.

(A) The global topology of the constructed N-linked glycosylation pathways. Circles stand for glycan species and arrows for enzymatic reactions from substrates to products. The size of each circle represents the impact on the pathways in the absence of the corresponding glycan. Circles and arrows are colored according to the positions in the pathways and the catalyzing enzymes, respectively. We depict the structures of some early and terminal glycans using the symbols in Figure 1. Glycan synthetic modules are labelled in the ascending order of the number of the participating glycans. (B) In the absence of each glycan, the number of extinguished downstream glycans is shown on the horizontal axis, and the vertical axis shows the number of such absent glycans leading to the same effect. The criterion of the central region is indicated by the arrow at the horizontal axis (Materials and Methods). (C) The number of glycans participating in each module. Module indices on the horizontal axis follow those appearing in (A). At the top in the horizontal direction, we show the number of GlcNAc on mannose branches that each terminal glycan has in a corresponding module.

Figure 3. Regulation of glycan synthesis.

(A) Enhancement of glycan production in specific modules is illustrated with a part of the pathways in Figure 2(A). The down-regulation of GnTIII, GnTIV, and FucT weakens the reactions crossed by red lines, thus strengthening the other reactions toward the 1st and 16th modules, as highlighted. (B) The modules to be enhanced or suppressed under combinations of enzyme up- and down-regulation. Here we show only the cases where six or fewer modules become enhanced, and exclude the indices of the modules that are never enhanced in these cases. Each row displays a unique pattern of module enhancement resulting from certain regulatory combinations, and is labelled as shown in Table S1. Enhanced modules are colored blue or green, and suppressed ones are indicated in white. The row including greens is for the case demonstrated in (A). (C) From the lists of modules to be enhanced together with a given module on the horizontal axis, we enumerated the minimum number of such co-enhanced modules as shown in the vertical axis. Blue is for less than or equal to 6 in the minimum number, obtained from the module enhancement pattern shown in (B).