Role of N-glycosylation in trafficking of apical membrane proteins in epithelia

Abstract

Polarized distribution of plasma membrane transporters and receptors in epithelia is essential for vectorial functions of epithelia. This polarity is maintained by sorting of membrane proteins into apical or basolateral transport containers in the trans-Golgi network and/or endosomes followed by their delivery to the appropriate plasma membrane domains. Sorting depends on the recognition of sorting signals in proteins by specific sorting machinery. In the present review, we summarize experimental evidence for and against the hypothesis that N-glycans attached to the membrane proteins can act as apical sorting signals. Furthermore, we discuss the roles of N-glycans in the apical sorting event per se and their contribution to folding and quality control of glycoproteins in the endoplasmic reticulum or retention of glycoproteins in the plasma membrane. Finally, we review existing hypotheses on the mechanism of apical sorting and discuss the potential roles of the lectins, VIP36 and galectin-3, as putative apical sorting receptors.

Fig. 1.

Simplified schema showing the pathways and enzymes involved in synthesis of high-mannose, hybrid, and complex N-glycans in the Golgi. High-mannose N-glycans imported from the endoplasmic reticulum (ER) to the Golgi can remain unchanged or can undergo various transformations under the influence of Golgi mannosidases and glycosyltransferases. Six different N-acetylglucosamine-glycosyltransferases (GnTs) present in the Golgi (I–VI) can add N-acetylglucosamine (GlcNAc) residues to the 3-mannosyl core of N-glycans (green rectangle) and thereby produce diverse carbohydrate structures. The addition of GlcNAc residues by GnT-I, -II, -IV, -V, and -VI, but not by GnT-III, allows elongation of the chains, referred to as “antennae.” Elongation of the chains is accomplished by addition of monosaccharide linkages catalyzed by other various glycosyltransferases (dashed arrows). GnT-I is a critical regulatory enzyme for formation of both hybrid and complex-type N-glycans. GnT-II acts downstream of GnT-I to catalyze the formation of the complex-type N-glycans. GnTs-IV, -V, and -VI promote branching of N-glycans by adding GlcNAc into the positions shown by different shades of blue. Individual N-glycans can be modified by one or more of these enzymes, resulting in N-glycans that contain from 1–5 antennae. In contrast, GnT-III (red) stops branching by adding a bisecting GlcNAc to any of the N-glycans, thereby preventing the downstream action of GnTs-II, -IV, -V, and -VI. In the example depicted, the effect of this enzyme on the product of GnT-I results in the formation of a 1-antennary N-glycan of the hybrid type. Branching also can be prevented by exposure of cells to inhibitors of the Golgi mannosidase I and II, deoxymannojirimycin (dMM) and swainsonine, resulting in preservation of the high-mannose or hybrid-type structure of N-glycans, respectively. A modified version of this figure is in Ref. .

Fig. 2.

H-K-ATPase β-subunits expressed on the apical surface of LLC-PK₁ cells are complex-type glycosylated, while the intracellular nonmature fraction contains high-mannose-type subunits. The YFP-linked β-subunit of the H-K-ATPase (YFP-β) stably expressed in LLC-PK₁ cells is detected in total cell lysates in 2 forms: EndoH resistant and EndoH sensitive. Only the EndoH-resistant complex-type form reaches the apical surface, as detected by apical surface biotinylation. The intracellular form of YFP-β is converted by EndoH to the deglycosylated protein that has the same gel mobility as YFP-β in cell lysates prepared after exposure of the cells to tunicamycin, the inhibitor of N-glycosylation,. The gel mobility of the EndoH-sensitive form of YFP-β is the same as that of YFP-β from the cells exposed to dMM (preserves the high-mannose structure of glycoproteins) and greater than that of YFP-β from the cells exposed to swainsonine (preserves the hybrid structure of glycoproteins). These results indicate that the intracellular fraction of YFP-β contains high-mannose N-glycans and likely represents the ER-resident form. Symbols for monosaccharide residues and abbreviations correspond to those in the legend to Fig. 1.