Biochimie 85 (2003) 323–330 www.elsevier.com/locate/biochi
Involvement of glycosylation in the intracellular trafficking of glycoproteins in polarized epithelial cells G. Huet a,*, V. Gouyer a, D. Delacour a, C. Richet a, J.P. Zanetta b, P. Delannoy b, P. Degand a b
a Unité INSERM 560, place de Verdun, 59045 Lille cedex, France Unité de Glycobiologie structurale et fonctionnelle, UMR CNRS n° 8576, Laboratoire de chimie biologique, Université des sciences et technologies de Lille, 59655 Villeneuve d’Ascq, France
Received 28 October 2002; accepted 6 February 2003
Abstract The surface of epithelial cells is composed of apical and basolateral domains with distinct structure and function. This polarity is maintained by specific sorting mechanisms occurring in the Trans-Golgi Network. Peptidic signals are responsible for the trafficking via clathrin-coated vesicles by means of an interaction with an adaptor complex (AP). The basolateral targeting is mediated by AP-1B, which is specifically expressed in epithelial cells. In contrast, the apical targeting is proposed to occur via apical raft carriers. It is thought that apically targeted glycoproteins contain glycan signals that would be responsible for their association with rafts and for apical targeting. However, the difficulty in terms of acting specifically on a single step of glycosylation did not allow one to identify such a specific signal. The complete inhibition of the processing of N-glycans by tunicamycin often results in an intracellular accumulation of unfolded proteins in the Golgi. Similarly, inhibition of O-glycosylation can be obtained by competitive substrates which gave a complex pattern of inhibition. Therefore, it is still unknown if glycosylation acts in an indirect manner, i.e. by modifying the folding of the protein, or in a specific manner, such as an association with specific lectins. © 2003 Éditions scientifiques et médicales Elsevier SAS and Société française de biochimie et biologie moléculaire. All rights reserved. Keywords: Glycosylation; Polarized cells; Trafficking
1. Introduction Epithelial cells are constituted by two distinct surfaces that differ in functional properties. In intestinal cells for example, the apical surface forms a brush border facing the intestinal lumen, which is involved in intestinal digestion, while the basolateral surface mediates cellular adhesion and signalization. These two distinct surfaces are separated by tight junctions. Consequently, the apical and basolateral surface have completely different compositions. The apical surface contains glycoproteins specifically involved in intestinal digestion and uptake of nutriment, and the basolateral surface contains adhesion molecules and receptors mediating signal transduction. In addition, the apical domain differs from the basolateral domain in terms of its lipid and carbohydrate composition. The apical membrane is enriched in sphingolipids, glycosphingolipids and cholesterol and * Corresponding author. Tel.: +33-3-20-29-88-60; fax: +33-3-20-53-85-62. E-mail address: [email protected]
glycosylphosphatidylinositol-anchored (GPI) glycoproteins. The generation and maintenance of two different domains implies a strict regulation of the targeting of components to each surface of the cell. 2. Trafficking pathways in polarized epithelial cells 2.1. There are two types of intracellular traffıcking pathways There are two possibilities by which components can reach their correct surface, i.e. a direct or indirect route  (Fig. 1). In the direct route (biosynthetic pathway), components are sorted in the Trans-Golgi Network (TGN) in carrier vesicles that make their way towards the apical or basolateral surface. In the indirect route (transcytosis pathway), components reach one surface (usually the basolateral surface), are then endocytosed and delivered to the other surface via early endosomes and recycling endosomes. Thus, sorting of glycoproteins is expected to occur both in the TGN and in the
© 2003 Éditions scientifiques et médicales Elsevier SAS and Société française de biochimie et biologie moléculaire. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 0 3 0 0 - 9 0 8 4 ( 0 3 ) 0 0 0 5 6 - 7
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cally in epithelial cells and shown to be involved in sorting to the basolateral membrane . 2.3. Sorting to the apical surface
Fig. 1. Representation of an intestinal cell characterized by apical (brush border of microvilli) and basolateral plasma membranes. The transport to the basolateral domain occurs through clathrin-coated carrier vesicles by means of a specific interaction of a peptidic signal with AP1-B adaptor complex. This trafficking pathway is targeted by NSF/SNAP/SNARE machinery. The transport to the apical domain occurs through apical raft carriers. Glycans have been proposed as prerequisite determinants for the association with raft and apical targeting. This pathway is NSF-independent, but regulated by proteins of SNARE complex.
endosomes. Usually, both direct and indirect routes occur in polarized cells. However, the importance of each route widely varies according to the cell-type. For example, trafficking to the apical surface mainly occurs through the direct pathway in the renal cells Madin-Darby Canine Kidney (MDCK cells), and through the indirect pathway in the intestinal Caco-2 cells. 2.2. Sorting to the basolateral surface Sorting of proteins towards the basolateral membrane is encoded by peptidic signals containing Tyr or Leu-Leu motifs, localized in the cytoplasmic domain of basolateral proteins . Some of these peptidic signals are identical to those mediating endocytosis in clathrin-coated vesicles. Such peptidic signals are recognized by an adaptor complex (AP) which plays a role both in the assembly of the clathrin coat and in the trafficking of the clathrin-coated vesicles . Different types of AP complexes are specifically involved in the transport of clathrin-coated vesicles towards the endosomal/lysosomal system or the plasma membrane in anterograde or retrograde pathways . The destination of a protein is thus dependent upon the specific binding of its peptidic signal sequence to a complementary AP complex. AP-1B is an AP complex which was recently found specifi-
The apical delivery has been proposed to involve the recruitment of apical glycoproteins within lipid rafts which would then bud from TGN into apical carrier vesicles . Rafts are membrane microdomains enriched in sphingolipids, glycosphingolipids and cholesterol particularly on the outer leaflet . Sphingolipids contain long saturated fatty acyl chains which tightly associate into packed assemblies within the more fluid environment of cis-unsaturated acyl chain phospholipids. Cholesterol plays a role in stabilizing such lipid microdomains . The liquid-ordered phase constituted by the lipids of microdomains confers them a property of insolubility in non-ionic detergents at 4 °C . Rafts (also called DIGs, detergent-insoluble glycosphingolipid complexes) can thus be isolated after detergent extraction and floatation ultracentrifugation on a discontinuous density gradient . Apical GPI-anchored proteins and transmembrane glycoproteins have been isolated by this procedure of detergent insolubility showing their association with lipid rafts [11–13]. Based on these observations, sorting signals for apical delivery were researched on these membrane proteins specifically enriched in lipid rafts. Several types of sorting signals for the apical surface have been proposed. 2.4. Regulation of biosynthetic pathways Vesicle docking and fusion are ensured by the proteins of SNARE machinery, comprising soluble cytosolic factors (soluble N-ethyl maleimide-sensitive factor (NSF) and soluble NSF attachment protein (SNAP)), and complementary SNAP-receptors (SNAREs) (respectively, localized on the target destination (t-SNARE) and on the carrier vesicle (v-SNARE)). The regulation of this biosynthetic pathway of carrier vesicles is controlled by small GTPases of the Rab family . Consequently, basolateral targeting is controlled by NSF/SNAP/SNARE mechanism involving t-SNAREs, syntaxin 4 and SNAP 23, and v-SNARE toxin-sensitive vesicleassociated membrane protein (VAMP), and by Rab 8 [15–17]. The apical route is insensitive to N-ethyl maleimide [18,19]. Several proteins have been shown to be involved in the regulation of apical raft carriers: t-SNAREs syntaxin 3 and SNAP 23, v-SNARE toxin-insensitive VAMP (Ti-VAMP or VAMP7) and annexin XIIIb [20–24]. 3. Glycan signal sequences for apical targeting 3.1. GPI anchor Due to the presence of GPI-anchored glycoproteins at the apical membrane of polarized cells  and the insolubility of GPI-anchored proteins in non-ionic detergent at 4 °C, the
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role of the GPI anchor as a sorting signal was first proposed. However, the function of the GPI anchor as an apical targeting determinant is questioned by several studies. The ectodomains of the GPI-anchored decay acceleration factor (DAF), placental alcaline phosphatase (PLAP) and Thy-1 were secreted apically when attachment of the GPI anchor had been abrogated [26–28]. More recently, a chimaeric construct of the rat growth hormone (rGH), a soluble protein secreted both apically and basolaterally, with the GPI anchor did not result into a delivery to the apical membrane in MDCK cells , in such a way that the role of the GPI anchor as an apical targeting signal is now debated. 3.2. N-glycans The putative role of N-glycans in apical targeting was studied through the use of drugs affecting the processing of N-glycans or of mutations of N-glycosylation sites of glycoproteins. 3.2.1. Inhibition in the processing of N-glycans The treatment of MDCK cells with tunicamycin, a drug which blocks the transfer of Glc3Man9GlcNAc2 from dolichol phosphate to Asn , led to a random secretion of apically secreted 80-kD sulphated glycoprotein complex and erythropoietin [31,32]. Nevertheless, data with tunicamycin were generally difficult to interpret because tunicamycin treatment often results in an intracellular accumulation of non-correctly folded proteins . The ecto-nucleotide pyrophosphatase phosphodiesterase (NPP3) becomes in part intracellularly accumulated in tunicamycin-treated MDCK and Caco-2 cells, but remains, for the other part, targeted to the apical plasma membrane . Studies performed using deoxymannojirimycin (an inhibitor of mannosidase I) and swainsonine (an inhibitor of mannosidase II) in Caco-2 cells, show that the processing of N-glycans was found to be important for the apical delivery of dipeptidylpeptidase IV (DPP-IV), but not for that of sucrase-isomaltase (SI) or aminopeptidase N [35,36]. 3.2.2. Addition of N-glycosylation sites The role of N-glycans in apical targeting signal was shown through the modification of the unglycosylated protein rGH by the addition of two N-glycosylation sites which resulted in the almost exclusive secretion of GH in the apical medium . The addition of N-glycosylation sites on GPI-anchored rGH also resulted in a predominant apical delivery . On the other hand, the role of N-glycans in apical targeting of transmembrane proteins was shown on different protein models lacking the basolateral sorting signal . In the absence of N-glycosylation and a basolateral sorting signal, the proteins accumulated in the Golgi complex not only in polarized MDCK cells but also in non-polarized CHO cells, indicating that N-glycans were efficient signals for cell surface targeting in both polarized and non-polarized cells. In this regard, it must be specified that N-glycans play an impor-
tant role in the folding process involving a unique chaperone system found in the ER, the so-called calnexin-calreticulin cycle [39,40]. Furthermore, cytoplasmic basolateral determinants were dominant over the carbohydrate apical sorting signal. In conclusion, it is still not clear if N-glycosylation plays a role through the correct folding of the proteins or through another specific mechanism. 3.3. O-glycans Other data showed that O-glycosylation could act as an apical targeting signal on both secreted and transmembrane glycoproteins. These data emerged from experiments that were carried out using an inhibitor of O-glycosylation or after the deletion of potential O-glycosylation sites. 3.3.1. Inhibition in the processing of O-glycans O-glycosylation can be inhibited by GalNAca-O-bn, a synthetic analogue of N-acetylgalactosamine, the first sugar always added to serine or threonine residues in the O-glycosylation process. The elongation of O-glycan chain occurs on this competitive acceptor instead of occurring on the natural substrate. Treatment of mucus-secreting HT-29 cells (HT-29 MTX) by GalNAca-O-bn induced an inhibition in the constitutive and secretagogue-induced secretion of mucins, which are highly O-glycosylated glycoproteins [41,42]. In addition, GalNAca-O-bn treatment also inhibited the apical targeting of several membrane brush border glycoproteins: a transmembrane glycoprotein (DPP-IV), a glycoprotein anchored through GPI moiety (carcinoembryonic antigen, CEA), and a mucin-like membrane glycoprotein (MUC1) in differentiated polarized HT-29 cells of mucinsecreting and enterocytic phenotypes [43,44]. These glycoproteins did not reach their membrane apical localization, but remained stored in the cytoplasm. In contrast, no change was found for the distribution of two basolateral N-glycoproteins, gp120 and gp525. In Caco-2 cells, GalNAca-O-bn also induced a selective alteration in the apical targeting of the brush border glycoproteins SI  and DPP-IV . 3.3.2. Deletion of O-glycosylation sites The p75 neurotrophic receptor (p75NTR) is a type I membrane protein primarily delivered to the apical plasma membrane when transfected in MDCK or Caco-2 cells . Deletion of a juxtamembrane region containing several O-glycosylation sites induced the targeting of the mutant protein to the basolateral domain. Constructs with the ectodomain of p75NTR showed that the juxtamembrane domain was efficient for an apical secretion in MDCK cells but not in Caco-2 cells, showing that the membrane anchor was a prerequisite associated determinant for the apical targeting [47,48]. For another glycoprotein, SI, expressed at the apical brush border of enterocytes, the stalk domain containing O-glycosylation sites and the membrane anchor constituted essential determinants for raft association and apical delivery
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. Fusion of the O-glycosylated stalk region and the transmembrane domain of human SI with rat GH allowed to induce an apical localization under conditions fulfilling an appropriate spatial requirement . Recently, both N- and O-glycans were reported to be involved in the apical targeting of DPP-IV . Nevertheless, if these many studies showed that N and/or O-glycosylation are prerequisite determinants for apical targeting, the mechanisms by which glycosylation could control intracellular trafficking are still unknown. Several authors proposed the hypothesis of a putative lectin, but this hypothesis is not confirmed, and the question remains to determine if glycosylation actually represents an apical targeting signal or acts through the folding of proteins and exposition of appropriate determinants.
GalNAca-O-bn was investigated in mucin-secreting HT-29 MTX cells. GalNAca-O-bn particularly induced a dramatic decrease (by 13-fold) in the relative amount of sialic acid, whereas no change was found in the relative amount of Gal residues. This result showed that the sialylation of mucins was primarily affected by GalNAca-O-bn in HT-29 MTX cells. Structural investigations have determined that oligosaccharides of HT-29 MTX mucins consisted of short sialylated structures (two to seven residues), mainly of core types 1 and 2, and that incorporation of sialic acid occurred primarily via an a2,3-linkage to a terminal Gal residue. The major structure was the monosialylated trisaccharide of core type 1, i.e. Neu5Aca2-3Galb1-3GalNAc (41%) . Consequently, the inhibition of the incorporation of Neu5Ac residues in HT-29 MTX mucins by GalNAca-O-bn could be related to an inhibition of ST3Gal I.
4. Cell-type specific effects of GalNAca-O-bn
4.1.2. Inhibition of the glycosylation of other glycoproteins by GalNAc␣-O-bn The effect of GalNAca-O-bn exposure upon the glycosylation of other glycoproteins was analysed particularly for brush-border associated glycoproteins and cell adhesion molecules in HT-29 cells. Different brush-border associated glycoproteins, i.e. DPP-IV, MUC1, and CEA, and basolateral glycoproteins i.e. integrins, gp525, and CD44 were examined for their glycosylation [43,44,54]. All these glycoproteins were found to contain a terminal sialylation by a2,3linked sialic acid. After permanent GalNAca-O-bn treatment, this terminal a2,3-sialylation was inhibited for some glycoproteins (DPP-IV, MUC1, CEA, CD44) but not for others (integrin b4 and a6, gp525). Among the glycoproteins with inhibited sialylation, the appearance of T antigen expression could be shown for three glycoproteins (DPP-IV, MUC1, and CD44). This antigen is a structure specifically O-linked to Thr or Ser residues, and its substitution by a2,3linked sialic acid is only obtained through the enzymatic activity of ST3Gal I. These observations again evidenced that GalNAca-O-bn treatment primarily inhibited the elongation of O-glycans by ST3Gal I in HT-29 cells. In contrast, N-glycosylated glycoproteins appeared to show variable sensitivity to GalNAca-O-bn treatment. GalNAca-O-bn was also found to inhibit the glycosylation of membrane proteins in other cell-types: an inhibition of O-glycosylation of the brush border glycoprotein SI in Caco-2 cells , an inhibition of the sialylation of CD44 in B16BL6 melanoma cells , and an inhibition of the sialylation of DPP-IV in Caco-2 and MDCK cells [36,45].
Several investigations showed that the effect of GalNAcaO-bn on O-glycosylation and on intracellular trafficking varied according to the cell-type. This cell-type specific effect was correlated with the metabolization of GalNAca-O-bn inside the cell. 4.1. Inhibition of the glycosylation by GalNAc␣-O-bn The effect of GalNAca-O-bn was initially studied on secreted mucins, which are highly O-glycosylated glycoproteins. 4.1.1. Inhibition of the glycosylation of secreted mucins by GalNAc␣-O-bn In all mucin-secreting cell lines treated by GalNAca-Obn, mucins were not only characterized by an increase in the expression of Tn antigen (GalNAc-Thr/Ser), but also by an increase in the expression of T antigen (Galb1-3GalNAc). The latter observation was surprising because GalNAcaO-bn was initially used as a competitive inhibitor of the activity of core-1 b3-Gal-T through the formation of the disaccharide Galb1-3GalNAca-O-bn. In fact, GalNAcaO-bn did not efficiently inhibit core-1 b3-Gal-T in vivo on the different cultured cells because the activity of this enzyme was very high in these cells and could transfer a Gal residue on both the endogenous (mucins) and exogenous (GalNAca-O-bn) substrates. In contrast, the high amount of the formed disaccharide Galb1-3GalNAca-O-bn acted as an efficient inhibitor of the elongation of the T antigen structures of mucins . In parallel to the increased expression of core region carbohydrate antigens Tn and T, GalNAcaO-bn was found to decrease the expression of peripheral carbohydrate antigens, in a cell line-specific manner: sialyl Lexand sulphomucin in LS174T cells , sialyl Lex in Kato III cells , terminal fucose (i.e. H-antigen) in Caco-2 cells , and sialyl T antigen in HT-29 MTX cells . A chemical analysis of mucins synthesized in the presence of
4.2. Target glycosyltransferases to GalNAc␣-O-bn Investigations on different cell-types (LS174T, Kato III, Caco-2 and HT-29 cell lines) showed that GalNAca-O-bn comes through the membrane and undergoes intracellular metabolization [52,53,56,57]. The disaccharide Galb13GalNAca-O-bn accounts for mainly GalNAca-O-bn derivatives synthesized intracellularly. Data emerging from the
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Fig. 2. Structure and hypothetical biosynthesis scheme of the major GalNAca-O-bn derivatives in HT-29 cells. The indicated glycosyltransferases have been proposed according to their known specificity in the biosynthesis of human oligosaccharides. The sugar residues transferred are indicated in bold letters. The Arrows leading to compound #2, 3 and 7b are dotted to indicate that the pathway is not clearly defined.
study of the glycosylation of mucins in LS174T and Caco-2 cells showed that GalNAca-O-bn could inhibit several types of glycosyltransferases: N-acetylglucosaminyltransferases, sialyltransferases, fucosyltransferases and sulphotransferases . In HT-29 cells, 11 different GalNAca-O-bn derivatives were identified using MALDI-TOF, GC/MS and [1H]-NMR analyses. Their possible synthesis pattern (shown in Fig. 2)  indicated that the formed Galb1-3GalNAca-O-bn behaved as an acceptor substrate for several other glycosyltransferases: Galb1-3GalNAc a2,3-sialyltransferases: ST3Gal I  and ST3Gal II [60,61] and core-2 b1,6-Nacetyl-glucosaminyltransferase I  and II . The high
amount of the oligosaccharide structure Neu5Aca2-3Galb13-GalNAc (compound #5), synthesized through the catalytic action of ST3Gal I, was connected to the strong inhibition of the incorporation of sialic acid through ST3Gal I in endogenous substrates. Furthermore, some of these initially processed compounds were further elongated showing that other glycosyltransferases could be inhibited in GalNAca-O-bn treated HT-29 cells, and in particular different types of sialyltransferases (Table 1). Finally, the complexity and the cell-specificity of the inhibition pattern generated by GalNAca-O-bn treatment still did not allow the specification of a glycan determinant that would be involved in apical targeting.
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Table 1 Enzymes potentially inhibited by GalNAc-a-O-bn treatment in HT-29 cells Enzymes Core-1 b3-Gal-T Core-2 b6-GlcNAc-T-I
UDP-Gal: GalNAc-Rb1,3-galactosyltransferase UDP-GlcNAc: Galb1-3GalNAc-R(GlcNAc to GalNAc) b1,6-N-acetylglucosaminyltransferase Core-2 b6-GlcNAc-T-II UDP-GlcNAc: Galb1-3GalNAc-R(GlcNAc to GalNAc) b1,6-N-acetylglucosaminyltransferase b4-Gal-T UDP-Gal: GlcNAc-Rb1,4-galactosyltransferase
EC number EC 188.8.131.52 EC 184.108.40.206
Accession # AF155582 M97347
References  
EC 2.4.1.-; (myeloid enzyme)
X55415 AF038660 AF038661 AF038662 AF038663 AF038664 M58596
EC 220.127.116.11; (plasma enzyme) L01698
X78031 U08112 L29555 X96667 U63090 L23767 NM006278 NM006100 Y11339 Y11340 AJ251053
        
GDP-Fuc: Galb1-4GlcNAc (Fuc to GlcNAc) a1,3-fucosyltransferase GDP-Fuc: Galb1-4GlcNAc (Fuc to GlcNAc) a1,3-fucosyltransferase GDP-Fuc: Galb1-4GlcNAc (Fuc to GlcNAc) a1,3-fucosyltransferase GDP-Fuc: Galb1-4GlcNAc (Fuc to GlcNAc) a1,3-fucosyltransferase GDP-Fuc: Neu5Aca2-3Galb1-4GlcNAc a1,3-fucosyltransferase
ST3Gal I ST3Gal II
CMP-Neu5Ac: Galb1-3GalNAc a2,3-sialyltransferase CMP-Neu5Ac: Galb1-3GalNAc a2,3-sialyltransferase
EC 18.104.22.168 EC 2.4.99.-
CMP-Neu5Ac: Galb1-4GlcNAc a2,3-sialyltransferase
ST3Gal VI ST6GalNAc I
CMP-Neu5Ac: Galb1-4GlcNAc a2,3-sialyltransferase CMP-Neu5Ac: R-GalNAca1-O-Ser/Thr a2,6-sialyltransferase
EC 2.4.99.EC 22.214.171.124
CMP-Neu5Ac: (Neu5Aca2-3)0–1Galb1-3GalNAca1-O-Ser/Thr a2,6-sialyltransferase CMP-Neu5Ac: Neu5Aca2-3Galb1-3GalNAc a2,6-sialyltransferase CMP-Neu5Ac: Neu5Aca2-3Galb1-3GalNAc a2,6-sialyltransferase
Fuc-TV Fuc-TVI Fuc-TIX
ST6GalNAc III ST6GalNAc IV
EC 126.96.36.199 EC 2.4.99.-
a a3-Fuc-T: GDP-Fuc: Galb1-4GlcNAc (Fuc to GlcNAc) a1,3-fucosyltransferase, including Fuc-TIV (myeloid enzyme) FUT4-encoded a1,3fucosyltransferase, Fuc-TV FUT5-encoded a1,3-fucosyltransferase, Fuc-TVI (plasma enzyme, EC 188.8.131.52) FUT6-encoded a1,3-fucosyltransferase, and Fuc-TIX FUT9-encoded a1,3-fucosyltransferase.
5. Conclusions and perspectives A large amount of data have been obtained about the knowledge of intracellular trafficking in polarized epithelial cells during the past few years. The existence of specific signals and specific mechanisms targeting one of the other surfaces of the cell is now evident. However, although large amount of data have shown that glycosylation was necessary for apical targeting, the mechanism by which glycosylation regulates this pathway is still unclear. The hypothesis of an interaction with a hypothetical lectin in the TGN has been proposed, but remains to be demonstrated. Data also revealed the existence of several mechanisms dependingon several signals for apical targeting. Another complexity also comes from the observations that, in the same protein, sorting signals for apical or basolateral domain act in a hierarchical manner according to their binding affinity.
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