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1.
Spodsberg N Jacob R Alfalah M Zimmer KP Naim HY 《The Journal of biological chemistry》2001,276(26):23506-23510
The impaired sorting profile to the apical membrane of human intestinal sucrase-isomaltase is the underlying cause in the pathogenesis of a novel phenotype of intestinal congenital sucrase-isomaltase deficiency. Molecular characterization of this novel phenotype reveals a point mutation in the coding region of the sucrase-isomaltase (SI) gene that results in an amino acid substitution of a glutamine by arginine at residue 117 of the isomaltase subunit. This substitution is located in a domain revealing features of a trefoil motif or a P-domain in immediate vicinity of the heavily O-glycosylated stalk domain. Expression of the mutant SI phenotype in epithelial Madin-Darby canine kidney cells reveals a randomly targeted SI protein to the apical and basolateral membranes confirming an exclusive role of the Q117R mutation in generating this phenotype. Unlike wild type SI, the mutant protein is completely extractable with Triton X-100 despite the presence of O-glycans that serve in the wild type protein as an apical sorting signal and are required for the association of SI with detergent-insoluble lipid microdomains. Obviously the O-glycans are not adequately recognized in the context of the mutant SI, most likely due to altered folding of the P-domain that ultimately affects the access of the O-glycans to a putative sorting element. 相似文献
2.
极性化上皮细胞的质膜因其所含蛋白质、脂质等组分不同,可以分为细胞膜顶端和细胞膜基底侧端两个区域,而新合成的蛋白质向这两个区域的有效分拣是上皮细胞维持其自身极性及正常功能所必需的。细胞膜基底侧端蛋白质的分拣主要由位于该蛋白质胞质区的信号肽所介导,关于这方面的研究是比较深入的;而细胞膜顶端蛋白质的分拣机制目前尚未阐明,因而显得比较复杂。近年来,糖类分子作为生物体内细胞识别和调控过程的信息分子日益受到关注,人们通过干扰聚糖合成、基因突变以及构建糖基化缺陷细胞株等实验方法,逐渐地认识到糖类分子在极性化上皮细胞的蛋白质分拣调节中起重要作用。由于糖分子本身结构非常复杂,而且目前缺乏研究糖类分子的有效手段,使得糖生物学的研究远远落后于蛋白质和核酸的研究。从而导致探讨糖类分子在蛋白质分拣过程的具体机制相对来说比较困难。本综述拟简要概括糖类分子中N-聚糖和O-聚糖在极性化上皮细胞的蛋白质分拣过程中的作用,以及两种聚糖在此过程中行使分拣信号功能的可能机制。 相似文献
3.
O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts. 总被引:9,自引:0,他引:9
The plasma membrane of polarised epithelial cells is characterised by two structurally and functionally different domains, the apical and basolateral domains. These domains contain distinct protein and lipid constituents that are sorted by specific signals to the correct surface domain [1]. The best characterised apical sorting signal is that of glycophosphatidylinositol (GPI) membrane anchors [2], although N-linked glycans on some secreted proteins [3] and O-linked glycans [4] also function as apical sorting signals. In the latter cases, however, the underlying sorting mechanisms remain obscure. Here, we have analysed the role of O-glycosylation in the apical sorting of sucrase-isomaltase (SI), a highly polarised N- and O-glycosylated intestinal enzyme, and the mechanisms underlying this process. Inhibition of O-glycosylation by benzyl-N-acetyl-alpha-D-galactosaminide (benzyl-GalNAc) was accompanied by a dramatic shift in the sorting of SI from the apical membrane to both membranes. The sorting mechanism of SI involves its association with sphingolipid- and cholesterol-rich membrane rafts because this association was eliminated when O-glycosylation was inhibited by benzyl-GaINAc. The results demonstrate for the first time that O-linked glycans mediate apical sorting through association with lipid rafts. 相似文献
4.
We have analyzed the respective roles of the stalk and/or the O-glycosylation sites in apical sorting by producing partially deleted mutants in this region of the human receptor for neurotrophins (P75(NTR)). The mere presence of O-glycosylations was not sufficient for efficient delivery to the apical surface since changing the stalk domain of P75(NTR) for the heavily O-glycosylated stalk from human decay-accelerating factor led to random distribution of the chimera. The presence of O-glycosylations, however, was a prerequisite for exit from the ER and protection from intracellular cleavage since a P75(NTR) containing the non O-glycosylated stalk of the human placental alkaline phosphatase was not transported to the cell surface but was cleaved and secreted from the basolateral side. Deletion of the membrane-proximal part of the stalk showed a more dramatic reversal of polarity of P75(NTR) than the deletion of the distal part. Furthermore, moving the first putative O-glycosylation site (T216) two amino acids away from the membrane resulted in a loss of apical polarity of P75(NTR), suggesting that an important clue for apical sorting resides in this part of the stalk. This loss of apical polarity paralleled a loss of association of P75(NTR) mutants with Lubrol rafts. These data indicate that the position of O-glycans in the proximal part of the stalk domain of P75(NTR) is crucial for apical sorting and may regulate association with apical rafts. 相似文献
5.
N-Glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in Madin-Darby canine kidney cells.
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Glycosyl-phosphatidylinositol (GPI)- anchored proteins are preferentially transported to the apical cell surface of polarized Madin-Darby canine kidney (MDCK) cells. It has been assumed that the GPI anchor itself acts as an apical determinant by its interaction with sphingolipid-cholesterol rafts. We modified the rat growth hormone (rGH), an unglycosylated, unpolarized secreted protein, into a GPI-anchored protein and analyzed its surface delivery in polarized MDCK cells. The addition of a GPI anchor to rGH did not lead to an increase in apical delivery of the protein. However, addition of N-glycans to GPI-anchored rGH resulted in predominant apical delivery, suggesting that N-glycans act as apical sorting signals on GPI-anchored proteins as they do on transmembrane and secretory proteins. In contrast to the GPI-anchored rGH, a transmembrane form of rGH which was not raft-associated accumulated intracellularly. Addition of N-glycans to this chimeric protein prevented intracellular accumulation and led to apical delivery. 相似文献
6.
Different classes of glycans are implicated as mediators of apical protein sorting in the secretory pathway of epithelial cells, but recent research indicates that sorting to the apical and basolateral surfaces may occur before completion of glycan synthesis. We have previously shown that a proteoglycan (PG) core protein can obtain different glycosaminoglycan (GAG) structures in the apical and basolateral secretory routes (Tveit H, Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells. J Biol Chem. 280:29596-29603) of epithelial Madin-Darby canine kidney (MDCK) cells. We have now also determined the detailed N-glycan structures acquired by a single glycoprotein species in the same apical and basolateral secretory pathways. For this purpose, rat growth hormone (rGH) with two N-glycan sites (rGH-2N) inserted into the rGH portion (NAS and NFT) was fused to green fluorescent protein (GFP) and expressed in MDCK cells. Immunoisolated rGH variants were analyzed for site occupancy and N-glycan structure by mass spectrometry. The extent of NAS and NFT site occupancy was different, but comparable for rGH-2N secreted apically and basolaterally. Microheterogeneity existed for the glycans attached to each N-glycan site, but no major differences were observed in the apical and basolateral pathways. Transfer of the GAG modification domain from the PG serglycin to the fusion site of rGH-2N and GFP allowed polymerization of GAG chains onto the novel protein variant and influenced the microheterogeneity of the N-glycans toward more acidic glycans, but did not alter the relative site occupancy. In conclusion, no major differences were observed for N-glycan structures obtained by the expressed model proteins in the apical and basolateral secretory pathways of epithelial MDCK cells, but insertion of a GAG attachment domain shifted the N-glycans to more acidic structures. 相似文献
7.
The O-glycosylated Stalk Domain Is Required for Apical Sorting of Neurotrophin Receptors in Polarized MDCK Cells 总被引:15,自引:2,他引:13
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Charles Yeaman Annick H. Le Gall Anne N. Baldwin Laure Monlauzeur Andre Le Bivic Enrique Rodriguez-Boulan 《The Journal of cell biology》1997,139(4):929-940
Delivery of newly synthesized membrane-spanning proteins to the apical plasma membrane domain of polarized MDCK epithelial cells is dependent on yet unidentified sorting signals present in the luminal domains of these proteins. In this report we show that structural information for apical sorting of transmembrane neurotrophin receptors (p75NTR) is localized to a juxtamembrane region of the extracellular domain that is rich in O-glycosylated serine/threonine residues. An internal deletion of 50 amino acids that removes this stalk domain from p75NTR causes the protein to be sorted exclusively of the basolateral plasma membrane. Basolateral sorting stalk-minus p75NTR does not occur by default, but requires sequences present in the cytoplasmic domain. The stalk domain is also required for apical secretion of a soluble form of p75NTR, providing the first demonstration that the same domain can mediate apical sorting of both a membrane-anchored as well as secreted protein. However, the single N-glycan present on p75NTR is not required for apical sorting of either transmembrane or secreted forms. 相似文献
8.
Jacob R Alfalah M Grünberg J Obendorf M Naim HY 《The Journal of biological chemistry》2000,275(9):6566-6572
The distinct protein and lipid constituents of the apical and basolateral membranes in polarized cells are sorted by specific signals. O-Glycosylation of a highly polarized intestinal brush-border protein sucrase isomaltase is implicated in its apical sorting through interaction with sphingolipid-cholesterol microdomains. We characterized the structural determinants required for this mechanism by focusing on two major domains in pro-SI, the membrane anchor and the Ser/Thr-rich stalk domain. Deletion mutants lacking either domain, pro-SI(DeltaST) (stalk-free) and pro-SI(DeltaMA) (membrane anchor-free), were constructed and expressed in polarized Madin-Darby canine kidney cells. In the absence of the membrane anchoring domain, pro-SI(DeltaMA) does not associate with lipid rafts and the mutant is randomly delivered to both membranes. Therefore, the O-glycosylated stalk region is not sufficient per se for the high fidelity of apical sorting of pro-SI. Pro-SI(DeltaST) does not associate either with lipid rafts and its targeting behavior is similar to that of pro-SI(DeltaMA). Only wild type pro-SI containing both determinants, the stalk region and membrane anchor, associates with lipid microdomains and is targeted correctly to the apical membrane. However, not all sequences in the stalk region are required for apical sorting. Only O-glycosylation of a stretch of 12 amino acids (Ala(37)-Pro(48)) juxtapose the membrane anchor is required in conjunction with the membrane anchoring domain for correct targeting of pro-SI to the apical membrane. Other O-glycosylated domains within the stalk (Ala(49)-Pro(57)) are not sufficient for apical sorting. We conclude that the recognition signal for apical sorting of pro-SI comprises O-glycosylation of the Ala(37)-Pro(48) stretch and requires the presence of the membrane anchoring domain. 相似文献
9.
Conversion of a secretory protein into a transmembrane protein results in its transport to the Golgi complex but not to the cell surface 总被引:42,自引:0,他引:42
We have carried out experiments designed to ask if it is possible to convert a secretory protein into an integral membrane protein by appending the membrane spanning domain of an integral membrane protein to its carboxy terminus. We first obtained expression of a cDNA clone encoding rat growth hormone (rGH) in eucaryotic cells, and found that this protein was secreted. We then constructed and expressed a hybrid gene encoding rGH fused to the membrane spanning and cytoplasmic domains of the vesicular stomatitis virus (VSV) glycoprotein (G). This fusion protein was anchored in microsomal membranes in the expected transmembrane configuration. The fusion protein was transported to the Golgi apparatus, and was esterified to palmitic acid, but it was not transported to the cell surface. We suggest that the sorting signal which allows rapid secretion of soluble rGH does not function when the protein is bound to the membrane. 相似文献
10.
Delacour D Cramm-Behrens CI Drobecq H Le Bivic A Naim HY Jacob R 《Current biology : CB》2006,16(4):408-414
The central aspect of epithelial cells is their polarized structure, characterized by two distinct domains of the plasma membrane, the apical and the basolateral membrane. Apical protein sorting requires various signals and different intracellular routes to the cell surface. The first apical targeting motif identified is the membrane anchoring of a polypeptide by glycosyl-phosphatidyl-inositol (GPI). A second group of apical signals involves N- and O-glycans, which are exposed to the luminal side of the sorting organelle. Sucrase-isomaltase (SI) and lactase-phlorizin hydrolase (LPH), which use separate transport platforms for trafficking, are two model proteins for the study of apical protein sorting. In contrast to LPH, SI associates with sphingolipid/cholesterol-enriched membrane microdomains or "lipid rafts". After exit form the trans-Golgi network (TGN), the two proteins travel in distinct vesicle populations, SAVs (SI-associated vesicles) and LAVs (LPH-associated vesicles) . Here, we report the identification of the lectin galectin-3 delivering non-raft-dependent glycoproteins in the lumen of LAVs in a carbohydrate-dependent manner. Depletion of galectin-3 from MDCK cells results in missorting of non-raft-dependent apical membrane proteins to the basolateral cell pole. This suggests a direct role of galectin-3 in apical sorting as a sorting receptor. 相似文献
11.
Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses an apical sorting signal in polarized MDCK cells. 总被引:12,自引:4,他引:8
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The influenza virus neuraminidase (NA), a type II transmembrane protein, is directly transported to the apical plasma membrane in polarized MDCK cells. By using deletion mutants and chimeric constructs of influenza virus NA with the human transferrin receptor, a type II basolateral transmembrane protein, we investigated the location of the apical sorting signal of influenza virus NA. When these mutant and chimeric proteins were expressed in stably transfected polarized MDCK cells, the transmembrane domain of NA, and not the cytoplasmic tail, provided a determinant for apical targeting in polarized MDCK cells and this transmembrane signal was sufficient for sorting and transport of the ectodomain of a reporter protein (transferrin receptor) directly to the apical plasma membrane of polarized MDCK cells. In addition, by using differential detergent extraction, we demonstrated that influenza virus NA and the chimeras which were transported to the apical plasma membrane also became insoluble in Triton X-100 but soluble in octylglucoside after extraction from MDCK cells during exocytic transport. These data indicate that the transmembrane domain of NA provides the determinant(s) both for apical transport and for association with Triton X-100-insoluble lipids. 相似文献
12.
The Natronobacterium pharaonis HtrII (NpHtrII) transducer interacts with its cognate photoactive sensory rhodopsin receptor, NpSRII, to mediate phototaxis responses. NpHtrII is predicted to have two transmembrane helices and a large cytoplasmic domain and to form a homodimer. Single cysteines were substituted into an engineered cysteine-less NpHtrII at 38 positions in its transmembrane domain. Oxidative disulfide cross-linking efficiencies of the monocysteine mutants were measured with or without photoactivation of NpSRII. The rapid cross-linking rates at several positions support that NpHtrII is a dimer when functionally expressed in the Halobacterium salinarum membrane. Thirteen positions in the second transmembrane segment (TM2) exhibited significant light-induced increases in cross-linking efficiency, and they define a single face traversing the length of the segment when modeled as an alpha-helix. Four positions in this helix showing light-induced decreases in efficiency are clustered on the cytoplasmic side of the protein. One of the monocysteine mutants, G83C, showed loss of phototaxis responses, and analysis of double mutants showed that the G83C mutation alters the dark structure of the TM2-TM2' region of NpHtrII. In summary, the results reveal conformationally active regions in the second transmembrane segment of NpHtrII and a face along the length of TM2 that becomes more available for TM2-TM2' cross-linking upon receptor photoactivation. The data also establish that one residue in TM2, Gly83, is critical for maintaining the proper conformation of NpHtrII for signal relay from the photoactivated receptor to the kinase-binding region of the transducer. 相似文献
13.
Gorski JP Liu FT Artigues A Castagna LF Osdoby P 《The Journal of biological chemistry》2002,277(21):18840-18848
14.
Mutations in the Middle of the Transmembrane Domain Reverse the Polarity of Transport of the Influenza Virus Hemagglutinin in MDCK Epithelial Cells 总被引:15,自引:1,他引:14
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Sasa Lin Hussein Y. Naim A. Chapin Rodriguez Michael G. Roth 《The Journal of cell biology》1998,142(1):51-57
The composition of the plasma membrane domains of epithelial cells is maintained by biosynthetic pathways that can sort both proteins and lipids into transport vesicles destined for either the apical or basolateral surface. In MDCK cells, the influenza virus hemagglutinin is sorted in the trans-Golgi network into detergent-insoluble, glycosphingolipid-enriched membrane domains that are proposed to be necessary for sorting hemagglutinin to the apical cell surface. Site- directed mutagenesis of the hemagglutinin transmembrane domain was used to test this proposal. The region of the transmembrane domain required for apical transport included the residues most conserved among hemagglutinin subtypes. Several mutants were found to enter detergent-insoluble membranes but were not properly sorted. Replacement of transmembrane residues 520 and 521 with alanines converted the 2A520 mutant hemagglutinin into a basolateral protein. Depleting cell cholesterol reduced the ability of wild-type hemagglutinin to partition into detergent-insoluble membranes but had no effect on apical or basolateral sorting. In contrast, cholesterol depletion allowed random transport of the 2A520 mutant. The mutant appeared to lack sorting information but was prevented from reaching the apical surface when detergent-insoluble membranes were present. Apical sorting of hemagglutinin may require binding of either protein or lipids at the middle of the transmembrane domain and this normally occurs in detergent-insoluble membrane domains. Entry into these domains appears necessary, but not sufficient, for apical sorting. 相似文献
15.
Markus Lerner Dieter Lemke Helga Bertram Hermann Schillers Hans Oberleithner Michael J Caplan Jürgen Reinhardt 《Cellular physiology and biochemistry》2006,18(1-3):75-84
The human non-gastric H,K-ATPase, ATP1AL1, belongs to the gene family of P-type ATPases. Consistent with their physiological roles in ion transport, members of this group, including the Na,KATPase and the gastric and non-gastric H,K-ATPases, are differentially polarized to either the basolateral or apical plasma membrane in epithelial cells. However, their polarized distribution is highly complex and depends on specific sorting signals or motifs which are recognized by the subcellular targeting machinery. For the gastric H,K-ATPase it has been suggested that the 4(th) transmembrane spanning domain (TM4) and its flanking regions induce conformational sorting motifs which direct the ion pump exclusively to the epithelial apical membrane. Here, we show in transfected Madin-Darby canine kidney (MDCK) cells that the related non-gastric H,KATPase, ATP1AL1, does contain similar sorting motifs in close proximity to TM4. A short extracellular loop between TM3 and TM4 is critical for this pump's apical delivery. A single point mutation in the corresponding region redirects ATP1AL1 to the basolateral membrane. In conclusion, our work provides further evidence that the cellular distribution of P-type ATPases is determined by conformational sorting motifs. 相似文献
16.
In polarized epithelial cells aminopeptidase N is targeted to the apical membrane. The aim of this study was to determine whether a sorting signal is necessary for its correct transport to the apical membrane and, if so, to localize this sorting signal to one of the domains of the transmembrane protein. Anchor-minus aminopeptidase N, consisting of the hemagglutinin signal peptide including its cleavage site, and the ectoplasmic domain of human aminopeptidase N were stably expressed in Madin-Darby canine kidney cells cultured on polycarbonate filters. By measurement of the enzymatic activity it was found that the anchor-minus aminopeptidase N was secreted in a polarized manner to the apical side. As a reference the secretion of the secretory granule protein, cystatin C, was likewise studied. Cystatin C was found to be secreted in a nonpolarized manner to both domains. Our data thus show that human aminopeptidase N carries an apical sorting signal and that it is localized on the ectodomain of the enzyme. 相似文献
17.
Jacob R Preuss U Panzer P Alfalah M Quack S Roth MG Naim H Naim HY 《The Journal of biological chemistry》1999,274(12):8061-8067
Lactase-phlorizin hydrolase (LPH) is an apical protein in intestinal cells. The location of sorting signals in LPH was investigated by preparing a series of mutants that lacked the LPH cytoplasmic domain or had the cytoplasmic domain of LPH replaced by sequences that comprised basolateral targeting signals and overlapping internalization signals of various potency. These signals are mutants of the cytoplasmic domain of the influenza hemagglutinin (HA), which have been shown to be dominant in targeting HA to the basolateral membrane. The LPH-HA chimeras were expressed in Madin-Darby canine kidney (MDCK) and colon carcinoma (Caco-2) cells, and their transport to the cell surface was analyzed. All of the LPH mutants were targeted correctly to the apical membrane. Furthermore, the LPH-HA chimeras were internalized, indicating that the HA tails were available to interact with the cytoplasmic components of clathrin-coated pits. The introduction of a strong basolateral sorting signal into LPH was not sufficient to override the strong apical signals of the LPH external domain or transmembrane domains. These results show that basolateral sorting signals are not always dominant over apical sorting signals in proteins that contain each and suggest that sorting of basolateral from apical proteins occurs within a common compartment where competition for sorting signals can occur. 相似文献
18.
Studies of how epithelial surface polarity into apical and basolateral domains is generated and maintained have proposed that carbohydrate modifications serve as apical targeting signals for proteins by interacting with lectin sorters. However, the experimental evidence in support of N-glycans, O-glycans and sialic acids mediating apical transport is still very controversial. This could be partly due to the fact that in most studies exogenously expressed proteins were analysed. One has, therefore, examined the role of carbohydrate moieties in apical targeting of the endogenous secretory protein osteopontin in MDCK cells. It was found, however, that sorting of osteopontin does not require N-glycosylation of the protein itself nor that of other factors involved in the sorting process. Incubation of cells with the inhibitor of O-glycosylation benzyl- f -GalNAc reduced the molecular weight of osteopontin by blocking sialic acid addition to O-glycans. Interestingly, also impairment of sialylation had no effect on polar secretion of the protein. Thus, the results show that both N-glycans and sialic acids are not essential sorting signals, suggesting that inner core carbohydrates and/or a proteinaceous signal mediate apical targeting of osteopontin. 相似文献
19.
Studies of how epithelial surface polarity into apical and basolateral domains is generated and maintained have proposed that carbohydrate modifications serve as apical targeting signals for proteins by interacting with lectin sorters. However, the experimental evidence in support of N-glycans, O-glycans and sialic acids mediating apical transport is still very controversial. This could be partly due to the fact that in most studies exogenously expressed proteins were analysed. One has, therefore, examined the role of carbohydrate moieties in apical targeting of the endogenous secretory protein osteopontin in MDCK cells. It was found, however, that sorting of osteopontin does not require N-glycosylation of the protein itself nor that of other factors involved in the sorting process. Incubation of cells with the inhibitor of O-glycosylation benzyl-alpha-GaINAc reduced the molecular weight of osteopontin by blocking sialic acid addition to O-glycans. Interestingly, also impairment of sialylation had no effect on polar secretion of the protein. Thus, the results show that both N-glycans and sialic acids are not essential sorting signals, suggesting that inner core carbohydrates and/or a proteinaceous signal mediate apical targeting of osteopontin. 相似文献
20.
Renold A Cescato R Beuret N Vogel LK Wahlberg JM Brown JL Fiedler K Spiess M 《The Journal of biological chemistry》2000,275(13):9290-9295
Polarized sorting of membrane proteins in epithelial cells is mediated by cytoplasmic basolateral signals or by apical signals in the transmembrane or exoplasmic domains. Basolateral signals were generally found to be dominant over apical determinants. We have generated chimeric proteins with the cytoplasmic domain of either the asialoglycoprotein receptor H1 or the transferrin receptor, two basolateral proteins, fused to the transmembrane and exoplasmic segments of aminopeptidase N, an apical protein, and analyzed them in Madin-Darby canine kidney cells. Whereas both cytoplasmic sequences induced endocytosis of the chimeras, only that of the transferrin receptor mediated basolateral expression in steady state. The H1 fusion protein, although still largely sorted to the basolateral side in biosynthetic surface transport, was subsequently resorted to the apical cell surface. We tested whether the difference in sorting between trimeric wild-type H1 and the dimeric aminopeptidase chimera was caused by the number of sorting signals presented in the oligomers. Consistent with this hypothesis, the H1 signal was fully functional in a tetrameric fusion protein with the transmembrane and exoplasmic domains of influenza neuraminidase. The results suggest that basolateral signals per se need not be dominant over apical determinants for steady-state polarity and emphasize an important contribution of the valence of signals in polarized sorting. 相似文献