Glycosaminoglycan Chains Affect Exocytic and Endocytic Protein Traffic

Protein glycosylation such as N- and O-linked glycans as well as glycosaminoglycans (GAGs) have been shown to contribute to polarized sorting in epithelial cells. Here, we analyzed the effect of GAGs more generally on protein traffic also in non-polarized cells. Using short sequence tags of 10–17 amino acids encoding known GAG attachment sites, we have converted the asialoglycoprotein receptor H1, which constitutively cycles between the plasma membrane and endosomes, into a proteoglycan. Expressed in HeLa cells, the receptor was almost completely modified with a chondroitin sulfate chain and could be efficiently labeled by [³⁵S]sulfation. GAG attachment altered the steady-state distribution of the receptor by inhibiting endocytosis, while recycling was not affected. The reduced internalization is not the result of immobilization by interaction with the extracellular matrix, because fluorescence recovery after photobleaching did not detect an increased immobile fraction nor even a significant change in mobility. GAG chains furthermore accelerated Golgi-to-cell surface transport of H1. The same acceleration of export was also observed for a GAG-tagged version of the secretory protein α1-protease inhibitor, suggesting that this effect acts generally on proteoglycans, possibly by directing them into distinct carriers. Our results show novel roles of GAGs in protein sorting also in non-polarized cells.     

Chondroitin Sulfate Accelerates Trans‐Golgi‐to‐Surface Transport of Proteoglycan Amyloid Precursor Protein

The amyloid precursor protein (APP) is a membrane protein implicated in the pathogenesis of Alzheimer's disease. APP is a part‐time proteoglycan, as splice variants lacking exon 15 are modified by a chondroitin sulfate glycosaminoglycan (GAG) chain. Investigating the effect of the GAG chain on the trafficking of APP in non‐polarized cells, we found it to increase the steady‐state surface‐to‐intracellular distribution, to reduce the rate of endocytosis and to accelerate transport kinetics from the trans‐Golgi network (TGN) to the plasma membrane. Deletion of the cytosolic domain resulted in delayed surface arrival of GAG‐free APP, but did not affect the rapid export kinetics of the proteoglycan form. Protein‐free GAG chains showed the same TGN‐to‐cell surface transport kinetics as proteoglycan APP. Endosome ablation experiments were performed to distinguish between indirect endosomal and direct pathways to the cell surface. Surprisingly, TGN‐to‐cell surface transport of both GAG‐free and proteoglycan APP was found to be indirect via transferrin‐positive endosomes. Our results show that GAGs act as alternative sorting determinants in cellular APP transport that are dominant over cytoplasmic signals and involve distinct sorting mechanisms.      

Apical transport of osteopontin is independent of N-glycosylation and sialylation

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- &#102 -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.     

Apical transport of osteopontin is independent of N-glycosylation and sialylation.

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.     

Early embryonic death of mice deficient in gamma-adaptin

Intracellular protein transport and sorting by vesicles in the secretory and endocytic pathways requires the formation of a protein coat on the membrane. The heterotetrameric adaptor protein complex 1 (AP-1) promotes the formation of clathrin-coated vesicles at the trans-Golgi network. AP-1 interacts with various sorting signals in the cytoplasmic tails of cargo molecules, thus indicating a function in protein sorting. We generated mutants of the gamma-adaptin subunit of AP-1 in mice to investigate its role in post-Golgi vesicle transport and sorting processes. gamma-Adaptin-deficient embryos develop until day 3.5 post coitus and die during the prenidation period, revealing that AP-1 is essential for viability. In heterozygous mice the amount of AP-1 complexes is reduced to half of controls. Free beta1- or micro1 chains were not detectable, indicating that they are unstable unless they are part of AP-1 complexes. Heterozygous mice weigh less then their wild-type littermates and show impaired T cell development.     

Role of Us9 Phosphorylation in Axonal Sorting and Anterograde Transport of Pseudorabies Virus

Alphaherpes viruses, such as pseudorabies virus (PRV), undergo anterograde transport in neuronal axons to facilitate anterograde spread within hosts. Axonal sorting and anterograde transport of virions is dependent on the viral membrane protein Us9, which interacts with the host motor protein Kif1A to direct transport. Us9-Kif1A interactions are necessary but not sufficient for these processes, indicating that additional cofactors or post-translational modifications are needed. In this study, we characterized two conserved serine phosphorylation sites (S51 and S53) in the PRV Us9 protein that are necessary for anterograde spread in vivo. We assessed the subcellular localization of phospho-Us9 subspecies during infection of neurons and found that the phospho-form is detectable on the majority, but not all, of axonal vesicles containing Us9 protein. In biochemical assays, phospho-Us9 was enriched in lipid raft membrane microdomains, though Us9 phosphorylation did not require prior lipid raft association. During infections of chambered neuronal cultures, we observed only a modest reduction in anterograde spread capacity for diserine mutant Us9, and no defect for monoserine mutants. Conversely, mutation of the kinase recognition sequence residues adjacent to the phosphorylation sites completely abrogated anterograde spread. In live-cell imaging analyses, anterograde transport of virions was reduced during infection with a recombinant PRV strain expressing GFP-tagged diserine mutant Us9. Phosphorylation was not required for Us9-Kif1A interaction, suggesting that Us9-Kif1A binding is a distinct step from the activation and/or stabilization of the transport complex. Taken together, our findings indicate that, while not essential, Us9 phosphorylation enhances Us9-Kif1A-based transport of virions in axons to modulate the overall efficiency of long-distance anterograde spread of infection.     

AP-1A and AP-3A lysosomal sorting functions

Heterotetrameric adaptor-protein complexes AP-1A and AP-3A mediate protein sorting in post-Golgi vesicular transport. AP-1A and AP-3A have been localized to the trans -Golgi network, indicating a function in protein sorting at this compartment. AP-3A appears to mediate trans -Golgi network-to-lysosome and also endosome-to-lysosome protein sorting. AP-1A is thought to be required for both trans -Golgi network-to-endosome transport and endosome-to- trans -Golgi network transport. However, the recent discovery of a role for monomeric GGA (Golgi localized γ-ear containing, ARF binding protein) adaptor proteins in trans -Golgi network to endosome protein transport has brought into question the long-discussed trans -Golgi network-to-endosome sorting function of AP-1A. Murine cytomegalovirus gp48 contains an unusual di-leucine-based lysosome sorting signal motif and mediates lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes, preventing exposure of major histocompatibility complex class I at the plasma membrane. We analyzed lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes in cell lines deficient for AP-1A, AP-3A and both, to determine their sorting functions. We find that AP1-A and AP3-A mediate distinct and sequential steps in the lysosomal sorting. Both sorting functions are required to prevent MHC class I exposure at the plasma membrane at steady-state.     

Immunocytochemical study of tissue distribution and hormonal control of chondroitin-, dermatan- and keratan sulfates from rodent uterus

The distribution patterns of rat and mouse uterine glycosaminoglycans (GAGs), as well as their modulation by estradiol (E2) and/or progesterone (P), were investigated using monoclonal antibodies (MABs) directed against chondroitin- (CS)/dermatan sulfates (DS), keratan sulfate (KS) and a trophoblast GAG. The localization of GAGs in relation to collagens (I, IV and VI) and fibronectin was also analyzed. We found that uterine GAGs are differentially distributed in the endometrium and myometrium, in a pattern that is species-related. CS-containing proteoglycans (PGs) occur between collagen bundles and fibroblasts, at the periphery of the latter, and in basement membrane zones (BMZs), in a pattern resembling that of collagen VI. BMZs contain preferentially CS-PGs bearing 4-sulfated disaccharides adjacent to the core protein. DS-PGs are mostly associated with collagen bundles. E2 and/or P elicit distinct modifications on the above described pattern, which are also species-related. The simultaneous administration of E2 and P changes the prevalent sulfation of the disaccharides adjacent to the core protein of stromal CS-PGs. In the mouse, an unsulfated intracellular epitope appears following E2 (or E2P) administration, mostly in epithelial cells. In the rat, KS and the trophoblast GAG are E2-dependent and down-regulated by P. The functional significance of the hormone-induced GAG changes, namely the possible role of the E2-dependent KS in implantation, are discussed.     

Specific sides to multifaceted glycosaminoglycans are observed in embryonic development

Ubiquitously found in the extracellular matrix and attached to the surface of most cells, glycosaminoglycans (GAGs) mediate many intercellular interactions. Originally described in 1889 as the primary carbohydrate in cartilage and then in 1916 as a coagulation inhibitor from liver, various GAGs have since been identified as key regulators of normal physiology. GAGs are critical mediators of differentiation, migration, tissue morphogenesis, and organogenesis during embryonic development. While GAGs are simple polysaccharide chains, many GAGs acquire a considerable degree of complexity by extensive modifications involving sulfation and epimerization. Embryos that lack specific GAG modifying enzymes have distinct developmental defects, illuminating the importance of GAG complexity. Revealing how these complex molecules specifically function in the embryo has often required additional approaches, the results of which suggest that GAG modifications might instructively mediate embryonic development.     

Mutations in the Middle of the Transmembrane Domain Reverse the Polarity of Transport of the Influenza Virus Hemagglutinin in MDCK Epithelial Cells

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.     

植物种子贮藏蛋白质及其细胞内转运与加工

高等植物种子成熟过程中贮存大量的贮藏蛋白质作为种子发芽和初期生长的重要营养来源。根据溶解性不同,种子贮藏蛋白质可分为白蛋白、球蛋白、醇溶蛋白和谷蛋白4类。在种子胚发育过程中,醇溶蛋白在粗面内质网合成后形成蛋白质聚集体,直接出芽形成蛋白体并贮存其中。白蛋白、球蛋白和谷蛋白在粗面内质网以分子量较大的前体形式合成后,根据各自的分选信号进入特定的运输囊泡,经由受体依赖型运输/聚集体形式运输转运至蛋白质贮藏型液泡中,然后经过液泡加工酶等的剪切转换为成熟型贮藏蛋白质并贮存其中。蛋白质的合成、分选、转运和加工等过程影响种子蛋白质的品质及含量。该文对种子贮藏蛋白质的分类和运输、加工以及这些过程对种子蛋白质品质和含量的影响进行了概述。     

植物种子贮藏蛋白质及其细胞内转运与加工

高等植物种子成熟过程中贮存大量的贮藏蛋白质作为种子发芽和初期生长的重要营养来源。根据溶解性不同, 种子贮藏蛋白质可分为白蛋白、球蛋白、醇溶蛋白和谷蛋白4类。在种子胚发育过程中, 醇溶蛋白在粗面内质网合成后形成蛋白质聚集体, 直接出芽形成蛋白体并贮存其中。白蛋白、球蛋白和谷蛋白在粗面内质网以分子量较大的前体形式合成后, 根据各自的分选信号进入特定的运输囊泡, 经由受体依赖型运输/聚集体形式运输转运至蛋白质贮藏型液泡中, 然后经过液泡加工酶等的剪切转换为成熟型贮藏蛋白质并贮存其中。蛋白质的合成、分选、转运和加工等过程影响种子蛋白质的品质及含量。该文对种子贮藏蛋白质的分类和运输、加工以及这些过程对种子蛋白质品质和含量的影响进行了概述。     

Sulfated glycosaminoglycans in protein aggregation diseases

Protein aggregation diseases are characterized by intracellular or extracellular deposition of misfolded and aggregated proteins. These aggregated deposits contain multiple proteinaceous and non-protein components that are thought to play critical roles in the etiology and pathogenesis of protein aggregation diseases in vivo. One of these components, the sulfated glycosaminoglycans (GAGs), includes heparan sulfate, chondroitin sulfate, and keratan sulfate. The sulfated GAGs are negatively charged heteropolysaccharides expressed in all mammalian tissues. Enzymatically generated structural patterns and the degree of sulfation in GAGs determine GAGs’ specific interactions with their protein ligands. Here, we review the potential roles of the sulfated GAGs in the pathogenesis and progression of protein aggregation diseases from a perspective of their sulfation modification. We also discuss the possibility of sulfated GAGs as therapeutic targets for protein aggregation diseases.     

Effects of HIV-1 Nef on retrograde transport from the plasma membrane to the endoplasmic reticulum

HIV-1 Nef protein down-regulates several important immunoreceptors through interactions with components of the intracellular sorting machinery. Nef expression is also known to induce modifications of the endocytic pathway. Here, we analyzed the effects of Nef on retrograde transport, from the plasma membrane to the endoplasmic reticulum using Shiga toxin B-subunit (STxB). Nef expression inhibited access of STxB to the endoplasmic reticulum, but did not modify the surface expression level of STxB receptor, Gb₃, nor its internalization rate as measured with a newly developed assay. Mutation of the myristoylation site or of a di-leucine motif of Nef involved in the interaction with the clathrin adaptor complexes AP1 and AP2 abolished the inhibition of retrograde transport. In contrast, mutations of Nef motifs known to interact with PACS-1, βCOP or a subunit of the v-ATPase did not modify the inhibitory activity of Nef on retrograde transport. Ultrastructural analysis revealed that Nef was present in clusters located on endosomal or Golgi membranes together with internalized STxB. Furthermore, in strongly Nef-expressing cells, STxB accumulated in endosomal structures that labeled with AP1. Our observations show that Nef perturbs retrograde transport between the early endosome and the endoplasmic reticulum. The potential transport steps targeted by Nef are discussed .     

Retromer recycles vacuolar sorting receptors from the trans-Golgi network

Receptor-mediated sorting processes in the secretory pathway of eukaryotic cells rely on mechanisms to recycle the receptors after completion of transport. Based on this principle, plant vacuolar sorting receptors (VSRs) are thought to recycle after dissociating of receptor–ligand complexes in a pre-vacuolar compartment. This recycling is mediated by retromer, a cytosolic coat complex that comprises sorting nexins and a large heterotrimeric subunit. To analyse retromer-mediated VSR recycling, we have used a combination of immunoelectron and fluorescence microscopy to localize the retromer components sorting nexin 1 (SNX1) and sorting nexin 2a (SNX2a) and the vacuolar sorting protein VPS29p. All retromer components localize to the trans -Golgi network (TGN), which is considered to represent the early endosome of plants. In addition, we show that inhibition of retromer function in vivo by expression of SNX1 or SNX2a mutants as well as transient RNAi knockdown of all sorting nexins led to accumulation of the VSR BP80 at the TGN. Quantitative protein transport studies and live-cell imaging using fluorescent vacuolar cargo molecules revealed that arrival of these VSR ligands at the vacuole is not affected under these conditions. Based on these findings, we propose that the TGN is the location of retromer-mediated recycling of VSRs, and that transport towards the lytic vacuole downstream of the TGN is receptor-independent and occurs via maturation, similar to transition of the early endosome into the late endosome in mammalian cells.     

N-linked carbohydrates act as lumenal maturation and quality control protein tags

Protein modifications such as ubiquitination and phosphorylation commonly serve as sorting tags that control the trafficking and stability of a protein within the cytosol. In recent years, N-linked glycans have emerged as key protein modifications for eukaryotic secretory proteins. These modifications support the recruitment of molecular chaperones and sorting receptors, which recognize specific glycoforms. Therefore, glycanases and carbohydrate transferases work in concert with lectin chaperones and receptors to aid in the maturation and quality control of glycoproteins.     

Membrane domains and polarized trafficking of sphingolipids

The plasma membrane of polarized cells consists of distinct domains, the apical and basolateral membrane, that are characterized by a distinct lipid and protein content. Apical protein transport is largely mediated by (glyco)sphingolipid--cholesterol enriched membrane microdomains, so called rafts. In addition changes in the direction of polarized sphingolipid transport appear instrumental in cell polarity development. Knowledge is therefore required of the mechanisms that mediate sphingolipid sorting and the complexity of the trafficking pathways that are involved in polarized transport of both sphingolipids and proteins. Here we summarize specific biophysical properties that underly mechanisms relevant to sphingolipid sorting, cargo recruitment and polarized trafficking, and discuss the central role of a subapical compartment, SAC or common endosome (CE), as a major intracellular site involved in polarized sorting of sphingolipids, and in development and maintenance of membrane polarity.     

Enhanced Glycosylation and Sulfation of Secretory Proteoglycans Is Coupled to the Expression of a Basic Secretory Protein

We have used coexpression of a salivary basic proline-rich protein (PRP) along with a proline-rich proteoglycan (PRPg) in pituitary AtT-20 cells to examine the regulation of glycosaminoglycan (GAG) biosynthesis and the storage of these secretory products for regulated secretion. The basic PRP caused a dose-dependent increase in sulfation of PRPg and also increased the extent to which PRPg polypeptide backbones are modified by a GAG chain. The sulfation of an endogenous proteoglycan was similarly increased in the presence of basic PRP; however, other sulfated secretory products of AtT-20 cells were unaffected. These results imply that enzymes functioning in elongation and sulfation of proteoglycans are coordinately regulated and that their activities respond to a change in the milieu of the intracellular transport pathway. Analysis of the regulated secretion of both the basic PRP and PRPg has indicated that while the presence of the GAG chain improves the storage of PRPg, the presence of PRPg does not increase the storage of basic PRP. Therefore, sulfation of GAGs does not appear to be a primary factor in regulated secretory sorting.     

Envelope-dependent,cyclophilin-independent effects of glycosaminoglycans on human immunodeficiency virus type 1 attachment and infection

Cell surface glycosaminoglycans (GAGs), in particular heparan sulfate (HS), have been proposed to mediate the attachment of human immunodeficiency virus type 1 (HIV-1) to target cells prior to virus entry, and both the viral gp120 envelope protein and virion-associated cyclophilin A (CypA) have been shown to directly interact with HS and its analogues. To determine the role of GAGs in HIV attachment and infection, we generated HIV-susceptible derivatives of CHO cell lines that either express high levels of GAGs (CHO-K1) or lack GAGs (pgsA745). Using a panel of HIV-1 envelopes, we found that cell surface GAG-mediated effects on virion attachment and infection vary in an envelope strain-dependent but coreceptor-independent manner. In fact, cell surface GAG-mediated enhancement of infection is confined to isolates that contain a highly positively charged V3-loop sequence, while infection by most strains is apparently inhibited by the presence of GAGs. Moreover, the enhancing and inhibitory effects of polycations and polyanions on HIV-1 infection are largely dependent on the presence of cell surface GAGs. These observations are consistent with a model in which GAGs influence in vitro HIV-1 infection primarily by modifying the charge characteristics of the target cell surface. Finally, the effects of GAGs on HIV-1 infection are observed to an equivalent extent whether CypA is present in or absent from virions. Overall, these data exclude a major role for GAGs in mediating the attachment of many HIV-1 strains to target cells via interactions with virion-associated gp120 or CypA.     

Occurrence of L-iduronic acid and putative D-glucuronyl C5-epimerases in prokaryotes

Glycosaminoglycans (GAGs) are polysaccharides that are typically present in a wide diversity of animal tissue. Most common GAGs are well-characterized and pharmaceutical applications exist for many of these compounds, e.g. heparin and hyaluronan. In addition, also bacterial glycosaminoglycan-like structures exist. Some of these bacterial GAGs have been characterized, but until now no bacterial GAG has been found that possesses the modifications that are characteristic for many of the animal GAGs such as sulfation and C5-epimerization. Nevertheless, the latter conversion may also occur in bacterial and archaeal GAGs, as some prokaryotic polysaccharides have been demonstrated to contain L-iduronic acid. However, experimental evidence for the enzymatic synthesis of L-iduronic acid in prokaryotes is as yet lacking. We therefore performed an in silico screen for D-glucuronyl C5-epimerases in prokaryotes. Multiple candidate C5-epimerases were found, suggesting that many more microorganisms are likely to exist possessing an L-iduronic acid residue as constituent of their cell wall polysaccharides.