The heparan sulfate-specific epitope 10E4 is NO-sensitive and partly inaccessible in glypican-1

The monoclonal antibody 10E4, which recognizes an epitope supposed to contain N-unsubstituted glucosamine, is commonly used to trace heparan sulfate proteoglycans. It has not been fully clarified if the N-unsubstituted glucosamine is required for antibody recognition and if all heparan sulfates carry this epitope. Here we show that the epitope can contain N-unsubstituted glucosamine and that nitric oxide-generated deaminative cleavage at this residue in vivo can destroy the epitope. Studies using flow cytometry and confocal immunofluorescence microscopy of both normal and transformed cells indicated that the 10E4 epitope was partially inaccessible in the heparan sulfate chains attached to glypican-1. The 10E4 antibody recognized mainly heparan sulfate degradation products that colocalized with acidic endosomes. These sites were greatly depleted of 10E4-positive heparan sulfate on suramin inhibition of heparanase. Instead, there was increased colocalization between 10E4-positive heparan sulfate and glypican-1. When both S-nitrosylation of Gpc-1 and heparanase were inhibited, detectable 10E4 epitope colocalized entirely with glypican-1. In nitric oxide-depleted cells, there was both an increased signal from 10E4 and increased colocalization with glypican-1. In suramin-treated cells, the 10E4 epitope was destroyed by ascorbate-released nitric oxide with concomitant formation of anhydromannose-containing heparan sulfate oligosaccharides. Immunoisolation of radiolabeled 10E4-positive material from unperturbed cells yielded very little glypican-1 when compared with specifically immunoisolated glypican-1 and total proteoglycan and degradation products. The 10E4 immunoisolates were either other heparan sulfate proteoglycans or heparan sulfate degradation products.     

S-Nitrosylation of secreted recombinant human glypican-1

Glypican-1 is a glycosylphosphatidylinositol anchored cell surface S-nitrosylated heparan sulfate proteoglycan that is processed by nitric oxide dependent degradation of its side chains. Cell surface-bound glypican-1 becomes internalized and recycles via endosomes, where the heparan sulphate chains undergo nitric oxide and copper dependent autocleavage at N-unsubstituted glucosamines, back to the Golgi. It is not known if the S-nitrosylation occurs during biosynthesis or recycling of the protein. Here we have generated a recombinant human glypican-1 lacking the glycosylphosphatidylinositol-anchor. We find that this protein is directly secreted into the culture medium both as core protein and proteoglycan form and is not subjected to internalization and further modifications during recycling. By using SDS-PAGE, Western blotting and radiolabeling experiments we show that the glypican-1 can be S-nitrosylated. We have measured the level of S-nitrosylation in the glypican-1 core protein by biotin switch assay and find that the core protein can be S-nitrosylated in the presence of copper II ions and NO donor. Furthermore the glypican-1 proteoglycan produced in the presence of polyamine synthesis inhibitor, α-difluoromethylornithine, was endogenously S-nitrosylated and release of nitric oxide induced deaminative autocleavage of the HS side chains of glypican-1. We also show that the N-unsubstituted glucosamine residues are formed during biosynthesis of glypican-1 and that the content increased upon inhibition of polyamine synthesis. It cannot be excluded that endogenous glypican-1 can become further S-nitrosylated during recycling.     

Copper-dependent autocleavage of glypican-1 heparan sulfate by nitric oxide derived from intrinsic nitrosothiols

Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 371-376). Increased polyamine uptake correlates with an increased number of positively charged N-unsubstituted glucosamine units in the otherwise polyanionic heparan sulfate chains of glypican-1. During intracellular recycling of glypican-1, there is an NO-dependent deaminative cleavage of heparan sulfate at these glucosamine units, which would eliminate the positive charges (Ding, K., Sandgren, S., Mani, K., Belting, M., and Fransson, L.-A. (2001) J. Biol. Chem. 276, 46779-46791). Here, using both biochemical and microscopic techniques, we have identified and isolated S-nitrosylated forms of glypican-1 as well as slightly charged glypican-1 glycoforms containing heparan sulfate chains rich in N-unsubstituted glucosamines. These glycoforms were converted to highly charged species upon treatment of cells with 1 mm l-ascorbate, which releases NO from nitrosothiols, resulting in deaminative cleavage of heparan sulfate at the N-unsubstituted glucosamines. S-Nitrosylation and subsequent deaminative cleavage were abrogated by inhibition of a Cu(2+)/Cu(+) redox cycle. Under cell-free conditions, purified S-nitrosylated glypican-1 was able to autocleave its heparan sulfate chains when NO release was triggered by l-ascorbate. The heparan sulfate fragments generated in cells during this autocatalytic process contained terminal anhydromannose residues. We conclude that the core protein of glypican-1 can slowly accumulate NO as nitrosothiols, whereas Cu(2+) is reduced to Cu(+). Subsequent release of NO results in efficient deaminative cleavage of the heparan sulfate chains attached to the same core protein, whereas Cu(+) is oxidized to Cu(2+).     

Constitutive and vitamin C-induced, NO-catalyzed release of heparan sulfate from recycling glypican-1 in late endosomes

The recycling heparan sulfate (HS)-containing proteoglycan glypican-1 (Gpc-1) is processed by nitric oxide (NO)-catalyzed deaminative cleavage of its HS chains at N-unsubstituted glucosamines. This generates anhydromannose (anMan)-containing HS degradation products that can be detected by a specific antibody. Here we have attempted to identify the intracellular compartments where these products are formed. The anMan-positive degradation products generated constitutively in human bladder carcinoma cell line (T24) or fibroblasts appeared neither in caveolin-1-associated vesicles nor in lysosomes. In Niemann-Pick C-1 (NPC-1) fibroblasts, where deaminative degradation is abrogated, formation of anMan-positive products can be restored by ascorbate. These products colocalized with Rab7, a marker for late endosomes. When NO-catalyzed degradation of HS was depressed in mouse neuroblastoma cell line (N2a) by using 3-beta[2(diethylamino) ethoxy]androst-5-en-17-one (U18666A), both ascorbate and dehydroascorbic acid restored formation of anMan-positive products that colocalized with Rab7. In T24 cells, constitutively generated anMan-positive products colocalized with both Rab7 and Rab9, whereas Gpc-1 colocalized with Rab9, a marker for transporting endosomes. Inhibition of endosomal acidification, which blocks transfer from early (Rab5) to late (Rab7) endosomes, abrogated deaminative degradation of HS. This could also be overcome by the addition of ascorbate, which induced formation of anMan-positive degradation products that colocalized with Rab7. After (35)S-sulfate labeling, similar degradation products were recovered in Rab7-positive vesicles. We conclude that NO-catalyzed degradation of HS may begin in early endosomes but is mainly taking place in late endosomes.     

Prion,amyloid beta-derived Cu(II) ions,or free Zn(II) ions support S-nitroso-dependent autocleavage of glypican-1 heparan sulfate

Copper are generally bound to proteins, e.g. the prion and the amyloid beta proteins. We have previously shown that copper ions are required to nitrosylate thiol groups in the core protein of glypican-1, a heparan sulfate-substituted proteoglycan. When S-nitrosylated glypican-1 is then exposed to an appropriate reducing agent, such as ascorbate, nitric oxide is released and autocatalyzes deaminative cleavage of the glypican-1 heparan sulfate side chains at sites where the glucosamines are N-unsubstituted. These processes take place in a stepwise manner, whereas glypican-1 recycles via a caveolin-1-associated pathway where copper ions could be provided by the prion protein. Here we show, by using both biochemical and microscopic techniques, that (a) the glypican-1 core protein binds copper(II) ions, reduces them to copper(I) when the thiols are nitrosylated and reoxidizes copper(I) to copper(II) when ascorbate releases nitric oxide; (b) maximally S-nitrosylated glypican-1 can cleave its own heparan sulfate chains at all available sites in a nitroxyl ion-dependent reaction; (c) free zinc(II) ions, which are redox inert, also support autocleavage of glypican-1 heparan sulfate, probably via transnitrosation, whereas they inhibit copper(II)-supported degradation; and (d) copper(II)-loaded but not zinc(II)-loaded prion protein or amyloid beta peptide support heparan sulfate degradation. As glypican-1 in prion null cells is poorly S-nitrosylated and as ectopic expression of cellular prion protein restores S-nitrosylation of glypican-1 in these cells, we propose that one function of the cellular prion protein is to deliver copper(II) for the S-nitrosylation of recycling glypican-1.     

Heparan sulfate degradation products can associate with oxidized proteins and proteasomes

The S-nitrosylated proteoglycan glypican-1 recycles via endosomes where its heparan sulfate chains are degraded into anhydromannose-containing saccharides by NO-catalyzed deaminative cleavage. Because heparan sulfate chains can be associated with intracellular protein aggregates, glypican-1 autoprocessing may be involved in the clearance of misfolded recycling proteins. Here we have arrested and then reactivated NO-catalyzed cleavage in the absence or presence of proteasome inhibitors and analyzed the products present in endosomes or co-precipitating with proteasomes using metabolic radiolabeling and immunomagnet isolation as well as by confocal immunofluorescence microscopy. Upon reactivation of deaminative cleavage in T24 carcinoma cells, [(35)S]sulfate-labeled degradation products appeared in Rab7-positive vesicles and co-precipitated with a 20 S proteasome subunit. Simultaneous inhibition of proteasome activity resulted in a sustained accumulation of degradation products. We also demonstrated that the anhydromannose-containing heparan sulfate degradation products are detected by a hydrazide-based method that also identifies oxidized, i.e. carbonylated, proteins that are normally degraded in proteasomes. Upon inhibition of proteasome activity, pronounced colocalization between carbonyl-staining, anhydro-mannose-containing degradation products, and proteasomes was observed in both T24 carcinoma and N2a neuroblastoma cells. The deaminatively generated products that co-precipitated with the proteasomal subunit contained heparan sulfate but were larger than heparan sulfate oligosaccharides and resistant to both acid and alkali. However, proteolytic degradation released heparan sulfate oligosaccharides. In Niemann-Pick C-1 fibroblasts, where deaminative degradation of heparan sulfate is defective, carbonylated proteins were abundant. Moreover, when glypican-1 expression was silenced in normal fibroblasts, the level of carbonylated proteins increased raising the possibility that deaminative heparan sulfate degradation is involved in the clearance of misfolded proteins.     

Modulations of glypican-1 heparan sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase, and nitric oxide/nitrite cleavage sites

Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Persson, S., and Fransson, L.-A. (1999) Biochem. J. 338, 317-323; Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2001) Proc. Natl. Acad. Sci. U. S. A., in press). Here, we have analyzed the effect of polyamine deprivation on the structure and polyamine affinity of the heparan sulfate chains in various glypican-1 glycoforms synthesized by a transformed cell line (ECV 304). Heparan sulfate chains of glypican-1 were either cleaved with heparanase at sites embracing the highly modified regions or with nitrite at N-unsubstituted glucosamine residues. The products were separated and further degraded by heparin lyase to identify sulfated iduronic acid. Polyamine affinity was assessed by chromatography on agarose substituted with the polyamine spermine. In heparan sulfate made by cells with undisturbed endogenous polyamine synthesis, free amino groups were restricted to the unmodified, unsulfated segments, especially near the core protein. Spermine high affinity binding sites were located to the modified and highly sulfated segments that were released by heparanase. In cells with up-regulated polyamine uptake, heparan sulfate contained an increased number of clustered N-unsubstituted glucosamines and sulfated iduronic acid residues. This resulted in a greater number of NO/nitrite-sensitive cleavage sites near the potential spermine-binding sites. Endogenous degradation by heparanase and NO-derived nitrite in polyamine-deprived cells generated a separate pool of heparan sulfate oligosaccharides with an exceptionally high affinity for spermine. Spermine uptake in polyamine-deprived cells was reduced when NO/nitrite-generated degradation of heparan sulfate was inhibited. The results suggest a functional interplay between glypican recycling, NO/nitrite-generated heparan sulfate degradation, and polyamine uptake.     

Glypican-1 is a vehicle for polyamine uptake in mammalian cells: a pivital role for nitrosothiol-derived nitric oxide

Polyamines (putrescine, spermidine, and spermine) are essential for growth and survival of all cells. When polyamine biosynthesis is inhibited, there is up-regulation of import. The mammalian polyamine transport system is unknown. We have previously shown that the heparan sulfate (HS) side chains of recycling glypican-1 (Gpc-1) can sequester spermine, that intracellular polyamine depletion increases the number of NO-sensitive N-unsubstituted glucosamines in HS, and that NO-dependent cleavage of HS at these sites is required for spermine uptake. The NO is derived from S-nitroso groups in the Gpc-1 protein. Using RNA interference technology as well as biochemical and microscopic techniques applied to both normal and uptake-deficient cells, we demonstrate that inhibition of Gpc-1 expression abrogates spermine uptake and intracellular delivery. In unperturbed cells, spermine and recycling Gpc-1 carrying HS chains rich in N-unsubstituted glucosamines were co-localized. By exposing cells to ascorbate, we induced release of NO from the S-nitroso groups, resulting in HS degradation and unloading of the sequestered polyamines as well as nuclear targeting of the deglycanated Gpc-1 protein. Polyamine uptake-deficient cells appear to have a defect in the NO release mechanism. We have managed to restore spermine uptake partially in these cells by providing spermine NONOate and ascorbate. The former bound to the HS chains of recycling Gpc-1 and S-nitrosylated the core protein. Ascorbate released NO, which degraded HS and liberated the bound spermine. Recycling HS proteoglycans of the glypican-type may be plasma membrane carriers for cargo taken up by caveolar endocytosis.     

N-unsubstituted glucosamine in heparan sulfate of recycling glypican-1 from suramin-treated and nitrite-deprived endothelial cells. mapping of nitric oxide/nitrite-susceptible glucosamine residues to clustered sites near the core protein

We have analyzed the content of N-unsubstituted glucosamine in heparan sulfate from glypican-1 synthesized by endothelial cells during inhibition of (a) intracellular progression by brefeldin A, (b) heparan sulfate degradation by suramin, and/or (c) endogenous nitrite formation. Glypican-1 from brefeldin A-treated cells carried heparan sulfate chains that were extensively degraded by nitrous acid at pH 3.9, indicating the presence of glucosamines with free amino groups. Chains with such residues were rare in glypican-1 isolated from unperturbed cells and from cells treated with suramin and, surprisingly, when nitrite-deprived. However, when nitrite-deprived cells were simultaneously treated with suramin, such glucosamine residues were more prevalent. To locate these residues, chains were first cleaved at linkages to sulfated l-iduronic acid by heparin lyase and released fragments were separated from core protein carrying heparan sulfate stubs. These stubs were then cleaved off at sites linking N-substituted glucosamines to d-glucuronic acid. These fragments were extensively degraded by nitrous acid at pH 3.9. When purified proteoglycan isolated from brefeldin A-treated cells was incubated with intact cells, endoheparanase-catalyzed degradation generated a core protein with heparan sulfate stubs that were similarly sensitive to nitrous acid. We conclude that there is a concentration of N-unsubstituted glucosamines to the reducing side of the endoheparanase cleavage site in the transition region between unmodified and modified chain segments near the linkage region to the protein. Both sites as well as the heparin lyase-sensitive sites seem to be in close proximity to one another.     

A novel role for nitric oxide in the endogenous degradation of heparan sulfate during recycling of glypican-1 in vascular endothelial cells

We show here that the endothelial cell-line ECV 304 expresses the heparan sulfate proteoglycan glypican-1. The predominant cellular glycoform carries truncated side-chains and is accompanied by heparan sulfate oligosaccharides. Treatment with brefeldin A results in accumulation of a glypican proteoglycan with full-size side-chains while the oligosaccharides disappear. During chase the glypican proteoglycan is converted to partially degraded heparan sulfate chains and chain-truncated proteoglycan, both of which can be captured by treatment with suramin. The heparan sulfate chains in the intact proteoglycan can be depolymerized by nitrite-dependent cleavage at internally located N-unsubstituted glucosamine moieties. Inhibition of NO-synthase or nitrite-deprivation prevents regeneration of intact proteoglycan from truncated precursors as well as formation of oligosaccharides. In nitrite-deprived cells, formation of glypican proteoglycan is restored when NO-donor is supplied. We propose that, in recycling glypican-1, heparan sulfate chains are cleaved at or near glucosamines with unsubstituted amino groups. NO-derived nitrite is then required for the removal of short, nonreducing terminal saccharides containing these N-unsubstituted glucosamine residues from the core protein stubs, facilitating re-synthesis of heparan sulfate chains.     

Defective nitric oxide-dependent, deaminative cleavage of glypican-1 heparan sulfate in Niemann-Pick C1 fibroblasts

Exit of recycling cholesterol from late endosomes is defective in Niemann-Pick C1 (NPC1) and Niemann-Pick C2 (NPC2) diseases. The traffic route of the recycling proteoglycan glypican-1 (Gpc-1) may also involve late endosomes and could thus be affected in these diseases. During recycling through intracellular compartments, the heparan sulfate (HS) side chains of Gpc-1 are deaminatively degraded by nitric oxide (NO) derived from preformed S-nitroso groups in the core protein. We have now investigated whether this NO-dependent Gpc-1 autoprocessing is active in fibroblasts from NPC1 disease. The results showed that Gpc-1 autoprocessing was defective in these cells and, furthermore, greatly depressed in normal fibroblasts treated with U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), a compound widely used to induce cholesterol accumulation. In both cases, autoprocessing was partially restored by treatment with ascorbate which induced NO release, resulting in deaminative cleavage of HS. However, when NO-dependent Gpc-1 autoprocessing is depressed and heparanase-catalyzed degradation of HS remains active, a truncated Gpc-1 with shorter HS chains would prevail, resulting in fewer NO-sensitive sites/proteoglycan. Therefore, addition of ascorbate to cells with depressed autoprocessing resulted in nitration of tyrosines. Nitration was diminished when heparanase was inhibited with suramin or when Gpc-1 expression was silenced by RNAi. Gpc-1 misprocessing in NPC1 cells could thus contribute to neurodegeneration mediated by reactive nitrogen species.     

Copper-dependent co-internalization of the prion protein and glypican-1

Heparan sulfate chains have been found to be associated with amyloid deposits in a number of diseases including transmissible spongiform encephalopathies. Diverse lines of evidence have linked proteoglycans and their glycosaminoglycan chains, and especially heparan sulfate, to the metabolism of the prion protein isoforms. Glypicans are a family of glycosylphosphatidylinositol-anchored, heparan sulfate-containing, cell-associated proteoglycans. Cysteines in glypican-1 can become nitrosylated by endogenously produced nitric oxide. When glypican-1 is exposed to a reducing agent, such as ascorbate, nitric oxide is released and autocatalyses deaminative cleavage of heparan sulfate chains. These processes take place while glypican-1 recycles via a non-classical, caveolin-associated pathway. We have previously demonstrated that prion protein provides the Cu2+ ions required to nitrosylate thiol groups in the core protein of glypican-1. By using confocal immunofluorescence microscopy and immunomagnetic techniques, we now show that copper induces co-internalization of prion protein and glypican-1 from the cell surface to perinuclear compartments. We find that prion protein is controlling both the internalization of glypican-1 and its nitric oxide-dependent autoprocessing. Silencing glypican-1 expression has no effect on copper-stimulated prion protein endocytosis, but in cells expressing a prion protein construct lacking the copper binding domain internalization of glypican-1 is much reduced and autoprocessing is abrogated. We also demonstrate that heparan sulfate chains of glypican-1 are poorly degraded in prion null fibroblasts. The addition of either Cu2+ ions, nitric oxide donors, ascorbate or ectopic expression of prion protein restores heparan sulfate degradation. These results indicate that the interaction between glypican-1 and Cu2+-loaded prion protein is required both for co-internalization and glypican-1 self-pruning.     

Glypicans

A family of lipid-linked heparan sulfate (HS) proteoglycans, later named glypicans, were identified some 15 years ago. The discoveries that mutations in genes involved in glypican assembly cause developmental defects have brought them into focus. Glypicans have a characteristic pattern of 14 conserved cysteine residues. There are also two-three attachment sites for HS side-chains near the membrane anchor. The HS side-chains consist of a repeating disaccharide back-bone that is regionally and variably modified by epimerization and different types of sulfations, creating a variety of binding sites for polycationic molecules, especially growth factors. Recycling forms of glypican-1 are potential vehicles for transport of cargo into and through cells. The glypican-1 core protein is S-nitrosylated and nitric oxide released from these sites cleave the HS chains at glucosamine units lacking N-substitution. This processing is necessary for polyamine uptake.     

Coordinated regulation of caveolin-1 and Rab11a in apical recycling compartments of polarized epithelial cells

Recent studies have identified caveolin-1, a protein best known for its functions in caveolae, in apical endocytic recycling compartments in polarized epithelial cells. However, very little is known about the regulation of caveolin-1 in the endocytic recycling pathway. To address this question, in the current study we compared the relationship between compartments enriched in sub-apical caveolin-1 and Rab11a, a well-defined marker of apical recycling endosomes, using polarized MDCK cells as a model. We show that caveolin-1-containing vesicles define a compartment that partially overlaps with Rab11a, and that the distribution of subapical caveolin-1 and Rab11a shows a similar dependence on microtubule disruption. Mutants of the Rab11a effector, Rab11-FIP2 also altered the localization of caveolin-1. These findings indicate that caveolin-1 is coordinately regulated with Rab11a within the apical recycling system of polarized epithelial cells, suggesting that the two proteins are components of the same pathway.     

Involvement of glycosylphosphatidylinositol-linked ceruloplasmin in the copper/zinc-nitric oxide-dependent degradation of glypican-1 heparan sulfate in rat C6 glioma cells

The core protein of glypican-1, a glycosylphosphatidylinositol-linked heparan sulfate proteoglycan, can bind Cu(II) or Zn(II) ions and undergo S-nitrosylation in the presence of nitric oxide. Cu(II)-to-Cu(I)-reduction supports extensive and permanent nitrosothiol formation, whereas Zn(II) ions appear to support a more limited, possibly transient one. Ascorbate induces release of nitric oxide, which catalyzes deaminative degradation of the heparan sulfate chains on the same core protein. Although free Zn(II) ions support a more limited degradation, Cu(II) ions support a more extensive self-pruning process. Here, we have investigated processing of glypican-1 in rat C6 glioma cells and the possible participation of the copper-containing glycosylphosphatidylinositol-linked splice variant of ceruloplasmin in nitrosothiol formation. Confocal microscopy demonstrated colocalization of glypican-1 and ceruloplasmin in endosomal compartments. Ascorbate induced extensive, Zn(II)-supported heparan sulfate degradation, which could be demonstrated using a specific zinc probe. RNA interference silencing of ceruloplasmin expression reduced the extent of Zn(II)-supported degradation. In cell-free experiments, the presence of free Zn(II) ions prevented free Cu(II) ion from binding to glypican-1 and precluded extensive heparan sulfate autodegradation. However, in the presence of Cu(II)-loaded ceruloplasmin, heparan sulfate in Zn(II)-loaded glypican-1 underwent extensive, ascorbate-induced degradation. We propose that the Cu(II)-to-Cu(I)-reduction that is required for S-nitrosylation of glypican-1 can take place on ceruloplasmin and thereby ensure extensive glypican-1 processing in the presence of free Zn(II) ions.     

Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling

The small GTPase Rab5 regulates the early endocytic pathway of transferrin (Tfn), and Rab5 deactivation is required for Tfn recycling. Rab5 deactivation is achieved by RabGAP5, a GTPase-activating protein, on the endosomes. Here we report that recruitment of RabGAP5 is insufficient to deactivate Rab5 and that developmentally regulated GTP-binding protein 2 (DRG2) is required for Rab5 deactivation and Tfn recycling. DRG2 was associated with phosphatidylinositol 3-phosphate–containing endosomes. It colocalized and interacted with EEA1 and Rab5 on endosomes in a phosphatidylinositol 3-kinase–dependent manner. DRG2 depletion did not affect Tfn uptake and recruitment of RabGAP5 and Rac1 to Rab5 endosomes. However, it resulted in impairment of interaction between Rab5 and RabGAP5, Rab5 deactivation on endosomes, and Tfn recycling. Ectopic expression of shRNA-resistant DRG2 rescued Tfn recycling in DRG2-depleted cells. Our results demonstrate that DRG2 is an endosomal protein and a key regulator of Rab5 deactivation and Tfn recycling.     

The amyloid precursor protein (APP) of Alzheimer disease and its paralog, APLP2, modulate the Cu/Zn-Nitric Oxide-catalyzed degradation of glypican-1 heparan sulfate in vivo

Processing of the recycling proteoglycan glypican-1 involves the release of its heparan sulfate chains by copper ion- and nitric oxide-catalyzed ascorbate-triggered autodegradation. The Alzheimer disease amyloid precursor protein (APP) and its paralogue, the amyloid precursor-like protein 2 (APLP2), contain copper ion-, zinc ion-, and heparan sulfate-binding domains. We have investigated the possibility that APP and APLP2 regulate glypican-1 processing during endocytosis and recycling. By using cell-free biochemical experiments, confocal laser immunofluorescence microscopy, and flow cytometry of tissues and cells from wild-type and knock-out mice, we find that (a) APP and glypican-1 colocalize in perinuclear compartments of neuroblastoma cells, (b) ascorbate-triggered nitric oxidecatalyzed glypican-1 autodegradation is zinc ion-dependent in the same cells, (c) in cell-free experiments, APP but not APLP2 stimulates glypican-1 autodegradation in the presence of both Cu(II) and Zn(II) ions, whereas the Cu(I) form of APP and the Cu(II) and Cu(I) forms of APLP2 inhibit autodegradation, (d) in primary cortical neurons from APP or APLP2 knock-out mice, there is an increased nitric oxide-catalyzed degradation of heparan sulfate compared with brain tissue and neurons from wild-type mice, and (e) in growth-quiescent fibroblasts from APLP2 knock-out mice, but not from APP knock-out mice, there is also an increased heparan sulfate degradation. We propose that the rate of autoprocessing of glypican-1 is modulated by APP and APLP2 in neurons and by APLP2 in fibroblasts. These observation identify a functional relationship between the heparan sulfate and copper ion binding activities of APP/APLP2 in their modulation of the nitroxyl anion-catalyzed heparan sulfate degradation in glypican-1.     

The disaccharide composition of heparins and heparan sulfates

Heparin and heparan sulfate can be cleaved selectively at their N-sulfated glucosamine residues by direct treatment with nitrous acid at pH 1.5. These polymers can also be cleaved selectively at their N-acetylated glucosamine residues by first N-deacetylating with hydrazine and then treating the products with nitrous acid at pH 4. These procedures have been combined and optimized for the conversion of these glycosaminoglycan chains into their disaccharide units. A modified hydrazinolysis procedure in which the glycosaminoglycans were heated with hydrazine:water (70:30) containing 1% hydrazine sulfate gave rapid rates of N-deacetylation and minimal conversion of the uronic acid residues to their hydrazide derivatives. Under these conditions, N-deacetylation was complete in 4 h and the beta-eliminative cleavage of the polymer chains that occurs during hydrazinolysis (P. N. Shaklee and H. E. Conrad (1984) Biochem. J. 217, 187-197) was eliminated. Treatment of the N-deacetylated polymer with nitrous acid at pH 3 for 15 h at 25 degrees C then gave simultaneous cleavage at the N-unsubstituted glucosamine residues and the N-sulfated glucosamine residues. These deamination conditions minimized, but did not eliminate, the side reaction in which nitrous acid-reactive glucosamine residues undergo ring contraction without glucosaminide bond cleavage. Thus, the disaccharides were obtained in a yield of 90% of those originally present in the glycosaminoglycan chains. Since the ring contraction side reaction occurs randomly at the diazotized glucosamine residues, the disaccharides formed in the pH 3 nitrous acid reaction were recovered in proportions equal to those in the original glycosaminoglycan chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mouse polyomavirus enters early endosomes, requires their acidic pH for productive infection, and meets transferrin cargo in Rab11-positive endosomes

Mouse polyomavirus (PyV) virions enter cells by internalization into smooth monopinocytic vesicles, which fuse under the cell membrane with larger endosomes. Caveolin-1 was detected on monopinocytic vesicles carrying PyV particles in mouse fibroblasts and epithelial cells (33). Here, we show that PyV can be efficiently internalized by Jurkat cells, which do not express caveolin-1 and lack caveolae, and that overexpression of a caveolin-1 dominant-negative mutant in mouse epithelial cells does not prevent their productive infection. Strong colocalization of VP1 with early endosome antigen 1 (EEA1) and of EEA1 with caveolin-1 in mouse fibroblasts and epithelial cells suggests that the monopinocytic vesicles carrying the virus (and vesicles containing caveolin-1) fuse with EEA1-positive early endosomes. In contrast to SV40, PyV infection is dependent on the acidic pH of endosomes. Bafilomycin A1 abolished PyV infection, and an increase in endosomal pH by NH4Cl markedly reduced its efficiency when drugs were applied during virion transport towards the cell nucleus. The block of acidification resulted in the retention of a fraction of virions in early endosomes. To monitor further trafficking of PyV, we used fluorescent resonance energy transfer (FRET) to determine mutual localization of PyV VP1 with transferrin and Rab11 GTPase at a 2- to 10-nm resolution. Positive FRET between PyV VP1 and transferrin cargo and between PyV VP1 and Rab11 suggests that during later times postinfection (1.5 to 3 h), the virus meets up with transferrin in the Rab11-positive recycling endosome. These results point to a convergence of the virus and the cargo internalized by different pathways in common transitional compartments.     

Invasion of host cells by JC virus identifies a novel role for caveolae in endosomal sorting of noncaveolar ligands

Invasion of glial cells by the human polyomavirus, JC virus (JCV), leads to a rapidly progressing and uniformly fatal demyelinating disease known as progressive multifocal leukoencephalopathy. The endocytic trafficking steps used by JCV to invade cells and initiate infection are not known. We demonstrated that JCV infection was inhibited by dominant defective and constitutively active Rab5-GTPase mutants that acted at distinct steps in endosomal sorting. We also found that labeled JCV colocalized with labeled cholera toxin B and with caveolin-1 (cav-1) on early endosomes following internalization by clathrin-dependent endocytosis. JCV entry and infection were both inhibited by dominant defective mutants of eps15 and Rab5-GTPase. Expression of a dominant-negative scaffolding mutant of cav-1 did not inhibit entry or infection by JCV. A single-cell knockdown experiment using cav-1 shRNA did not inhibit JCV entry but interfered with a downstream trafficking event important for infection. These data show that JCV enters cells by clathrin-dependent endocytosis, is transported immediately to early endosomes, and is then sorted to a caveolin-1-positive endosomal compartment. This latter step is dependent on Rab5-GTPase, cholesterol, caveolin-1, and pH. This is the first example of a ligand that enters cells by clathrin-dependent endocytosis and is then sorted from early endosomes to caveosomes, indicating that caveolae-derived vesicles play a more important role than previously realized in sorting cargo from early endosomes.