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Originally published In Press as doi:10.1074/jbc.M409179200 on January 27, 2005

J. Biol. Chem., Vol. 280, Issue 14, 13913-13920, April 8, 2005
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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*

Roberto Cappai{ddagger}§, Fang Cheng||, Giuseppe D. Ciccotosto{ddagger}, B. Elise Needham{ddagger}, Colin L. Masters{ddagger}, Gerd Multhaup**, Lars-Åke Fransson||{ddagger}{ddagger}, and Katrin Mani||

From the ||Department of Cell and Molecular Biology, Section for Cell and Matrix Biology, Lund University, Biomedical Center C13, SE-221 84, Lund, Sweden, {ddagger}Department of Pathology and §Center for Neuroscience, The University of Melbourne, Victoria 3010, The Mental Health Research Institute of Victoria, Parkville, Victoria 3052, Australia, and **Institute for Chemistry/Biochemistry, Free University of Berlin, Thielallee 63, D-14195, Berlin, Germany

Received for publication, August 11, 2004 , and in revised form, January 24, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Processing of the amyloid precursor protein (APP)1 of Alzheimer disease (AD) involves several proteases and regulatory proteins, collectively designated {alpha}-, {beta}-, and {gamma}-secretases (Fig. 1). {beta}- and {gamma}-Cleavages lead to the generation of amyloid-{beta} (A{beta}) peptides A{beta}1–40 and A{beta}1–42 (1, 2). The A{beta} peptides are believed to cause the neurotoxicity associated with AD via an increase in A{beta} levels (3). Eventually, A{beta} accumulates into amyloid fibrils and finally senile plaques, the key pathological hallmark of AD. However, the APP paralogue amyloid precursor-like protein 2 (APLP2) does not contain the A{beta} sequence and hence does not contribute to amyloid formation. Both APP and APLP2 bind metal ions and heparin, but the physiological significance of this property is unclear. Nevertheless, several in vitro studies have shown that APP and its {alpha}-secretory form are involved in neuronal growth and survival and in neurite outgrowth (for reviews, see Refs. 4–7).



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  		FIG. 1.
Schematic structure of the amyloid-{beta} precursor protein APP695. It is a type I transmembrane protein with a large ectodomain containing both metal- and HS-binding subdomains (amino acid positions are indicated). Cu(II) can be reduced to Cu(I). The binding of zinc increases the affinity for HS. The Cu(II)-binding A{beta} peptide is generated from a segment that is partially embedded in the membrane (1–7).

 
The primary transcript of the APP gene undergoes extensive alternative splicing. The isoform APP695 is expressed at high levels in central nervous system neurons (8). APP-deficient mice (APP-/-) show reactive gliosis, especially in the cerebral cortex and the hippocampus, and decreased locomotor activity (9). With age, they develop deficits in cognitive function and impairment in long-term potentiation. Mice that die prematurely show a loss of presynaptic vesicle marker proteins (10). Moreover, there is an increase of copper in the cerebral cortex of APP-/- mice (11).

As shown in Fig. 1, APP has an N-terminal copper-binding domain with nanomolar affinity (12). Cu(II) bound to this site is reduced to Cu(I) in vitro (13, 14). Structural analysis of this copper-binding site by using NMR reveals a surface location that favors Cu(I) coordination (15). Downstream from the N-terminal copper-binding domain is a zinc binding motif, also with nanomolar affinity (16), and upstream from the copper-binding domain is a potential heparin/heparan sulfate (HS) binding motif with similar affinity (see Fig. 1) (17). The presence of micromolar concentrations of Zn(II) ions increases the binding of heparin/HS to APP 2–4-fold (16, 18). The crystal structure of the N-terminal domain of APP (19) exhibits both positively charged and hydrophobic surfaces that could bind both the HS side chains and the core protein of a proteoglycan (PG).

Results of in vitro studies strongly suggest that HSPGs regulate the effects of APP on neurite outgrowth (17, 20, 21). When PG from neonatal mouse brain cells was subjected to affinity chromatography on an APP695-substituted matrix, the HSPG glypican-1 (Gpc-1) was the most strongly bound with an affinity in the nanomolar range (22).

Gpc-1 belongs to a family of glycosylphosphatidylinositol (GPI)-linked HSPG and is found at high levels in the brain where it is expressed by both neurons and glia cells. The core proteins of all glypicans have a characteristic pattern of 14 conserved Cys residues in their central domain. It has been generally assumed that the Cys residues participate in disulfide bond formation (for review, see Ref. 23). However, many of the Cys residues in Gpc-1 can become stably S-nitrosylated (SNO) by endogenously formed nitric oxide (NO) in a reaction that is dependent on Cu(II)-to-Cu(I) reduction (24). SNO formation is probably facilitated by the presence of N-unsubstituted glucosamine residues () in HS, which would favor the formation of thiol anions in Cys.

During intracellular recycling of Gpc-1 via a lipid raft/caveolin-1-associated endosome-to-Golgi pathway, NO is released from the SNO groups in the Gpc-1 core protein, resulting in deaminative cleavage of the HS chains at the residues (25). The deaminative cleavage generates anhydromannose (anMan), which remains at the reducing end of the HS oligosaccharide fragments (25). In both cell cultures and cell-free experiments, using purified Gpc-1, NO-release from SNO groups in Gpc-1 can be triggered by ascorbate (24).

The Cu(II) ions required for Gpc-1 S-nitrosylation could be delivered to Gpc-1 by a copper-loaded cuproprotein, such as APP/APLP2, A{beta}1–40, ceruloplasmin, or the prion protein (26, 27). Accordingly, Gpc-1 does not become S-nitrosylated in prion null fibroblasts. Moreover, Zn(II) ions inhibit Cu(II) ion-dependent S-nitrosylation, probably because they compete for the same binding site(s) in Gpc-1 (26). The addition of ceruloplasmin, which can bind Cu(II) ions and maintain a Cu(II)-Cu(I) redox cycle, leads to extensive Gpc-1 HS degradation, even in the presence of Zn(II) ions. A GPI-linked splice variant of ceruloplasmin colocalizes with Gpc-1 in rat C6 glioma cells (27).

APP can occur in different membrane domains of the cell surface, one associated with lipid rafts, another outside these domains (28, 29). The latter APP molecules are primarily processed via {alpha}-cleavage at the cell surface (4–7, 30), whereas those associated with lipid rafts are subject to {beta}-cleavage during endocytosis (4–7, 28). Because Gpc-1 is also lipid raft-associated (25, 29), APP and Gpc-1 may be cotransported during endocytosis and recycling.

We have investigated the possibility that APP and APLP2 regulate the processing of Gpc-1. By using both cell-free and cell culture experiments utilizing neurons and fibroblasts from wild-type and knock-out mice, we find that APP and APLP2 in neurons and APLP2 in fibroblasts are important modulators of NO-catalyzed and ascorbate-triggered HS degradation in Gpc-1.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Mouse N2a neuroblastoma cells were obtained from ATCC and maintained in minimal essential medium supplemented with 10% donor calf serum. Polyclonal antisera against mouse Gpc-1 and SNO-Cys as well as monoclonal antibodies (mAbs) recognizing SNO-Gpc-1 (mAb S1), a -containing HS epitope (mAb JM-403), anMan-terminating HS oligosaccharides (mAb AM) and suitably tagged secondary antibodies as well as radioactive precursors, enzymes, prepacked columns, Centriplus tubes, and chemicals were generated or obtained as described previously (24–26). A monoclonal antibody (mAb890) recognizing an epitope common to rhAPP695+1 and rhAPP770 was obtained from R&D systems. The zinc probe FluoZin-3 was from Molecular Probes, and iminodiacetic acid was from Fluka.

[3H]Glucosamine-labeled Gpc-1 was obtained from difluoromethylornithine- and brefeldin A-treated N2a cells as previously described for T24 cells (24). The difluoromethylornithine treatment was used to increase the content of units in HS and was carried out while cells grew to confluency, which usually took 1–2 days. The brefeldin A treatment was used to generate non-S-nitrosylated Gpc-1 PG (25). Recombinant ectodomains of APP695 (sAPP695{alpha}) and APLP2 (31) were produced as described (32). The proteins were saturated with divalent cations by exposure to 1 mM CuCl2 or ZnCl2 and then dialyzed against phosphate-buffered saline, pH 7.4. To convert APP695{alpha}/APLP2-Cu(II) to corresponding Cu(I) forms, solutions were incubated at 37 °C overnight (13).

Mice—The generation and initial characterization of APP-/- and APLP2-/- mice have been previously described (9, 33). The APP-/- and APLP2-/- mice were derived from the same strain as the wild-type mice (C57BL6J/129sv). Genetic background of each individual fetal pup was determined by PCR of tail DNA using primer sets as described previously (33).

Cells—Primary cortical neurons were prepared from mouse brain and maintained as described previously (34). Mouse embryonic fibroblasts were prepared from embryonic day 14 or 15 mice using sterile conditions. Individual embryos were placed into separate wells of a 6-well dish, and the head, liver, and gastrointestinal organs were dissected free and the remaining body was washed twice in fresh Hank's balanced salt solution and placed in a new well with fresh 2.5 ml of Hank's balanced salt solution. The tissue was finely minced with sterile scissors, 0.25% w/v trypsin was added, and the cell suspension was transferred to a 15-ml tube and kept in a 37 °C water bath with gentle shaking for 20 min. Trypsin inhibitor mixture (0.1% w/v lima bean trypsin inhibitor and 0.032% w/v DNase) was added to dissolve the DNA released from the cells. The cell suspension was then added to a sterile syringe, and the contents were passed through an 18-gauge syringe and triturated 30 times. The cell suspension was pelleted, and medium was replaced with culture medium (Dulbecco's minimal essential medium, containing high glucose and supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 10% fetal calf serum) and added to a 175-cm2 flask and placed in a 37 °C incubator in a 5% CO2 atmosphere. The culture medium was replaced after 24 h and then changed every 3–4 days thereafter.

Autocleavage of HS in Gpc-1—The high-molecular weight Gpc-1 precursor (approximately 1–3 nM) was exposed to various combinations of metal ions, recombinant ectodomains of APP695 or APLP2, and NO-donor (sodium nitroprusside) and triggered by ascorbate (1 mM for 10 min) to autodegrade its own HS chains, all in phosphate-buffered saline, pH 7.4, at 37 °C in the dark. The reactions were monitored by Superose 6 gel chromatography of the various NO-released HS fragments and the alkali-released stubs remaining on the core protein as described previously (24). Control degradation of units was carried out by HNO2 at pH 3.9.

Confocal Laser Scanning Immunofluorescence Microscopy—The various procedures including seeding of cells, fixation, the use of primary and secondary antibodies, generation of images by sequential scans, and data processing were the same as those used previously (24–26) or as recommended by the manufacturers. Cells were first precoated with 10% anti-mouse total Ig and 1% goat serum and then exposed to primary antibodies. The second antibody used was either goat anti-mouse total Ig when the primary antibody was a monoclonal or goat anti-rabbit IgG when the primary antibody was polyclonal. The second antibodies were tagged with either fluorescein isothiocyanate or Texas Red and appropriately combined for colocalization studies. In the controls, the primary antibody was omitted. The images shown were obtained at a focal plane that was at the center of the cell and of 0.3–0.5 µm thickness. Identical exposure settings were used for image capture. Images were digitized and transferred to Adobe PhotoShop for merging, annotation, and printing.

Frozen brain tissue (11) was cut in a cryostat into 8-µm sections and fixed in 2% (w/v) paraformaldehyde, 0.1% (v/v) Triton X-100 and then incubated overnight at 4 °C in blocking solution (PBS, 10% anti-mouse total Ig, and 1% goat serum) prior to confocal immunofluorescence microscopy as above.

Flow Cytometry—Mouse embryonic fibroblasts were seeded in 24-well plates and grown to near confluence in minimal essential medium containing glutamine, penicillin, streptomycin, and 10% fetal calf serum. Cells were rinsed with medium and detached using trypsin (0.5 ml of 0.05% (w/v) trypsin in PBS for 1 min). Trypsinization was terminated by replacing the trypsin solution with 0.5 ml of medium supplemented with 10% fetal bovine serum. Cells were recovered by gentle suspension and transferred to tubes, adding 1 vol of PBS containing 1% bovine serum albumin (w/v). Cells were then pelleted by centrifugation and resuspended in 0.2 ml of PBS after removal of the supernatant. Cells were fixed for 30 min in 1 ml of PBS containing 4% paraformaldehyde (w/v) while initially vortexing. Permeabilization was performed by incubation with 0.2% Triton X-100 in PBS (v/v) for 20 min. Immunostaining of the cells with the mAb specific for anMan-containing HS degradation products as the primary antibody and goat anti-mouse total Ig as the secondary antibody was performed as described for confocal microscopy. In the controls, the primary antibody was omitted. After each step, cells were recovered by centrifugation at 350 x g for 5 min. The cells were finally suspended in PBS containing 1% bovine serum albumin and analyzed for fluorescence in a fluorescence-assisted cell sorting instrument (Calibur, BD Biosciences) operated by Cell-Quest software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Gpc-1 and NO-catalyzed HS Degradation in Mouse N2a Neuroblastoma Cells—As shown in Fig. 2, A–C, Gpc-1 colocalized with -containing HS chains (JM-403) in N2a cells, indicating that Gpc-1 was substituted with NO-sensitive HS chains. This Gpc-1 also colocalized with APP at paranuclear sites (Fig. 2, D–F). Although SNO forms of various proteins were generally abundant (Fig. 2G), Gpc-1-SNO was scarce as indicated by the lack of signal from mAb S1 (Fig. 2H). Nevertheless, there was robust generation of anMan-containing HS oligosaccharides via NO-catalyzed cleavage (Fig. 2K) in compartments where Gpc-1 also occurred (Fig. 2, J and L). This suggested that NO and subsequently nitroxyl anion were not exclusively derived from stable SNO groups but, to a significant extent, via Zn(II) ion-supported transnitrosation (26). Therefore, we studied the effect of zinc-supplementation of metal ion-depleted cells.



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  		FIG. 2.
Immunolocalization of total Gpc-1, total S-nitrosylated protein, S-nitrosylated Gpc-1, HS, APP, and anMan-containing HS degradation products in mouse N2a neuroblastoma cells. The panels show confocal laser immunofluorescence staining for mouse Gpc-1 (GPC), a -specific epitope in HS (JM-403), APP, total S-nitrosylated protein (SNO-Cys), S-nitrosylated Gpc-1 (S1), and an anMan-containing epitope in HS degradation products (AM). Merged panels are indicated. Scale bar, 20 µm.

 
In N2a cells treated with the divalent metal ion chelator iminodiacetate, the generation of anMan-containing products by NO-catalyzed HS degradation was much reduced (Fig. 3A) and could not be elicited by ascorbate treatment (Fig. 3B). Degradation was restored by supplementation with zinc ions (Fig. 3C), and the products again colocalized with Gpc-1, their presumed substrate (Fig. 3D). In the course of zinc-supported NO-catalyzed cleavage of HS, Zn(II) ions should be released from Gpc-1 when ascorbate triggers NO release (26). We tested this in the following experiment.



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  		FIG. 3.
Effect of Zn(II) ions on NO-catalyzed ascorbate-triggered HS degradation in N2a cells. The panels show confocal laser immunofluorescence staining for an anMan-containing epitope in HS degradation products (AM) and mouse Gpc-1 (GPC) in 1 mM iminodiacetate (IDA)-treated cells (A), in 1 mM iminodiacetate-treated cells, and 1 mM ascorbate (Asc)-treated cells (B) or in 1 mM iminodiacetate-treated, 1 mM ZnCl2-treated, and 1 mM ascorbate-treated cells (C and D). Merged panel is indicated in D. Scale bar, 20 µm.

 
In unperturbed cells, free Zn(II) ions were barely detectable by the zinc-specific probe under the conditions used (Fig. 4A) and almost undetectable after exposure to NO-donor (Fig. 4B) in keeping with an increased binding of zinc to Gpc-1 in conjunction with SNO formation. Zn(II) ion release did not appear to be induced by exposure to ascorbate alone (cf. Fig. 4, A and C) but increased after treatment with both NO-donor and ascorbate (Fig. 4D). Taken together, these results showed that there is extensive zinc-supported NO-catalyzed degradation of the HS chains in Gpc-1 of N2a cells despite the fact that zinc is redox inert. As Gpc-1 and APP colocalized in N2a cells (Fig. 2, D–F) and because APP could potentially support a copper redox cycle, we investigated the effect of APP/APLP2 on cell-free NO-catalyzed HS autocleavage in Gpc-1 in the presence of zinc ions.



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  		FIG. 4.
Release of bound Zn(II) ion during NO-catalyzed ascorbate-triggered HS degradation in N2a cells. The panels show confocal laser immunofluorescence staining of cells for free zinc ion using 2.5 µM zinc probe (Zn-Pro) after no treatment (A), after treatment with 1 mM sodium nitroprusside (SNP) (B), 1 mM ascorbate (Asc) (C), and after treatment with both 1 mM SNP and 1 mM ascorbate (D). Scale bar, 20 µm.

 
APP/APLP2-supported Cell-free Autocleavage of HS in Gpc-1— In these experiments, we used a purified Gpc-1 that was [3H]GlcN-labeled in the HS chains and isolated from polyamine-deprived and brefeldin A-treated N2a cells. As shown previously (24–26), this gives rise to a non-S-nitrosylated Gpc-1 glycoform with long and -rich, i.e. NO-sensitive, HS chains.

To test the effect of APP and APLP2 on autocatalyzed HS degradation in cell-free experiments, purified Gpc-1 from N2a cells was incubated at 37 °C overnight in the presence of Zn(II) and Cu(II) ions, NO-donor, and ascorbate with or without added recombinant APP or APLP2 ectodomains. HS degradation was monitored by gel chromatography on Superose 6 of the mixture of NO-cleaved HS oligosaccharides and the remaining HS stubs released by alkali from the truncated Gpc-1. In the absence of added proteins, degradation was limited (cf. solid line with dotted line in Fig. 5A), as shown previously when Zn(II) and Cu(II) ions competed for the same binding sites in Gpc-1 (26, 27). With the addition of APP695{alpha}, extensive HS degradation was observed (Fig. 5B, solid line), almost to the same extent as with HNO2 at pH 3.9 (Fig. 5B, dotted line). Hence, APP supported extensive HS degradation with the same relative activity as prion protein (26) and ceruloplasmin (27).



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  		FIG. 5.
Effect of APP and APLP2 ectodomains on Cu/Zn-NO-catalyzed autocleavage of Gpc-1 HS in a cell-free system. Shown are chromatograms of [3H]HS chains or [3H]HS chain fragments and oligosaccharides derived from N2a Gpc-1 by alkaline borohydride treatment without previous treatment (dotted line in A) or after exposing Gpc-1 (1–3 nM) to 1 mM ZnCl2, 1 mM CuCl2, 1 mM sodium nitroprusside (SNP), and 1 mM L-ascorbate for 24 h (solid line in A), after exposing Gpc-1 to 1 mM ZnCl2, 1 mM CuCl2, 1 mM SNP, 1 mM L-ascorbate, and 1 µM APP695{alpha} (solid line in B) or after treatment of alkali-released [3H]HS chains with HNO2 at pH 3.9 (dotted line in B), after exposing Gpc-1 to 1 mM ZnCl2, 1 mM CuCl2, and 1 µM APP695{alpha} for 24 h and then to 1 mM SNP and 1 mM L-ascorbate for an additional 24 h (C), after exposing Gpc-1 to 1 mM ZnCl2, 1 mM CuCl2, 1 mM SNP, 1 mM ascorbate, and 1 µM APP695{alpha} pretreated with CuCl2 overnight and dialyzed (solid line in D), after exposing Gpc-1 to 1 mM ZnCl2, 1 mM CuCl2, 1 mM SNP, 1 mM ascorbate, 1 µM APP695{alpha}, and 1 µM APP695{alpha} pretreated with CuCl2 overnight and dialyzed (dotted line in D), after exposing Gpc-1 to 1 mM ZnCl2, 1 mM CuCl2, 1 mM SNP, and 1 µM APP695{alpha} (solid line in E), after exposing Gpc-1 to 1 mM CuCl2, 1 mM SNP, and 1 µM APP695{alpha} (dotted line in E), after exposing Gpc-1 to 1 mM ZnCl2, 1 mM CuCl2, 1 mM SNP, 1 µM APP695{alpha}, and 1 µM APP695{alpha} pretreated with CuCl2 overnight and dialyzed (F), after exposing Gpc-1 to 1 mM ZnCl2, 1 mM CuCl2, 1 mM SNP, 1 mM ascorbate, and 1 µM APLP2 ectodomain (G), after exposing Gpc-1 to 1 mM ZnCl2, 1 mM CuCl2, 1 mM SNP, 1 mM ascorbate, 1 µM APP695{alpha}, and 1 µM APLP2 ectodomain (H), or after exposing Gpc-1 to 1 mM ZnCl2, 1 mM CuCl2, 1 mM SNP, 1 mM ascorbate, 1 µM APP695{alpha}, and 1 µM APLP2 ectodomain pretreated with CuCl2 overnight and dialyzed (I). All of the incubations were performed for 24 h at 37 °C.

 
Because exposure of APP to Cu(II) ions should eventually result in the formation of APP-Cu(I), we incubated Gpc-1 with Zn(II) and Cu(II) ions and APP695{alpha} separately for 24 h and then added NO-donor and ascorbate. APP-Cu(I) will be generated during the first 24-h phase of the reaction. Under these conditions, HS degradation during the second phase was somewhat less pronounced (Fig. 5C), suggesting that reoxidation of APP-Cu(I) to APP-Cu(II) did not occur or that APP-Cu(I) inhibited the reaction. To test this more definitively, we incubated Gpc-1 with Zn(II) and Cu(II) ions, NO-donor, ascorbate, and preformed APP-Cu(I). In this case (Fig. 5D, solid line), HS degradation was much more limited. Furthermore, APP-Cu(I) inhibited the reaction even in the presence of equimolar amounts of APP-Cu(II) (Fig. 5D, dotted line).

Ascorbate was necessary as a trigger of APP-Cu(II)-supported autoprocessing of Gpc-1. Incubation of Gpc-1 with Zn(II) and Cu(II) ions, NO-donor, and APP695{alpha} resulted in a very limited degradation (Fig. 5E, solid line), and when zinc ions were omitted, there was no observable degradation (Fig. 5E, dotted line). Furthermore, HS degradation was not obtained when preformed APP695{alpha}-Cu(I) was substituted for ascorbate (Fig. 5F).

Interestingly, the APLP2 ectodomain in the Cu(II) form did not support ascorbate-triggered and NO-catalyzed HS degradation (Fig. 5G). Moreover, APLP2, both in the Cu(II) form (Fig. 5H) and in the Cu(I) form (Fig. 5I), inhibited degradation in the presence of equimolar amounts of APP-Cu(II).

In summary, in the presence of Zn(II) and Cu(II) ions, APP initially supported NO-catalyzed HS degradation in a cell-free system but, as Cu(II) was reduced to Cu(I), it became an inhibitor. In contrast, APLP2 was an inhibitor irrespective of the copper redox state.

NO-catalyzed HS Degradation in Brain Tissue and Cells from Wild-type, APP, or APLP2 Knock-out Mice—The extent of NO-catalyzed HS degradation was assessed by staining for anMan-containing HS oligosaccharides. In brain tissue from 12-week-old APP-/- mice, HS degradation was more pronounced than in tissue from wild-type mice (Fig. 6, A–B). In brain tissue from APLP2-/- mice, HS degradation was only slightly augmented (Fig. 6C). In primary cultures of cortical neurons from wild-type mice, staining for anMan-containing oligosaccharides was weak (Fig. 6D), but in neurons from APP-/- or APLP2-/- mice, the intensity of staining was increased approximately 2- and 1.5-fold, respectively, indicating that NO-dependent HS degradation was more pronounced (Fig. 6, E–F). Hence, both APP and APLP2 are important modulators of Gpc-1 autodegradation in neurons that correlates well with the extent of NO-catalyzed HS degradation in brain tissue from knock-out mice.



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  		FIG. 6.
Formation of anMan-containing HS degradation products in brain tissue (A–C), in primary cortical neurons (D–F), and in growth-quiescent fibroblasts (G–I) from wild-type (WT), APP, or APLP2 knock-out mice. The panels show confocal laser immunofluorescence images of frozen brain tissue sections from 12-week-old animals (A–C), of cultured primary neurons (D–F) and of confluent embryonic fibroblasts (G–I) from wild-type (A, D, and G), APP-/- (B, E, and H), or APLP2-/- mice (C, F, and I) stained for anMan-containing HS degradation products (AM). The intensity of cell staining in D–F was measured by using Adobe PhotoShop. Scale bar, 20 µm. Insets, results of flow cytometry of anMan-positive cells. The results are expressed in arbitrary fluorescence intensity units/cell.

 
In cultures of proliferating fibroblasts from wild-type mice, there was extensive HS degradation (result not shown), whereas there was minimal degradation in confluent growth-quiescent cultures as indicated by staining for anMan-containing degradation products (Fig. 6G). In growth-quiescent fibroblasts from APP-/- mice, there was also minimal HS degradation (Fig. 6H). However, in confluent fibroblast cultures from APLP2-/- mice, there was extensive NO-catalyzed HS degradation (Fig. 6I).

Quantification by flow cytometry was performed with near confluent fibroblasts, because fully confluent cell cultures could not easily be dispersed into single cells. The results (see insets in Fig. 6) revealed that HS degradation was increased approximately 2-fold in cells that did not express APLP2 as compared with wild-type cells or APP-deficient cells (Fig. 6, compare I with G and H). These findings indicate that APLP2 is an important modulator of Gpc-1 autoprocessing in fibroblasts.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Autocatalyzed HS degradation in Gpc-1 is dependent on several factors and requirements. Firstly, the core protein of Gpc-1 has to be S-nitrosylated. This requires endogenous generation of nitrosonium cation (NO+), a supply of Cu(II) ions, and probably also the presence of residues in the HS chains (24–25). The copper ions are reduced to Cu(I) when the thiol anions are converted to SNO. Whereas free Cu(II) ions are scarce in cells (35), there is an exchangeable pool that is probably regulated by various copper transporters. In fibroblasts, the prion protein is an important supplier of Cu(II). In prion null fibroblasts, Gpc-1 is poorly S-nitrosylated but the addition of copper ions and NO-donor or ectopic expression of the prion protein in these cells restores it (26). Secondly, NO release from SNO must be triggered by some reducing agent, such as ascorbate. Thirdly, the reduction of NO to nitroxyl anion (NO-), which is the HS-cleaving agent, requires reoxidation of Cu(I) to Cu(II) or oxidation of SH groups.

In the brain, free Zn(II) ions may be relatively abundant especially during synaptic activity. Because zinc is redox-inert and appears to compete with Cu(II) for binding to Gpc-1, the level of stable S-nitrosylation in Gpc-1 would be low, resulting in reduced capacity for HS autocleavage. Zinc-supported Gpc-1 autoprocessing will cease when all available SH groups have been converted to disulfides. To maintain the cyclic process, an agent that reduces the disulfides is required (see Fig. 7). Ascorbate is unable to reduce disulfides (25, 26), but Cu(I)-containing cuproproteins or microsomal cytochromes are potential candidates.



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  		FIG. 7.
Model of Zn(II)- and NO-catalyzed autocleavage of Gpc-1 HS chains. Top left, GPI-linked Gpc-1 proteoglycan with SH groups in the globular part of the protein core (circle) that contains bound Zn(II) ion and is substituted with three HS chains (unbroken long thick lines). When exposed to NO, Gpc-1 is converted to an isoform containing both thiolate and SNO (top right). When subsequently exposed to ascorbate, Gpc-1 releases NO and Zn(II) ion, ascorbate is oxidized to dehydroascorbate, thiols are oxidized to disulfides, and NO- is formed (bottom right). Ensuing NO-catalyzed deaminative cleavage of HS chains in the same molecule generates molecular nitrogen and anMan-containing HS fragments (broken thick lines) and Gpc-1 with truncated HS chains (bottom left). To maintain the cyclic process, disulfides have to be reduced to thiols.

 
The limitations imposed by the presence of zinc ions could be overcome if a Cu(II)-to-Cu(I) redox cycle took place on another adjacent cuproprotein (Fig. 8, CuP). GPI-linked ceruloplasmin can fulfill this function in glia cells (27). The present results suggest that APP can serve in a similar function in N2a neuroblastoma cells where it colocalizes with Gpc-1 in perinuclear compartments. Thereby, APP can support NO-catalyzed HS degradation in the presence of both Cu(II) and Zn(II) ions. However, APLP2 appears not to support degradation.



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  		FIG. 8.
Model of possible regulatory roles for APP/APLP2 in Cu/Zn-NO-catalyzed and ascorbate-triggered Gpc-1 HS degradation. Top left, GPI-linked, Gpc-1 proteoglycan with SH groups, intact HS chains, and bound Zn(II). Upon exposure to NO, an S-nitrosylated Gpc-1 still containing Zn(II) is formed (top right). During this step, Cu(II) in an adjacent cuproprotein (CuP), which could be APP but not APLP2, is reduced to Cu(I). When SNO- and Zn(II)-containing Gpc-1 is exposed to ascorbate, NO is released and reduced to NO-, ascorbate is oxidized to dehydroascorbate, and Cu(I) is oxidized to Cu(II) (bottom right). NO- cleaves the HS chains generating HS oligosaccharides, a Gpc-1 with truncated HS chains and molecular nitrogen (bottom left). In this scenario, Zn(II) is not involved and remains bound to Gpc-1 throughout and the copper redox cycle takes place on an adjacent cuproprotein. In a mixed scenario where there is the release of zinc ion (Fig. 7), a Cu(I)-containing cuproprotein (CuP) may be involved in the reduction of the disulfide bonds. However, in the presence of APP-Cu(I) or APLP2-Cu(II), the autodegradative process should be retarded or inhibited. The site of action could be at the indicated step or at earlier steps. Inhibition of S-nitrosylation of Gpc-1 by APP/APLP2-Cu(I) or oxidation of NO- to nitrosonium cation (NO+) by APLP2-Cu(II) are possible inactivation mechanisms.

 
Furthermore, APP and APLP2 modulate the extent of HS degradation in Gpc-1. Although the APP-Cu(II) form supports Gpc-1 autoprocessing, the APP-Cu(I) form and both the Cu(II) and Cu(I) forms of APLP2 inhibit autoprocessing in vitro (Fig. 8). Supporting in vivo results show that, in the absence of APP, HS degradation is unregulated and anMan-containing HS oligosaccharides accumulate in the brain and in primary cultures of cortical neurons. In APLP2-deficient neurons, there is also an increased HS degradation. In confluent cultures of fibroblasts, the absence of APLP2 results in excessive NO-catalyzed HS degradation, whereas the lack of APP has no effect. The balance between activators and inhibitors of Gpc-1 autoprocessing as well as the redox state of copper ions in APP and APLP2 may be different in neurons and fibroblasts. Both APP and APLP2 may be inhibitors in neurons, whereas APP may be an activator in fibroblasts. Because APLP2 does not contain a Cu-binding A{beta} segment, we propose that the N-terminal metal-binding domain in APP/APLP2 (12–14) is involved in the modulation of Gpc-1 autoprocessing. The Cu-binding A{beta} region of APP may only be involved in the stimulatory effect, as A{beta} peptide itself also supports Gpc-1 autoprocessing (26).

To obtain information on the physiological function of APP and APLP, several laboratories have generated various combinations of single, double, or triple knock-out mice (11, 19, 36, 37 and references therein). Overall, the results indicate that APLP2 has the dominant physiological role because even APP-/-/APLP1-/-/APLP2+/- mice display postnatal lethality (36). Although APP-/- mice are viable, APP may serve an essential role in the maintenance of synaptic function during aging. However, no apparent molecular abnormalities have been observed so far in APP-/- and APLP2-/- mice with the exception of an accumulation of copper in cortical neurons (11, 37). The results of the present study identify excessive NO-catalyzed degradation of HS in neurons of APP and APLP2 knock-out mice.

The present observations indicate, for the first time, that there is a functional relationship between the heparin/HS and copper-binding activities of the cysteine-rich region in APP and APLP2 in their modulation of the nitroxyl anion-catalyzed HS degradation in Gpc-1. Based on previous structural studies, this region is believed to be composed of a separate heparin-binding/growth factor domain (19) and a copper-binding domain (15) joined by a linker. The former domain should connect APP to HS in Gpc-1, and the second domain should be involved in modulating the copper-dependent redox reactions required for NO-catalyzed HS degradation. APP-Cu(I) and APLP2-Cu(II)/Cu(I) inhibit the autodegradative process. Inhibition of Gpc-1 S-nitrosylation by APP/APLP2-Cu(I) or inhibition of HS degradation via oxidation of the cleaving agent NO- to the inactive NO+ by APLP2-Cu(II) is a possible inactivation mechanism.

It has been reported that GPI-linked proteins play a role both for {beta}-secretase cleavage of APP and for A{beta} secretion (38). Moreover, HS interacts directly with {beta}-secretase (39) and uptake of exogenously added {beta}-secretase ectodomain is mediated by APP (40). It appears likely that the common carrier is the GPI-linked Gpc-1, which is binding to both {beta}-secretase and APP.

To release NO and trigger HS degradation, a reducing agent is required. The physiological trigger has not been identified, but ascorbate is a possible candidate. Mice can synthesize ascorbate in extraneuronal tissues and transport it into the brain via a specific carrier protein. Ablation of the gene for this transporter results in perinatal death by respiratory failure and intraparenchymal brain hemorrhage (41). Moreover, L-gulono-{gamma}-lactone oxidase knock-out mice, which are unable to synthesize ascorbate, require dietary supplementation considerably above the corresponding level that would be required to prevent scurvy in humans (42). Hence, the specific requirement for ascorbate in the brain could be due to its role as a trigger of HS degradation.

The neurotoxicity associated with AD is either a result of the process leading to accumulation of A{beta} peptide or caused by the A{beta} oligomers themselves (4–7). Widespread damage caused by NO-derived peroxynitrite has been observed in brain tissue from cases of AD (43). Furthermore, an involvement of Gpc-1 in the pathogenesis of AD has been suggested by the results of several previous studies (for review, see Ref. 44). Studies from this laboratory have shown that HS degradation during Gpc-1 recycling is carried out both by non-enzymatic (NO) and enzymatic (heparanase) processes that are independent of one another (25). Hence, when the timing is disturbed, such that heparanase removes the HS chains from Gpc-1 before release of NO is triggered, "idle" nitroxyl anions will be formed. These can then be converted to harmful peroxynitrite. Hence, sparsely glycanated, recycling Gpc-1 could continually produce toxic nitroxyl anion. Conversely, if there is uncontrolled NO-catalyzed HS degradation, accumulation of anMan-containing HS oligosaccharides is also potentially hazardous. If not immediately subject to reduction by nonspecific aldose reductases, the aldehyde-containing anMan residues could easily react with amino groups in proteins.


   FOOTNOTES
 
* The work was supported by grants from the Swedish Science Council (VR-M), the Cancer Fund, the Alzheimerfonden, the Tegger, Kock, and Österlund Foundations, and the Medical Faculty of Lund University (to L.-Å. F. and K. M.) and in part by the National Health and Medical Research Council of Australia (to R. C. and C. L. M.) and the Deutsche Forschungsgemeinschaft (to G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Lund University, BMC C13, SE-221 84, Lund, Sweden. Tel.: 46-46-222-8573; Fax.: 46-46-222-3128; E-mail: lars-ake.fransson{at}medkem.lu.se.

1 The abbreviations used are: APP, amyloid precursor protein; AD, Alzheimer disease; anMan, anhydromannose; APLP2, amyloid precursor-like protein 2; , glucosamine with free amino group; Gpc-1, glypican-1; A{beta}, amyloid-{beta}; GPI, glycosylphosphatidylinositol; mAb, monoclonal antibody; HS, heparan sulfate; NO, nitric oxide; PG, proteoglycan; SNO, S-nitroso group; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Hui Zheng and Sam Sisodia for the APP and APLP2 knock-out mice, respectively. We also thank Prof. Catharina Svanborg (Department of Microbiology, Immunology, and Glycobiology, Lund University) for use of microscope facilities, Prof. Dick Heinegård of this department for use of cryostat, Prof. Guido David (University of Leuven, Leuven, Belgium) and Dr. Gunnar Peijler (Uppsala University, Uppsala, Sweden) for generous gifts of monoclonal antibodies, and Dr. Mattias Belting for advice. The technical assistance of Birgitta Havsmark is greatly appreciated. We thank the animal house staff of the Department of Pathology, The University of Melbourne, for their assistance in maintaining the mice.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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