©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Chondroitin Sulfate Attachment Site of Appican Is Formed by Splicing Out Exon 15 of the Amyloid Precursor Gene (*)

Menelas N. Pangalos , Spiros Efthimiopoulos , Junichi Shioi , Nikolaos K. Robakis (§)

From the (1) Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, New York 10029

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Appicans are secreted and cell-associated chondroitin sulfate proteoglycans containing Alzheimer amyloid precursor (APP) as their core protein. Appicans are found in brain tissue, and in cell cultures their expression depends on both cell type and growth conditions. Here we report that the core protein of appicans derives from an APP mRNA lacking exon 15. Splicing out of this exon creates a new consensus sequence for the attachment of a chondroitin sulfate chain in the resulting APP product. Transfection of C6 glioma or 293 kidney fibroblast cells with APP cDNAs containing exon 15 produced no appican, while transfection with an APP cDNA lacking this exon induced high levels of appican production. Polymerase chain reactions indicated that appican-producing cells contained an APP mRNA species without exon 15, whereas cells without this mRNA produced no appican. Site-directed mutagenesis combined with immunoreactivity experiments showed that the chondroitin sulfate chain is attached to a serine residue 16 amino acids upstream of the amino terminus of the A sequence of APP. The attachment of a glycosaminoglycan chain close to the A sequence of APP may affect the proteolytic processing of APP and production of A. The proteoglycan nature of APP suggests that addition of the chondroitin sulfate glycosaminoglycan is important for the implementation of the biological function of these proteins.


INTRODUCTION

The amyloid precursor proteins (APPs)() are a family of type I transmembrane proteins involved in the development of Alzheimer disease (AD). They are the precursors of A peptide, which aggregates to form the amyloid depositions characteristic of AD pathology (1, 2, 3, 4) . The A sequence includes the last 28 extracytoplasmic residues and 11-15 amino acids of the adjacent transmembrane domain of APP. In addition, certain mutations of the APP gene seem sufficient for the induction of the AD phenotype (5, 6, 7) . The APP gene locus contains at least 18 exons, and several distinct APP mRNA species have been detected as a result of alternative exon splicing. Certain APP isoforms contain a 56-amino acid insert with high sequence homology to the Kunitz-type serine protease inhibitors (KPI), encoded by a single exon (8) . Additional APP mRNA species have also been identified where exon 14 is fused to exon 16 by splicing out the 54-bp exon 15 of the APP gene (L-APP (9) ). APPs are extensively modified post-translationally, a process that may affect both the production of A and the biological function of these proteins. Secreted APP, containing almost all of the extracytoplasmic sequence but without the transmembrane and cytoplasmic regions, is produced when membrane full-length APP is cleaved by the unidentified enzyme(s) secretase (for review see Ref. 10).

Chondroitin sulfate proteoglycans are a class of molecules consisting of a core protein to which one or more chondroitin sulfate (CS) glycosaminoglycan (GAG) chains are covalently attached. The CS GAG chains are bound via a xylose sugar to a serine residue of the core protein. Although the structural characteristics that determine the attachment site of a CS GAG chain are poorly understood, the serine residue part of the consensus sequence Ser-Gly, usually preceded by acidic amino acids, is the most commonly found site for CS attachment (11, 12) . Due to the large size of the GAG chains, proteoglycans can be considerably larger than their core proteins. These macromolecules are postulated to be involved in a number of key cellular events (for review see Ref. 12), including neuroprotection from excitotoxic agents (13) , and in brain they have been suggested to modulate cell adhesion, axonal growth, and neural patterning (14, 15) . Proteoglycans, including heparan, dermatan, and chondroitin sulfates, have been found in and around senile plaques and neurofibrillary tangles and have been suggested to play a role in the pathogenesis of AD (16, 17, 18) .

Protein sequencing and immunoreactivity studies showed that a novel CSPG secreted by C6 glioma cells and migrating on SDS-PAGE between 140 and 250 kDa had a KPI-containing 120-kDa secreted APP as its core protein (19) . This secreted CSPG is derived from a membrane-bound cellular full-length precursor, after the core APP is cleaved by secretase (20) . In cell cultures, expression of the APP proteoglycans (appicans) is cell-specific and depends on the growth conditions (21) . Appicans are found in the brain and in rat brain primary cultures are produced by glia, but not neurons (26) . Recent evidence suggested that the CS chain of APP is attached near to or within the A sequence of APP, but the resolution of the methodology used did not allow a distinction between these two possibilities (21) . In this study we present evidence that the core protein of appicans is derived from the translation of an APP mRNA missing exon 15 and that the CS GAG chain is attached to a serine residue located 16 amino acids upstream of the A sequence of APP.


EXPERIMENTAL PROCEDURES

Cell Cultures and Treatments

Rat C6 glioma (C6), mouse neuro-2a neuroblastoma (N2a), human SY5Y neuroblastoma (SY5Y), and human 293 embryonic kidney fibroblast (293) cells were obtained from the ATCC cell bank. Cultures were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum (JRH Biosciences). Medium was collected for analysis after 2-3 days of confluent cell growth. Cultures were maintained at 37 °C, as described (19, 21) .

Metabolic labeling and immunoprecipitation of media from semi-confluent C6 cells was performed as described previously (20) . Chondroitinase ABC or AC digestions and treatment of the resulting digests were performed as described (21) . SDS-electrophoresis, immunoblotting, and quantitation of the obtained signals by densitometry using a computerized Magiscan Joyce Lobel image analysis system were performed as described (19, 21) .

Antibodies

For APP detection, the following antisera and monoclonal antibodies were used (APP amino acid (aa) positions are based on APP): GID specific for aa 179-185 (22) , R7 specific for aa 296-315 of the KPI insert (23) , 5 specific for aa 444-592 of human APP (Athena Neurosciences), R3 and monoclonal antibody 1G6 specific for aa 628-652 (21) , R47 specific for aa 652-667 (19) (see also Fig. 1 C).


Figure 1: Immunoprecipitation of appican by different antibodies. A, C6 cultures were labeled with [S]sulfate for 22 h (see ``Experimental Procedures''). Conditioned medium was collected and heat-treated in the presence of 0.1% SDS, and 2.0 10 trichloroacetic acid-precipitable counts were immunoprecipitated with anti-APP antibodies GID ( lane1), R7 ( lane2), R47 ( lane3), R3 ( lane4) and 1G6 ( lane5). Samples were separated by SDS-PAGE and fluorography. Exposure times to x-ray film were adjusted to give similar APP signals with the different antibodies. Arrowhead represents unmodified APP. B, chondroitinase digestion was performed before immunoprecipitation to show disappearance of the appican smear. Lane1, minus chondroitinase; lane2, plus chondroitinase. Arrowhead and arrow represent unmodified APP and unmodified APP plus appican core protein, respectively. Samples were immunoprecipitated with R47 antiserum. Numbers at the right of A represent mobilities of molecular mass markers in kDa. C, diagrammatic representation of the APP regions recognized by the antibodies used in the present study.



Plasmid Construction and Transfections

APP, APP, and a KPI-containing L-APP (KL-APP) cDNA were subcloned into pRc-CMV between restriction sites HindIII and XbaI.

A construct, KL-APPSA, was engineered with serine 619, 16 amino acids upstream of A, mutated to an alanine. Briefly, the KL-APP/pRc-CMV plasmid was partially digested with HindIII and BglII, and a 6089-bp fragment (A) containing 3` KL-APP sequences and the intact pRc-CMV sequence was isolated. KL-APP/pRc-CMV was also digested with HindIII and BsmI to isolate a 1930-bp fragment product (B) containing 5` KL-APP sequences. Two synthetic APP oligonucleotides were made, APP-S2 (nucleotides 1931-2007) and APP-AS1 (antisense nucleotides 1929-2011), in which nucleotide 1978 was changed from T to G (nucleotide numbering based on KL-APP). These were then phosphorylated and annealed to form a double-stranded sticky end product (C). Fragments A, B, and C were ligated to give construct KL-APPSA/pRc-CMV and subsequently cloned and sequenced (Sequenase 2.0 kit, U. S. Biochemical Corp.) to confirm the mutation.

C6 and 293 cells were either stably transfected with CsCl-purified APP, APP, or KL-APP DNA (pRc-CMV) or transiently transfected with KL-APPSA/pRc-CMV, using the DOTAP protocol according to manufacturer's instructions (Boehringer Mannheim). Transfectants were selected and maintained in 400 µg/ml Geneticin (Life Technologies, Inc.).

Isolation of RNA, Reverse Transcription and Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated from C6, 293, N2a, and SY5Y as described (24) . Ten µg of total RNA from each cell line was reverse transcribed, using Moloney murine leukemia virus reverse transcriptase (250 units, U. S. Biochemical Corp.) and oligo(dT) as a primer (100-µl reaction volume) to make first strand cDNA. Three µl of each cDNA reaction mixture was used for amplification by PCR with Taq polymerase (2.5 units, Stratagene) using APP-specific primers 5`-TGTGATTTATGAGCGCA-3` (nucleotides 1672-1688) and 5`-GCCGTTCTGCTGCATCTTGGA-3` (nucleotides 2315-2335). The reaction volume of 100 µl contained 50 pmol of primers and 2.5 mM of each dNTP (Pharmacia Biotech Inc.) and was overlaid with 100 µl of mineral oil. Reactions were for 30 cycles: 0.5 min at 92 °C, 1 min at 48 °C, and 3 min at 72 °C. PCR products were separated on a 4% non-denaturing polyacrylamide gel and visualized by ethidium bromide staining.


RESULTS AND DISCUSSION

Immunoprecipitation of Appican with Various Antibodies

Recent studies using Western blotting (21) suggested that the CS GAG chain(s) of appican was attached proximal to or within the A sequence of APP, either at Ser-637 located 16 residues upstream from the N terminus of A or at Ser-660, which corresponds to A residue 8 (numbering according to APP). Each of these serines is part of a Ser-Gly sequence. To further characterize the appican APP core protein and to determine the exact site of attachment of the CS GAG chain, conditioned media from SO-labeled C6 cell cultures were immunoprecipitated using various antibodies. GID antibodies that recognize all APP isoforms and R7 antibodies specific to the KPI-containing APP (K-APP) immunoprecipitated approximately equal amounts of appican, in agreement with earlier results suggesting that most of the C6 appican derives from K-APP (19, 20, 21) . In addition, both of these antibodies immunoprecipitated the secreted unmodified APP (Fig. 1 A, lanes1 and 2). Both GID and R7 antibodies recognize the appican core protein after chondroitinase digestion (19, 21) . R47 antisera specific to K-APP sequence 653-668, which includes Ser-660, also immunoprecipitated appican, although less efficiently than GID (Fig. 1 A, lane3). R47 recognized the core APP produced after chondroitinase treatment of appican (Fig. 1 B, lane2). In contrast, neither monoclonal antibody 1G6 nor polyclonal R3, both specific to K-APP sequence 628-657, which includes serine 637, was able to immunoprecipitate appican although they recognized the unmodified secreted APP (Fig. 1 A, lanes4 and 5). In addition, these antibodies failed to recognize the appican core protein produced after chondroitinase treatment (data not shown). This failure may be due to the interference from the disaccharide stub that remains attached to the core protein after chondroitinase digestion of appican (19) . Taken together, these data suggested, although did not prove, that Ser-637 rather than Ser-660 was the major site for attachment of the CS GAG chain of appican.

Close inspection of the SDS-PAGE mobility of the appican core APP produced after chondroitinase digestion indicated that this protein migrated slightly faster than the secreted unmodified K-APP (Fig. 1 B), suggesting that these two proteins may differ in structure.

Transfection of Cells with APP Isoforms

To further define the APP isoform containing the CS chain(s), C6 cells that produce appican endogenously (19) or 293 cells that produce no endogenous appican (21) were stably transfected with the human cDNAs encoding APP, K-APP, or K-APP without exon 15 (KL-APP). Possible involvement of the latter isoform as an appican core protein was suggested by the observation that fusion of exon 14 to exon 16 results in the formation of the consensus sequence Glu-Xaa-Ser-Gly (Fig. 2 C), where Ser-619, corresponding to Ser-637 of K-APP, becomes an excellent candidate for the attachment of the CS chain (11, 12) . Absence of the 18 amino acids encoding exon 15 from the appican core protein was also consistent with the slightly faster mobility exhibited by this protein compared with the secreted unmodified K-APP (Fig. 1 B).


Figure 2: APP and appican overexpression in transfected C6 and 293 cells. Media from untransfected and transfected ( A) rat C6 or ( B) human 293 cells were collected after 3 days of confluent culture and separated by SDS-PAGE prior to immunoblotting with antibody 5. Volumes loaded were adjusted to account for differences in total cellular protein present in each culture, as determined by BCA assay of cell homogenates. Lane1, untransfected cells; lanes2 and 3, cells transfected with KL-APP; lane4, cells transfected with K-APP; and lane5, cells transfected with APP. Medium collected from cells transfected with KL-APP was digested with chondroitinase ABC ( lane3). Numbers on the right represent mobilities of molecular mass markers in kDa. C, splicing out of exon 15 results in fusion of exon 14 to exon 16 and formation of the consensus sequence EXSG for the attachment of a CS chain to the serine. This site is 16 residues upstream of the A sequence of APP. Openarrow indicates amino acid position of possible CS chain attachment



Colonies overexpressing each APP isoform were cultured in the presence of 10% fetal bovine serum for 2-3 days, and conditioned media were collected and analyzed by SDS-PAGE and immunoblotting. Appican or APP was detected with 5 antisera, specific to human APP (25) . Following stable transfection of C6 or 293 cells with either APP or APP cDNA, increased APP expression was clearly seen in both transfectants with no increase in appican secretion (Fig. 2 A, lanes4 and 5). At least 10 independent clones of each transfection were analyzed with identical results. In contrast, C6 cells transfected with the KL-APP cDNA showed a marked increase in the secretion of the 140-250-kDa diffuse staining characteristic of appican (Fig. 2 A, lane2) (see also Refs. 19-21). Similarly, 293 cells, which do not produce endogenous appican, secreted high amounts of appican into the media upon transfection with KL-APP (Fig. 2 B, lane2).

Treatment of media samples with chondroitinase ABC eliminated the appican staining, with a concomitant increase in the human KL-APP core protein (Fig. 2, A and B, lane3). Treatment with chondroitinase AC yielded similar results, showing that the GAG chains attached to the KL-APP core protein consist of chondroitin sulfate and not dermatan sulfate (data not shown; see also Ref. 19). Densitometric quantitation of the relative amounts of the transfected KL-APP core protein before and after chondroitinase treatment indicated that in all C6 transfected cell cultures more than 90% of the total secreted KL-APP was in the CSPG form. In contrast, in 293 cells only about 40-50% of the expressed KL-APP was in the PG form.

APP mRNA Expression in Different Cell Lines as Determined by RT-PCR

To examine whether there is any relationship between appican production and expression of APP mRNA lacking exon 15, we performed polymerase chain reactions using reverse-transcribed RNA from different cell lines. Of these cell lines, C6 and N2a synthesize appican endogenously, whereas SY5Y and 293 do not (21) . First strand cDNA was prepared from total RNA of these cultures, and PCR was then performed on these cDNA preparations using APP-specific primers, flanking exon 15 (see ``Experimental Procedures''). Under these conditions, the predicted PCR product derived from APP cDNA containing the 54-bp exon 15 is a transcript of 664 bp, while the predicted PCR product derived from APP cDNA without exon 15 is a transcript of 610 bp. Plasmids K-APP/pRc-CMV and KL-APP/pRc-CMV were used as controls (Fig. 3). The cDNA samples from the SY5Y neuroblastoma and 293 fibroblast cultures yielded a single PCR transcript of 660 bp, equal in size to the transcript derived from the K-APP plasmid DNA. In contrast, cDNA samples from C6 and N2a cells produced two PCR transcripts, one equal in size to that produced from the K-APP plasmid DNA and an additional smaller transcript of about 610 bp, identical in size to that obtained from the KL-APP plasmid DNA (Fig. 3). To ensure that exon 15 was absent from the lower molecular weight PCR product, we cloned and sequenced this transcript from C6 cells. The obtained sequence confirmed both the APP nature of the transcripts and the absence of exon 15 from the smaller transcript (data not shown). These data show that cells producing L-APP mRNA also produce appican, while cells that do not produce L-APP mRNA produce no appican. Combined with the immunoprecipitation and transfection experiments, these observations suggested that 1) the core protein of appican is the translation product of an APP mRNA lacking exon 15 and 2) Ser-619 of KL-APP (corresponding to Ser-637 of K-APP) is the attachment site of the CS GAG chain.


Figure 3: RT-PCR analysis of APP mRNA isoform expression in transformed cell lines. RT-PCR products were separated by 4% non-denaturing PAGE and analyzed by ethidium bromide staining. APP specific primers used flanked exon 15 APP DNA sequences. Products are shown from C6 glioma, SY5Y neuroblastoma, N2a neuroblastoma, and 293 fibroblasts. RT-PCR was also performed on control samples of K-APP/pRc-CMV (APP) and KL-APP/pRc-CMV (L-APP). Numbers on the right represent mobilities of molecular mass markers from phage digested with BstE II in bp.



Transfection of C6 and 293 Cells with Mutated KL-APP to Identify CS Attachment Site

To test the hypothesis that Ser-619 of KL-APP is the CS attachment site (Fig. 2 C), C6 and 293 cells were transfected with a mutated KL-APP cDNA, in which the T residue at coding position 1977 was substituted with a G (see ``Experimental Procedures''). The translation product of this mutated mRNA, designated KL-APPSA, will contain an alanine residue, instead of a serine, at position 619. Since this is the postulated GAG attachment site, the effect of such a mutation should be the inhibition of appican production. Indeed, Fig. 4 shows that although C6 and 293 cells transfected with KL-APP cDNA secreted appican, which appeared as the characteristic heterogenous staining between 140 and 250 kDa, transfection of these cells with KL-APPSA resulted in the secretion of only a homogenous 120-kDa molecule that was insensitive to chondroitinase ABC treatment. These results further confirm that Ser-619 of KL-APP, located 16 residues N-terminal to A sequence, is the primary attachment site of the CS GAG chain of appicans.


Figure 4: Site-directed mutagenesis of Ser-619 to alanine in KL-APP prevents CS chain attachment. C6 ( lanes1-4) and 293 cells ( lanes5-8) were transfected with either KL-APP ( lanes1, 2 and 5, 6) or KL-APPSA ( lanes 3, 4 and 7, 8). Medium was collected after 3 days of confluent culture, incubated in the presence (+) or absence (-) of chondroitinase ABC, and separated by SDS-PAGE prior to immunoblotting with human APP specific antibody 5. Numbers on the right represent mobilities of molecular mass markers in kDa.



Taken together, our results indicate the core proteins of appicans lack exon 15 of the APP gene. Splicing out of this exon creates a sequence -Glu-Gly-Ser-Gly-, which constitutes an excellent consensus sequence for the attachment of the CS chain (Fig. 2 C). The absence of appican production in cells transfected with KL-APPSA suggests that appicans contain only one CS chain. These data, as well as earlier data (19, 20, 21, 26) , suggest that most of the appican core proteins are derived from the K-APP isoforms, although isoforms missing both the KPI insert and exon 15 may also occur as appicans. However, the percent of APP that receives a CS chain appears to be dependent on the cell type. Thus, in C6 cells more than 90% of the produced transfected KL-APP was in appican form compared with only 40-50% in 293 cells. In addition, Chinese hamster ovary cells transfected with KL-APP contained 50-60% of the produced protein as appican.() Interestingly, leucocytes, where L-APPs without exon 15 were first described (9) , produced little or no appican.()

Appicans are CSPGs found in brain, and in vitro they are produced endogenously by neural cells (21, 26) . CSPGs have been suggested to have important functions in the central nervous system, including cell adhesion, guidance of growing axons (14, 15, 27) , and participation in the healing process following brain injury (28) . Interestingly, CSPGs have been found in both senile plaques and neurofibrillary tangles, suggesting that these molecules are involved in the pathology of AD (18) . The detection of appicans suggests that APP has additional biological functions deriving from its proteoglycan nature. Some of these functions may be involved in the development of AD. Recent evidence indicates that brain appican is produced by astrocytes (26) . Because these cells are found associated with neuritic plaques, and participate in the formation of brain scars following neuronal injury, astrocytic appican may well be involved in the development of these pathological structures. Furthermore, since proteoglycans have also been shown to bind A (29, 30) , appicans may interact with A in ways that result in either inhibition or stimulation of A aggregation. In addition, the attachment of the GAG chains close to the A sequence of APP could alter its proteolytic processing and thus affect production of A.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AG08200 and AG05138. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Tel.: 212-241-9380; Fax: 212-831-1947.

The abbreviations used are: APP, amyloid precursor protein; AD, Alzheimer disease; CSPG, chondroitin sulfate proteoglycan; CS, chondroitin sulfate; GAG, glycosaminoglycan; PG, proteoglycan; KPI, Kunitz-type protease inhibitor; aa, amino acids; RT, reverse transcription; PCR, polymerase chain reaction; CMV, cytomegalovirus; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).

M. Pangalos and N. K. Robakis, unpublished observations.

J. Shioi and N. K. Robakis, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Claudia Schmauss and Kai Liu for help with RNA preparation and PCR techniques, Dr. Kevin Felsenstein for providing plasmid KL-APP/pRc-CMV, and Dr. Ivan Lieberburg for providing antibody 5.


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