Yonago Acta medica 2008;51:1–9

Alteration of Concanavalin A Binding Glycoproteins in
Cerebro­spinal Fluid and Serum of Alzheimer’s Disease Patients
Yuki Hashimoto, Miyako Taniguchi, Miki Kitaura, Yuka Nakamura, Daiki Jimbo and
Katsuya Urakami
Department of Biological Regulation, School of Health Science, Tottori University Faculty of Medicine, Yonago 683-8503 Japan

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly. It is characterized pathologically by the formation of senile plaques and neurofibrilly tangles in thebrain. Diagnostic markers for detecting earlier stages of AD are needed. We measured
the intensity of concanavalin A (Con A) binding activities of glycoproteins of the cerebrospinal fluid (CSF) and serum of subjects to clarify the modification of core mannose since
we expected that aberrant glycosylation of glycoproteins might be useful as a new biomarker for detecting AD. CSF samples were collected from 15 patients with probable AD(AD group), 5 patients with probable dementia with Lewy bodies (DLB) (DLB group) and
8 controls without dementia (control group), whereas serum samples from 20 patients withprobable AD and 20 controls without dementia were also collected. Glycoproteins in the
CSF and serum were detected by lectin blotting using Con A. In the CSF of the AD group,
2 Con A binding glycoproteins were significantly higher compared with the control group.
Furthermore, using analysis of variance, 3 Con A binding glycoproteins detected fromthe CSF of the AD group showed significant differences among the 3 groups. The levelsof 3 Con A binding glycoproteins were significantly lower than in non-dementia controls
in the serum. These changes in Con A binding activities did not depend on the amount ofproteins. Therefore, the data indicate that the aberrance of protein glycosylation relates tothe pathology of AD, and has some promise as a new biomarker for the diagnosis of AD.
Key words: Alzheimer’s disease; concanavalin A; glycoprotein; glycosylation

Recently, donepezil hydrochloride, an inhibitor of acetylcholinesterase, was created forthe treatment of AD (Yamanishi et al., 1998). It
is used as a treatment for mild to moderate AD
patients in Japan (Homma et al., 2000), and it’s effectiveness in mild cognitive impairment, whichis the prodromal stage of AD, has been reported(Salloway et al., 2004). Clinical diagnosis of AD
is based on a clinical interview of the patient’s
medical history, neuropsychiatric examinations andneuroimaging studies. However, there is a matter

Alzheimer’s disease (AD) is an age-related dementia characterised pathologically by the appearanceof senile plaques composed primarily of aggregated forms of -amyloid protein and neurofibrillarytangles in the brain (Glenner and Wong, 1984;
Grundke-Iqbal et al., 1986). Since the 1990’s, AD
has become the most common cause of dementia
in Japan (Urakami et al., 1998). AD is not only a
medical care issue but also a social issue, becauseof the rapid aging of the population in Japan andother countries.

Abbreviations: AD, Alzheimer’s disease; Con A, concanavalin A; CSF, cerebrospinal fluid; DLB, dementia with Lewy
bodies1

Y. Hashimoto et al.

and Shaanan, 1997). Concanavalin A (Con A) is
one of the lectins that targets the core mannose(Ohyama et al., 1985). We examined the alteration of core mannose modification by measuring
Con A binding activities of glycoproteins in the
CSF and serum. In this study, we showed thedifference in glycosylation between the CSF andserum, and the ability of a new biomarker of AD.

of objectivity in current diagnostic criteria for AD.
Therefore, specific biological diagnostic markers
for detecting earlier stage of AD and specific distinction of AD are becoming more important.
The need to improve diagnostic accuracy
has prompted the search for diagnostic markers in
the cerebrospinal fluid (CSF) or serum of persons
with AD. To date, no single biochemical marker
has been found to yield sufficient sensitivity and/or specificity of detection. Nevertheless, the combination of multiple biomarkers such as tau, -amyloid 1-42 and -40 (Galasko et al., 1998; Kanai etal., 1998) may improve diagnostic accuracy. Additionally, there are new possibile biomarkers forthe diagnosis of AD such as apolipoprotein A-1,
cathepsin D, hemopexin, transthyretin and 2 pigment epithelium-derived factor isoforms (Abdi etal., 2006; Castano et al., 2006). Identification of
new biological markers, which can be used alone,
or in combination with other markers is highly
necessary.
Among the more than 100 types of posttranslational modifications, glycosylation is themost common. N-type glycosylation on asparagines and O-type glycosylation on serine or
threonine occur in over 50% of all proteins (Varki
et al., 1999). Altered glycosylation is reported
in several disease states (Lis and Sharon, 1986;
Collinge et al., 1996) and in AD (Ahmed andThornalley, 2002). Glycosylation of proteins withglucose and other saccharide derivatives leads tothe formation of fructosamines and advanced glycation endproducts (Day et al., 1979; Furth, 1988).
Formation of the endproducts has been linked
to the development of AD (Dukic-Stefanovic etal., 2001). Additionally, neural sialyltransferase,
which catalyses the addition of terminal sialic
acid onto newly synthesized proteins following
removal of core mannose sugars (Paulson andColley, 1989; Harduin-Lepers et al., 1995), is decreased in the brains of AD patients (Maguire andBreen, 1995).
Lectins are powerful tools for distinguishing differences in the carbohydrate compositionof glycoproteins (Lis and Sharon, 1986; Elgavish

Materials and Methods
Subjects and samples
Before collecting samples, informed consent was
obtained from the patients and their families, andethical investigation was approved. All subjects
were examined by a dementia specialist (KU).
Then, interviews of medical history, neuropsychiatric examinations and neuroimaging studies,such as computed tomography and/or magnetic
resonance imaging, single photon emission computed tomography were carried out for the diagnosis. AD diagnosis was done according to the
Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (American Psychiatric Association, 1994) and the National Institute of Neurological Communicative Disorders and Stroke
and the Alzheimer Disease and Related Disorders
Association Criteria (McKhann et al., 1984). Dementia with Lewy bodies (DLB) is characterized
by persistent visual hallucinations, fluctuating
cognitive impairment and parkinsonisms, andtypical senile dementia. The diagnosis for DLB
patients was done using the diagnosis in the consensus guidelines for the clinical and pathologic
diagnosis of DLB (McKeith, 2006). Non-dementia control subjects had no cognitive impairments
or diseases of the nervous system, yet may have
had some other diseases not related to dementia.
All samples were immediately frozen immediately after collecting until experimentation.
CSF samples were collected from 15 patientswith probable AD, 5 patients with probable DLB and
8 non-dementia controls (Table 1). The AD group2

Alteration of Con A binding glycoproteins in AD

consisted of 8 males and 7 females (mean age ± SD,
73.8 ± 5.2 years), the DLB group, 3 males and 2
females (77.2 ± 3.4 years) and the control group, 3
males and 5 females (73.3 ± 8.5 years).
Serum samples were collected from 20 probable AD patients and 20 non-dementia controls(Table 2). The AD group was made of 7 males
and 13 females (79.2 ± 6.8 years), and the control
group, 4 males and 16 females (71.6 ± 12.0 years).
This study did not use the same subject sample
between the CSF and serum.

Table 1. Characteristics of CSF samples
Gender		
(M/F)
Age*

Group
AD
[15]
DLB		[ 5]Control		[ 8]

8/7
3/2
3/5

Status73.8 ± 5.2 Probable
77.2 ± 3.4 Probable73.3 ± 8.5 No cognitive impairments
and diseases of the NS[ ], number of subjects.
* Mean ± SD.
AD, Alzheimer’s disease; CSF, cerebrospinal fluid; DLB,
dementia with Lewy bodies; F, female; M, male; NS, nervous system.

Table 2. Characteristics of serum samples

Pretreatment of samples
The quantity of total protein included in the CSF
and serum were determined by absorptiometry
using Protein Assay Kit II (Bio-Rad, Hercules,
CA), and albumin was eliminated by a Montage
Albumin Deplete Kit (Millipore, Billerica, MA).
Then, these were immediately stored at –80˚C until analysis.

Group

Gender		
(M/F)
Age*

StatusAD		[20] 7/13 79.2 ± 6.8 Probable
Control		[20] 4/16 71.6 ±12.0 No cognitive impairments
and diseases of the NS[ ], number of subjects.
* Mean ± SD.
AD, Alzheimer’s disease; F, female; M, male; NS, nervous
system.

Lectin blotting

Japan) for 1 h at room temperature with shaking.
The blocked membranes were then rinsed with
0.1% Tween-20 in phosphate-buffered saline, andeach was washed 3 times for 5 min with shaking
at room temperature. Then, the membranes wereincubated with 2 μg/mL Con A labeled biotin (JOil Mills, Tokyo, Japan) overnight at 4˚C. Finally, biotinylated Con A binding glycoproteins werereacted with Streptavidin-Conjugated Alkaline
Phosphatase (1:1000 dilution) (Promega, Madison,
WI) for 2 h at 25˚C and visualized with WesternBlue (Promega). The Con A binding activities ofeach band were analyzed by Multi Gauge version
2.1 software (Fuji Film, Tokyo). The differenceof staining of each membrane was compensatedby the background intensity of area took from theoriginal intensity of glycoprotein band.

We applied the technique of lectin blotting (Savage
et al., 1992) for detection of Con A binding glycoproteins. Each sample containing total proteinof 5 μg was mixed with a sample buffer to a final
concentration of 12 mM Tris-HCl (pH 6.8), 25%
glycerol, 2% SDS, 14.4 mM 2-mercaptoethanoland 0.1% bromophenol blue, and boiled in a boiling bath for 5 min. The mixtures and molecularweight markers (GE Healthcare, Buckinghamshire, United Kingdom) were separated by 5%
to 20% SDS-PAGE in a running buffer (pH 8.3)
containing 25 mM Tris, 192 mM glycine and0.1% SDS. SDS-PAGE was performed by 20
mA/56 cm 2 (area) and 1 mm (thickness) for 90
min. Separated proteins were electrophoretically transferred onto a polyvinylidene difluoride
membrane (Millipore) previously activated intoa transfer buffer containing 16 mM Tris, 120
mM glycine and 10% methanol at 100 mA for 1
h. The membrane was blocked in a Block Ace
Solution (Dainippon Sumitomo Pharma, Osaka,

Coomassie brilliant blue staining
Every protein in the SDS-PAGE gel was stainedwith coomassie brilliant blue solution containing3

Y. Hashimoto et al.

50% methanol, 0.5 mg/mL coomassie brilliant
blue and 10% acetic acid for 20 min at room temperature with shaking. Also, the gel had its color
removed with 25% ethanol and 8% acetic acid atroom temperature with shaking until positive protein bands appeared.

Results
Lectin blotting
We detected some Con A binding glycoproteins in
the CSF by lectin blotting, and tentatively named
them a (over 250 kDa) to g (about 25 kDa) in descending order of molecular weight (Fig. 1). Con
A binding activities of the 4 glycoproteins in the
CSF are shown in Fig. 2.
Figures 2-I to -IV indicate intensities of Con
A binding activities of CSF glycoproteins. The
levels of Con A binding activities of glycoproteinsa (6,477 ± 2,520) (mean ± SD) and g (9,650 ±5,557) from the CSF in the AD group were significantly higher than those of a (3,978 ± 2,133) andg (4,779 ± 1,746) in the control group (P < 0.05).
Con A binding glycoprotein a had 88.89% sensitivity and 66.67% specificity (cut off 5026) and g
had 77.78% sensitivity and 73.77% specificity (cutoff 5783) between the AD group and the control
group. Additionally, the Con A binding activityof glycoprotein b (between 250 and 160 kDa) in
the AD group was also higher than that of the
DLB group (P < 0.05) and that had 100.0% sensitivity and 66.67% specificity (cut off 7303) between those 2 groups, but there was no significant
difference between the AD and control groups.
The intensity of f (between 35 and 30 kDa) and gin the DLB group were significantly higher thanin the control group (P < 0.01) and those Con A
binding glycoproteins had 100.0% sensitivity and100.0% specificity (cut off 8010: f, 9503: g). The
intensities of CSF Con A binding glycoprotein b,f and g had significant difference among 3 groups(P < 0.05, analysis of variance).
We performed lectin blotting of Con A binding glycoproteins in the serum, and called A(between 250 and 160 kDa) to J (about 25 kDa)
in descending order of molecular weight (Fig. 3).
Figure 4 shows intensities of Con A binding activities of glycoproteins in the serum. In the AD
group, the Con A binding intensity of glycoprotein H (between 50 and 35 kDa) (6,234 ± 2,692),

Silver staining
The amount of CSF protein was lower than theserum protein, and the sensitivity of protein detection in coomassie brilliant blue staining was lower
than that of silver staining. We could not visualize the protein of CSF in coomassie brilliant blue
staining, and so we performed silver staining of
the protein in the gel. The gel was swamped into
50% methanol for 10 min at room temperature
with shaking then washed with distilled water for
10 min at room temperature. Sodium thiosulfate(0.02%) was used for intensifying the reaction for
3 min, followed by incubation with silver nitratesolution containing 1 mg/mL silver nitrate in
0.34% sodium hydroxide and 1.7% ammonia for
20 min at room temperature with shaking. Then,each gel was washed 4 times with distilled waterfor 10 min at room temperature, and developedwith 2% sodium carbonate in 0.004% formaldehyde at room temperature with shaking. To stop
the reaction, 7% acetic acid solution was used for
10 min at room temperature.
Statistical analysis and receiver operating
characteristic analysis
Intensities of Con A binding activities of glycoproteins were compared with Mann-Whitney’s U-test:
among the AD, the DLB and control groups for
the CSF; and between the AD and control groups
for the serum. We also compared the levels of
CSF Con A binding glycoproteins among the 3groups with analysis of variance. Con A binding
glycoproteins which showed significant differences by statistical analysis in the CSF and serum
were analyzed by receiver operating characteristic
analysis for indicating sensitivity and specificity.
4

Alteration of Con A binding glycoproteins in AD

Fig. 1. Banding patterns of concanavalin A (Con A) reactive glycoproteins in the cerebrospinal fluid (CSF) detectedby lectin blotting with Con A. Glycoproteins analyzed in this study (black arrow heads) are tentatively named a to g.
Lane M: molecular markers; AD, Alzheimer’s disease patients; control, non-dementia control subjects.

Fig. 2. Concanavalin A (Con A) binding activities of the cerebrospinal fluid (CSF) glycoprotein a, b, f and g determined by densitometry are seen in 2-I, -II, -III and IV, respectively. Shown are intensities of each samples. *P < 0.05,**P < 0.01: comparison between the Alzheimer’s disease (AD) group and the dementia with Lewy bodies (DLB)
group or control group, or both (Mann-Whitney’s U-test). [ ], number of subjects.

I (about 30 kDa) (5,765 ± 2,290) and J (about 25
kDa) (5291 ± 2089) were significantly lower thanthe control group: 8,851 ± 3,470 (P < 0.05), 8,817± 2,581 (P < 0.001), 7,564 ± 2,235 (P < 0.01),
respectively. Additionally, Con A binding glycoproteins H, I and J had 70.00% sensitivity and60.00% specificity (cut off 6454), 80.00% sensitivity and 75.00% specificity (cut off 6311) and
85.00% sensitivity and 65.00% specificity (cut off5657). Other Con A binding glycoproteins had
no significant differences between AD and othergroups (data not shown).
5

Y. Hashimoto et al.

Fig. 3. Banding patterns of concanavalin A (Con A) reactive glycoproteins in the serum detected by lectinblotting with Con A. Glycoproteins analyzed in this study (black arrow heads) are tentatively named A to J.
Lane M: molecular markers; AD, Alzheimer’s disease patients; control, non-dementia control subjects.

*
Semiquantification of glycoproteins in the
CSF and serum
We also analyzed protein levels of CSF and serum
glycoproteins which showed significant intergroupdifferences with lectin blotting. As proteins canbe stained directly with coomassie brilliant blue
or silver staining, we could compare the amountof glycoprotein. Differences in protein levels of
CSF Con A binding glycoproteins a, b, f and gwere not significant between groups which hadsignificant differences in Con A binding activities. Differences in the amount of coomasie brilliant blue-stained serum glycoproteins H, I and J
were not significant between the AD and control
groups.

*****Discussion
In AD patients, some Con A binding glycoproteins were altered in the CSF and serum. These
data indicate that the CSF Con A binding glycoproteins in a and g show the separation between
the AD group and the control group. However, inboth CSF and serum, glycoproteins with different
Con A binding activities were not distinguishedby the amount. From this we figured that the al-

Fig. 4. Concanavalin A (Con A) binding activities ofserum glycoproteins H, I and J determined by densitometry are seen in 4-I, -II and -III respectively. Shown areintensities of each samples. *P < 0.05, **P < 0.01, ***P
< 0.001: comparison between the Alzheimer’s disease(AD) group and control group (Mann-Whitney’s U-test).
[ ], number of subjects.

6Alteration of Con A binding glycoproteins in AD

Various molecular weights of Con A binding
glycoproteins from the CSF such as a, b, f and gwere found to have the potential of an aberrant
sugar chain. The change was found in Con A
binding glycoproteins with both large and small
molecular weights in the AD group and/or DLB
group but not in the control group in the CSF,
which is probably caused by a variation in glycosylation. However, altered Con A binding activities of serum glycoproteins were observed in glycoproteins with less than 40 kDa. In the serum of
AD patients, aberrant glycosylation suggested by
Con A binding activity was limited to the glycoproteins with smaller molecular weights.
The central nervous system is isolated fromthe blood circulation by the blood-brain barrier
and blood-cerebrospinal fluid barrier because of
its physiological importance. These barriers havebeen thought to protect the brain from various
harmful materials in the circulation as static walls(Segal, 2000; Begley and Brightman, 2003; Nitta
et al., 2003). In addition, membrane proteinspromote cell membrane permeability of a low
molecular weight compound, called a transporter,and these membrane proteins have been thought
to relate to the active efflux of harmful materials
from the brain (Kusuhara and Sugiyama, 2001;
Lee et al., 2001; Sun et al., 2003; Begley, 2004).
Therefore, we considered that aberrant glycosylation of glycoproteins with a relatively large molecular weight which was directly or secondarily
generated could not be excreted into the circulating blood because of the blood-brain barrier
and/or blood-cerebrospinal fluid barrier: theseglycoproteins might have been excreted into othermetabolic pathways instead of those. On theother hand, relatively smaller glycoproteins could
be passed through the blood-brain barrier and/or
blood-cerebrospinal fluid barrier into the circulating blood. Consequently, a metabolic pathway using blood flow might exist for those glycoproteinsas one possible pathway.
In the future, it will be necessary to identify
the glycoproteins altered in AD. However, wefound that glycosylation of Con A binding gly-

teration was due to a sugar chain rather than to theamount of glycoprotein. Additionally, the alteration of the sugar chain specifically occurring at the
mannose site was the reason for using Con A lectin in this study. Recently, there have been some
reports which suggest the relationship between
glycosylation and AD. An unusual glycoform ofacetylcholinesterase, which does not bind to Con A,
is increased in the AD brain and CSF (Sáez-Valero
et al., 1999, 2000). Wheat germ agglutinin-binding glycoproteins are decreased in the CSF of AD
patients (Fodero et al., 2001), and there is a relationship between aberrant glycosylation and thepathology of AD (Ahmed and Thornalley, 2002),
while several glycoproteins are altered in AD(Puchades et al., 2003). These results support ourfindings and suggest that a sugar chain of glycoproteins is altered in AD. Moreover, it was possible to distinguish glycoproteins f and g in the DLB
group from the control group. The glycoprotein bin the CSF might be separated the AD group fromthe DLB group. The CSF glycoproteins b, f andg indicated aberrant glycosylation also occur in
DLB, and this fact suggests that alteration of mannose glycosylation of Con A binding glycoprotein
might be a new biomarker in not only AD but also
other dementia such as DLB.
On the other hand, we found that there wasno significant difference in CSF glycoprotein dusing lectin blotting with Con A in this study,
although wheat germ agglutinin-binding activity was significantly lower than in the AD groupin previous study (data not shown). Wheat germ
agglutinin specifically binds to -N-acetylglucosamine and terminal sialic acid (Nagata andBurger, 1974; Monsigny et al., 1980). These
results suggested aberrances in glycosylation at-N-acetylglucosamine and/or sialic acid sites,
but not at the mannose site. In this way, using
some lectins in the same glycoprotein might beable to clarify the site of altered sugar chain.
In AD, aberrant glycosylation of glycoproteins was found ubiquitously in the CSF, but thosefound in the serum were limited to the glycoproteins with relatively smaller molecular weights.
7

Y. Hashimoto et al.

coproteins in the AD group was different in both
CSF and serum. Alteration of Con A binding activities might especially have some use as a new
biomarker in the serum. On the other hand, thisstudy has some difficult issues such as increasingof respective samples, identification of molecularweight of each Con A binding glycoprotein andinvestigation of DLB or other non-AD dementia inthe serum. In the immediate future, those problems will be solved and clarifying the mechanismof variant glycosylation in AD might contributenot only to the discovery of a new biomarker but
also to the resolution of a new pathology.

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Acknowledgments: We thank Dr. Kenji Wada, Dr.
Yosuke Wakutani and Dr. Kenji Nakashima of the Department of Neurology, Institute of Neurological Sciences, Tottori University Faculty of Medicine for providing
us the CSF and serum samples. We also would like tothank Mr. Tim Wiltshire, Tottori University Faculty of
Medicine for checking the English of this article.

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Received July 25, 2007; accepted January 4, 2008
Corresponding author: Yuki Hashimoto

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