Concanavalin Agglutinin Levels are Decreased in Peripheral Blood of Alzheimer’s Disease Patients

Abstract

Alzheimer’s disease (AD) seriously threatens patients’ lives and causes severe burden to the families. Early prevention and treatment can alleviate the development of the disease; therefore it is important to find new effective and non-traumatic biomarkers for early diagnosis. In this study, peripheral blood samples were collected from 24 AD patients and the same number of age- and gender-matched control subjects. Lectin reactive glycosylation levels including beta-D-galactosyl ricinus communis agglutinin 120 (RCA), peanut agglutinin (PNA), concanavalin agglutinin (Con A), alpha-L-fucosyl ulex europeus agglutinin (UEA), dolichos biflorus agglutinin (DBA), and galanthus nivalis (GNL), in the red blood cells of peripheral blood were examined by western blotting. We found that lectin levels were altered with aging and gender; some lectin levels were different between AD patients and the control subjects. Only Con A levels were significantly decreased in AD patients compared to age-matched control subjects. These results suggest that Con A levels in peripheral blood may be a potent diagnostic marker for AD.

Keywords

Alzheimer’s disease Con A glycoproteins lectin peripheral blood

INTRODUCTION

Alzheimer’s disease (AD) is the most frequent cause of dementia characterized by a progressive decline in cognitive function [1]. The key neuropathological hallmarks in AD brain are diffuse and neuritic extracellular amyloid plaques and intracellular neurofibrillary tangles [2 –4]. The new guidelines by the National Institute on Aging and the Alzheimer’s Association conceive AD as a three-staged disease process, including a preclinical stage, a stage of mild cognitive impairment (MCI), and the final stage of dementia [5]. AD is an irreversible disease, which is a serious threat to patients’ lives and severe burden to the families. Early prevention and treatment can alleviate the development of the disease, so it is crucial to find effective biomarkers for early diagnosis. Amyloid-β (Aβ) and tau are biomarkers in the cerebrospinal fluid (CSF) that correlate well with AD. Although they predict the onset of memory-related symptoms, there is no clear correlation with other symptoms of AD. Moreover, to obtain these markers, an invasive procedure is required [6]. MRI techniques are able to single out some groups of patients who are highly likely to develop into full-blown AD, but it is applicable only in a very small subset of the whole AD population. As a result, looking for noninvasive and effective diagnostic markers for AD is important for prevention and therapy of the disease.

Extensive data, which are available from epidemiological studies, suggest that insulin dysfunction is associated with an increased relative risk for AD. Many other studies have shown that glucose utilization is disrupted in AD brain and precedes the appearance of clinical symptoms [7]. Impaired glucose metabolism was also observed in the brains of early onset AD patients, known as MCI, and AD transgenic mice [8, 9]. We recently found that the deregulation of glucose metabolism leads to the generation of advanced glycation endproducts and induces Alzheimer-like tau pathology and memory deficits [10]. Furthermore, glucose uptake metabolism disorder also induces abnormal protein glycosylation [11].

Protein glycosylation, which is involved in the processes of proper protein folding, protein anchoring to cell membranes, and the delivery of proteins to organelles, is one of the most common post-translational modifications of proteins in eukaryotes. In AD patients, the above processes of protein folding, protein anchoring to cell membranes, and the delivery of proteins to organelles are impaired. Many studies indicated that protein glycosylation was altered in AD [12, 13]. Several proteins which are critical to AD pathogenesis, e.g., AβPP and β-secretase, are both modified by glycosylation, which suggests that glycosylation modification may play a role in the production of Aβ [14, 15]. Tau in AD brain is aberrantly glycosylated in addition to being hyperphosphorylated [16]. The cytoplasmic addition of N-acetylglucosamine (GlcNAc) to serine/threonine residues (O-GlcNAc) has been to shown to regulate tau phosphorylation levels. More interestingly, the aberrant tau glycosylation appears to precede and promote the abnormal tau hyperphosphorylation [17], which suggests that glycosylation may play a role in the formation and/or maintenance of neurofibrillary tangles [18]. Glycosylation of acetylcholinesterase (AChE) was altered in AD frontal cortex, but not in cerebellum, and associated with an increase in the proportion of Globular amphiphilic dimeric (G2a) and monomeric (G1a) isoforms, which may reflect the abnormal pattern of AChE glycosylation in AD CSF [19]. All of these studies imply that protein glycosylation plays an important role in the pathophysiology of AD.

Some researchers measured the levels of glycoproteins in CSF and found that wheat germ agglutinin (WGA) binding glycoproteins were significantly lower in the patients of AD than the controls [20]. It is known that lectins are abundant in peripheral blood. In order to identify potential diagnostic biomarkers for AD, six lectins, namely ricinus communis agglutinin (RCA), peanut agglutinin (PNA), concanavalin agglutinin (Con A), ulex europeus agglutinin (UEA), dolichos biflorus agglutinin (DBA) [21], and galanthus nivalis lectin (GNL), were used to recognize glycoproteins, which were selectively binding with the whole sugar molecule, or part of the sugar molecule through glycosidic linkages in the blood [22] from AD patients and age-matched controls. We found that lectin levels were altered with aging and gender and that only Con A levels significantly decreased in AD patients compared with age-matched subjects. The results suggest that Con A levels in peripheral blood may be a new potent diagnostic marker for AD.

MATERIALS AND METHODS

Blood samples were collected from patients treated in the Union Hospital, affiliated hospital of Tongji Medical College, Huazhong University of Science and Technology. The patients were clinically diagnosed with AD and were matched by age and gender to a control subject (Table 1). Diabetes mellitus was considered an exclusion criterion. After the samples were collected, they were quickly subjected to centrifugation at 60× g for 15 min at 4°C. Then, the supernatant was discarded, and the substratum as red blood cells was collected. Each component was stored at –80°C before use. The samples were grouped by age and gender, and detected only using the samples coming from the red blood cells section, as lectins were abundant in red blood cells.

Methods

The antibodies against lectins used in the study recognized the following residues: UEA, RCA, Con A, PNA, GNL, and DBA. All the antibodies were obtained from Vector Laboratories (Burlingame, CA). The majority of lectins listed above are present in AD brain and peripheral blood [23].

Western blotting

The section of red blood cells was mixed with sample buffer (3:1, v/v) containing 200 mM Tris-HCl (pH 7.6), 8% SDS, 40% glycerol, 40 mM dithiothreitol, boiled for 10 min, and then centrifuged at 12,000 g for 10 min at 25°C. The supernatant was stored at –80°C for western blot analysis. The total protein levels were adjusted to 1μg/μL using BCA protein assay kit. The proteins underwent electrophoresis in 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and the separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were then blocked with 5% nonfat milk dissolved in TBSTween-20 (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.2% Tween-20) for 1 h and probed with a primary antibody at 4°C overnight. The blots were then incubated with horseradish peroxidase streptavidin-conjugated second antibody (1:1000) from Vector Laboratories for 1 h at 37°C, and visualized with enhanced chemiluminescence. The protein bands were quantitatively analyzed using Kodak Digital Science 1D software (Eastman Kodak Company, New Haven, CT).

Statistical analysis

Data are presented as mean ± SE and analyzed using SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA). Statistical significance was determined by one-way ANOVA followed by LSD’s post hoc test or Student’s t-test. Significance was assessed as p < 0.05.

RESULTS

Table 1 shows the distribution of AD patients, and female patients and the controls were divided into three groups by age: ≤65 years, 65–75 years, and ≥75 years, each group with 4 people; male patients were divided into ≤80 years (n = 3), 80–85 years (n = 5), and ≥85 years (n = 4). Each patient was matched by age and gender to a control subject. The general cognitive function of each patient was assessed using Mini-Mental State Examination (MMSE) score. Diagnoses were made by a neurologist, who performed extensive behavioral, neuropsychological, and neuroimaging assessments. The AD patients were diagnosed according to the criteria of National Institute of Neurological and Communicative Diseases and Stroke/Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) [7]. The AD patients selected for our study include early, moderate, and severe stages of AD; the MMSE score is 12.3 ± 8.7. The control group consisted of patients who presented to the clinic with subjective complaints, underwent exactly the same diagnostic work-up as the AD patients, and were not diagnosed as AD or MCI. All participants with diabetes mellitus were excluded. Western blotting was used to detect the lectin protein levels of UEA, RCA, Con A, PNA, GNL, and DBA in the red blood cells samples. Figures 1 and 2 show the blots of UEA, RCA, Con A, PNA, GNL, and DBA protein in the samples of AD patients and controls, respectively. The prominent lectin-reactive glycoproteins with apparent molecular masses were chosen for quantitative analysis. For UEA, RCA, Con A, and DBA, the apparent band was around 130 kDa, but for PNA, the band was around 90 kDa, and GNL was about 150 kDa. The different molecular weights of proteins may be due to the lectins binding with different kind of proteins. It was not very easy to differentiate the exact proteins unless lectin-affinity chromatography and mass spectrum were used.

AD is an age-related neurodegenerative disorder characterized by gradual loss of normal functions [24]. Hence we detected whether the glycoproteins binding to the lectin levels were changed with aging in both female AD patients and control subjects; the relative lectin levels were expressed as the intensity which were normalized using the blots of the ≤65-year-old control group. Firstly, we found that the UEA level was remarkably decreased in the control participants (≥75 years old). However, in AD patients, UEA first showed an increase in the patients aged 65–75 years, then a decrease in the ≥75 years old patients compared with the patients aged ≤65 years. RCA level was decreased in the 65–75-year-old and ≥75-year-old compared with the ≤65-year-old control subjects. We also observed that RCA level was decreased in AD patients aged ≤65 years compared with age-matched control subjects. Con A level showed no significant change with aging neither in control subjects nor AD patients, while it was significantly decreased in AD patients compared with age-matched control subjects. PNA level in the ≥75 years group in control subjects also represented a decline. GNL level was decreased in the ≥75-year-old both in the control and AD subjects. No significant change of DBA level was observed in control subjects or AD patients (Figs. 1 –3).

In male subjects, the relative intensity of lectin levels was normalized using the blots of the ≤80-year-old control group. UEA level was remarkably decreased with aging in AD patients; it also showed a decline in the subjects 80–85 or ≥85 years old in AD compared to the age-matched control subjects. RCA level was decreased in ≥85 years old AD patients compared with those in AD patients aged 80–85 years old. Con A level was increased in the 80–85 or ≥85 years old group compared with ≤80-year-olds in control subjects. More importantly, it was decreased in AD patients aged ≤80 years, 80–85 years, and ≥85 years compared to those of the age-matched control subjects. PNA level was reduced only in the control group aged ≥85 years. GNL and DBA levels were not affected in male subjects either in control subject or AD patients (Figs. 1, 2, 4).

Lastly, we analyzed the lectin levels in total female and male control subjects and AD patients. We found that only Con A levels were decreased in AD patients compared to the controls in both female and male groups, while other lectin levels had no significant difference between AD patients and the control subjects (Figs. 5 and 6). All the results suggested that Con A level in peripheral blood may be a potential diagnostic marker forAD.

DISCUSSION

Protein glycosylation occurs in two general forms, N-linked glycosylation and O-linked glycosylation. N-linked glycosylation occurs in the endomembrane system and has a canonical serine/threonine (Ser/Thr)-X-asparagine (Asn) motif to which glycans are added to the Asn residue [25]. However, O-linked glycosylation sites do not have a well-characterized motif for glycan addition [26]. Decreased tau O-GlcNAcylation, which is the addition of carbohydrates to Ser or Thr residues, causes abnormal hyperphosphorylation of tau, which is probably induced by deficient brain glucose uptake metabolism in AD and other tauopathies [27]. Another hallmark of AD, three major isoforms of AβPP, derived by alternative splicing, contain 695, 751, and 770 amino acids that are heavily O-glycosylated and contain two N-linked glycosylation sites [28, 29]. An unusual glycosylation of AChE is also observed in both brains and CSF of AD patients [30]. The activity of sialyltransferase, which catalyzes the sialylation of glycoproteins, was reported to be down-regulated in AD brain [31]. These results indicated that protein glycosylation may be involved in ADpathogenesis.

Lectins, as powerful tools for distinguishing differences in the carbohydrate composition of glycoproteins [32, 33], have been incorporated into AD glycoproteomics studies including Con A, WGA, PNA, and Datura stramonium agglutinin. Con A has high affinity a-linked mannosyl and terminal glucosyl carbohydrate moieties that are the primary components of glycans associated with N-linked glycosylation [34]. However, it is important to note that Con A has a significant hydrophobic binding domain and has been shown to bind proteins that do not fit the criteria of canonical N-linked glycosylation [35, 36]. WGA binds to GlcNAc and sialic acid residues that are commonly added to proteins during O-linked glycosylation [37]. PNA is specific for terminal galactosyl residues, while Datura stramonium agglutinin targets β-linked GlcNAc that are found in N-linked glycans [38].

The levels of many WGA-reactive glycoproteins were reported to decrease in AD CSF. This decrease in staining is specific for WGA, as there was no significant difference between AD and non-AD when CSF glycoproteins are stained with several other lectins (Con A, RCA, and Lens culinaris agglutinin). As far as we know, little was reported about alteration of lectin reactive glycoproteins in peripheral blood. In the present study, western blotting was used to assess lectin levels in red blood cells. Six antibodies against lectins were applied in the research, the molecular weight of the apparent band of Con A, UEA, or RCA was around 130 kDa, indicating an identical protein may be affected in the three lectins.

We further subdivided the patients by age and gender, and the results showed that lectin levels were altered with aging and gender; some lectin levels were different between AD patients and control subjects. With aging, UEA, RCA, PNA, and GNL level remarkably decreased in the female control participants; only UEA and PNA levels decreased in the male control subjects. Female GNL level, and male UEA and RCA levels were decreased in AD patients with aging. DBA levels were not affected in male subjects either in the controls or AD patients. Con A level showed no significant change with aging neither in control subjects nor AD patients, while it was significantly decreased in AD patients compared to age-matched control subjects. These results showed that glycoprotein level was influenced not only in AD progression but also in normal aging, and Con A level in the red cells of peripheral blood may be a potential diagnostic marker for AD.

Con A was reported to bind with multiple functional proteins, e.g., glutamate dehydrogenase, glia fibrillary acidic protein, tropomyosin 3, Rab GDP-dissociation inhibitor XAP-4 (XAP4), and heat shock protein 90, in AD hippocampus. Among these proteins, XAP4 and HSP90 levels decreased; α-enolase, γ-enolase, and XAP-4 increased in AD inferior parietal lobule compared with normal controls [36]. In the present study, the exact protein that binds to Con A in the peripheral blood waits for further study. A variety of chromatographic techniques such as immunoaffinity, lectin affinity, multiple affinity removal system depletion, strong cation exchange, and reverse phase high-performance liquid chromatography may be helpful to solve this problem.

In conclusion, we found that Con A levels were decreased in AD patients compared to the control, which suggests that Con A level in peripheral blood may be a potent protein marker for diagnosis of AD.

Footnotes

ACKNOWLEDGMENTS

This work was supported by the grants from National Natural Science Foundation of China (No: 81303252, 81401047 and 81271402), and Program for New Century Excellent Talents in University (NCET-11-0175).

Authors’ disclosures available online ().

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