Kyoka Hoshi

A unique N-glycan on human transferrin in CSF: a possible biomarker for iNPH

Idiopathic normal pressure hydrocephalus (iNPH) is an elderly dementia caused by abnormal metabolism in the cerebrospinal fluid (CSF). The tap test is the current basis for confirming iNPH, but it shows very low sensitivity, indicating that many patients who might be cured by a shunt operation will be missed. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we found two transferrin isoforms: one had a unique N-glycan (Tf-1) whereas the other had N-glycan similar to that of serum transferrin (Tf-2). Glycan analyses revealed that Tf-1 had branching (biantennary) asialo- and agalacto-complex type N-glycans (N-acetylglucosamine [GlcNAc]-terminated glycans), which carried bisecting β1,4-N-acetylglucosamine and core α1,6-fucose. To examine glycoform whether changes in iNPH, we introduced the Tf-2/Tf-1 ratio as a diagnostic index, which minimized blot-to-blot variations in measurement. The Tf-2/Tf-1 ratios of iNPH patients are significantly higher than those of controls (p = 0.0019) and Alzheimers patients (p = 0.0010). This suggests that the Tf-2/Tf-1 ratio could distinguish iNPH from Alzheimers disease, and possibly other dementias. In conclusion, glycoform analysis has diagnostic potential in neurological diseases.

High Throughput ELISAs to Measure a Unique Glycan on Transferrin in Cerebrospinal Fluid: A Possible Extension toward Alzheimer's Disease Biomarker Development.

We have established high-throughput lectin-antibody ELISAs to measure different glycans on transferrin (Tf) in cerebrospinal fluid (CSF) using lectins and an anti-transferrin antibody (TfAb). Lectin blot and precipitation analysis of CSF revealed that PVL (Psathyrella velutina lectin) bound an unique N-acetylglucosamine-terminated N-glycans on “CSF-type” Tf whereas SSA (Sambucus sieboldiana agglutinin) bound α2,6-N-acetylneuraminic acid-terminated N-glycans on “serum-type” Tf. PVL-TfAb ELISA of 0.5 μL CSF samples detected “CSF-type” Tf but not “serum-type” Tf whereas SSA-TfAb ELISA detected “serum-type” Tf but not “CSF-type” Tf, demonstrating the specificity of the lectin-TfAb ELISAs. In idiopathic normal pressure hydrocephalus (iNPH), a senile dementia associated with ventriculomegaly, amounts of the SSA-reactive Tf were significantly higher than in non-iNPH patients, indicating that Tf glycan analysis by the high-throughput lectin-TfAb ELISAs could become practical diagnostic tools for iNPH. The lectin-antibody ELISAs of CSF proteins might be useful for diagnosis of the other neurological diseases.

Evaluation of blood-brain barrier function by quotient alpha2 macroglobulin and its relationship with interleukin-6 and complement component 3 levels in neuropsychiatric systemic lupus erythematosus

Although quotient of alpha2 macroglobulin (Qα2MG) was previously reported to be useful for the evaluation of blood–brain barrier (BBB) function, it is not commonly used. We therefore evaluated BBB function among the various subsets of neuropsychiatric systemic lupus erythematosus (NPSLE) using quotient Q α2MG. Furthermore, we determined the correlation between Q α2MG and cerebrospinal (CSF) interleukin (IL)-6 level and quotient complement component 3 (Q C3). To determine intrathecal production of C3, the C3 index (Q C3/Q α2MG) was also calculated. Fifty-six patients with SLE were included in this study. Of these, 48 were diagnosed with NPSLE, consisting of 30 diffuse NPSLE patients (acute confusional state (ACS): n = 14, non-ACS: n = 16) and 18 patients with focal NPSLE. CSF IL-6 concentration, and paired serum and CSF levels of α2MG and C3, were measured by enzyme-linked immuno solvent assay (ELISA). The Q α2MG, Q C3, and C3 index were then calculated. Q α2MG, Q C3, and IL-6 concentrations in the CSF were significantly elevated in NPSLE compared with non-NPSLE. Among the subsets of NPSLE, significant increases in Q α2MG, CSF IL-6, and Q C3 were observed in ACS compared with non-ACS or focal NPSLE. There was a positive correlation between CSF IL-6 level and Q α2MG, as well as between Q C3 and Q α2MG, in diffuse NPSLE. There were no significant differences in C3 index between NPSLE and non-NPSLE, as well as among the subgroups of NPSLE. Our study suggests that BBB disruption is present in ACS, and elevated levels of IL-6 and C3 in CSF in diffuse NPSLE, especially in ACS, might result from their entry to the CSF from the systemic circulation through the damaged BBB, as well as increased intrathecal production. Furthermore, Q α2MG might be useful for the evaluation of BBB integrity.

A unique glycan-isoform of transferrin in cerebrospinal fluid: A potential diagnostic marker for neurological diseases

BACKGROUND Cerebrospinal fluid (CSF) is sequestered from blood by the blood-brain barrier and directly communicates with brain parenchymal interstitial fluid, leading to contain specific biomarkers of neurological diseases. SCOPE OF REVIEW CSF contains glycan isoforms of transferrin (Tf): one appears to be derived from the brain and the other from blood. MAJOR CONCLUSIONS CSF contains two glycan-isoforms; brain-type Tf and serum-type Tf. Glycan analysis and immunohistochemistry suggest that serum-type Tf having α2, 6sialylated glycans is derived from blood whereas brain-type Tf having GlcNAc-terminated glycans is derived from the choroid plexus, CSF producing tissue. The ratio of serum-type/brain-type Tf differentiates Alzheimers disease from idiopathic normal pressure hydrocephalus, which is an elderly dementia caused by abnormal metabolism of CSF. The ratios in Parkinsons disease (PD) patients were higher than those of controls and did not appear to be normally distributed. Indeed, detrended normal Quantile-Quantile plot analysis reveals the presence of an independent subgroup showing higher ratios in PD patients. The subgroup of PD shows higher levels of CSF α-synuclein than the rest, indicating that PD includes two subgroups, which differ in levels of brain-type Tf and α-synuclein. GENERAL SIGNIFICANCE Glycosylation in central nervous system appears to be unique. The unique glycan may be a tag for glycoprotein, which is biosynthesized in the central nervous system. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.

Lectin-dependent inhibition of antigen–antibody reaction: application for measuring α2,6-sialylated glycoforms of transferrin

We developed a high-throughput Enzyme-linked immunosorbent assay (ELISA) for measuring α2,6-sialylated transferrin (Tf), based on inhibition of anti-Tf antibody binding to α2,6-sialylated Tf by a lectin, Sambucus sieboldiana Agglutinin (SSA). The inhibition was not observed with other glycoforms, such as periodate-treated, sialidase-treated and sialidase/galactosidase-treated Tf, suggesting that the assay was glycoform specific. This finding was applied to an automated latex-agglutination immunoassay, using SSA lectin as an inhibitor (SSA-ALI). The concentration of α2,6-sialylated Tf measured by SSA-ALI in human cerebrospinal fluid was correlated with that of ELISA (r2 = 0.8554), previously developed for measuring α2,6-sialylated Tf.

Subgroup differences in 'brain-type' transferrin and α-synuclein in Parkinson's disease and multiple system atrophy.

Two transferrin (Tf) glycan-isoforms were previously found in cerebrospinal fluid (CSF); one appears to be derived from serum (Tf-2) and the other from choroid plexus, a CSF-producing tissue (Tf-1). To analyse metabolic differences associated with the two isoforms, their ratio (Tf-2/Tf-1) was defined as the Tf index. Here we report that Tf indices of patients with tauopathies including Alzheimers disease (2.29 + 0.64) were similar to those of neurological controls (2.07 + 0.87) (P = 0.147). In contrast, Tf indices with Parkinsons disease (PD, 3.38 ± 1.87) and multiple system atrophy (MSA, 3.15 ± 1.72) were higher than those of the controls (2.07 ± 0.87), the P-values being < 0.001 and 0.024, respectively. Tf indices of PD and MSA did not appear to be normally distributed. Indeed, detrended normal Quantile-Quantile plot analysis revealed the presence of an independent subgroup showing higher Tf indices in PD and MSA. The subgroup of PD showed higher levels of CSF α-synuclein (38.3 ± 17.8 ng/ml) than the rest (25.3 ± 11.3 ng/ml, P = 0.012). These results suggest that PD (and MSA) includes two subgroups, which show different metabolism of CSF transferrin and α-synuclein.

In situ visualization of a glycoform of transferrin: localization of α2,6-sialylated transferrin in the liver

We previously found that a lectin, Sambucus sieboldiana agglutinin (SSA), bound to α2,6-sialylated glycan epitopes on transferrin and inhibited anti-transferrin antibody binding to the antigen in ELISA (SSA inhibition). Here we report that SSA inhibition is applicable to immunohistochemistry, localizing α2,6-sialylated transferrin in the liver. Immunohistochemistry using anti-transferrin polyclonal antibody revealed that transferrin was detected in hepatocytes near interlobular veins. Addition of SSA lectin markedly attenuated the staining. Sialidase treatment of a liver section abolished SSA binding and concomitantly cancelled SSA inhibition, suggesting that SSA binding to glycan epitopes on the section was essential for the inhibition. To examine the importance of proximity between antigen epitopes and SSA-binding (glycosylation) sites, we prepared two anti-peptide antibodies against partial amino acid sequences of transferrin. One antibody (Tf-596Ab) is against a peptide sequence, Cys596-Ala614, which is proximal to N-glycosylation sites (Asn-432 and Asn-630). The other (Tf-120Ab) is against a peptide sequence, Val120-Cys137, distal to the sites. The staining signals of Tf-596Ab were reduced by the addition of SSA, whereas those of Tf-120Ab were reduced only a little. This result suggests that proximity of the antigen epitope to SSA binding sites is critical for SSA inhibition in immunohistochemistry.

Lectin inhibits antigen–antibody reaction in a glycoform‐specific manner: Application for detecting α2,6sialylated‐carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is a glycoprotein marker, which is widely used for diagnosing various cancers, especially colon adenocarcinoma. In addition, CEA mediates homotypic adhesion of colon adenocarcinoma cells, which appears to favor hematogenous metastasis. CEA carries α2,6sialyl residues on its N‐glycans whereas a normal counterpart, normal fecal antigen‐2, does α2,3sialyl residues, suggesting that cancer‐specific α2,6sialylation on CEA may play a role for cell invasion and metastasis. A simple and rapid estimation of α2,6sialyled CEA in detergent extracts from formalin‐fixed colon adenocarcinoma by “lectin inhibition” is reported. In the lectin inhibition method, Sambucus sieboldiana Agglutinin (SSA) lectin, an α2,6sialic acid binder, was used as a glycoform‐specific inhibitor for antigen–antibody reaction in ELISA. Detergent extracts from colon adenocarcinoma showed a fair amount of ELISA signal in the absence of SSA whereas the signal was markedly reduced (45≈74%) in the presence of SSA, suggesting that the extracts contains α2,6sialyled CEA. The presence of α2,6sialyled CEA in the extracts was confirmed by lectin microarray, in which SSA, Sambucus nigra agglutinin, and Trichosanthes japonica agglutinin I lectins were used as α2,6sialyl binders. Thus lectin inhibition is a simple and rapid method for detecting α2,6sialyled CEA even in crude detergent extracts from formalin‐fixed adenocarcinoma tissue.

Lectin-Based Assay for Glycoform-Specific Detection of α2,6-sialylated Transferrin and Carcinoembryonic Antigen in Tissue and Body Fluid

Antibodies are useful for detecting glycoprotein antigens, but a conventional antibody recognizes only a protein epitope rather than a glycan. Thus, glycan isoform detection generally requires time- and labor-consuming processes such as lectin affinity column chromatography followed by sandwich ELISA. We recently found antigen-antibody reactions that were inhibited by lectin binding to glycans on the glycoprotein antigen, leading to a convenient glycoform-specific assay. Indeed, Sambucus sieboldiana agglutinin (SSA) lectin, a binder to sialylα2,6galactose residue, inhibited antibody binding to α2,6-sialylated transferrin (Tf) (SSA inhibition). SSA inhibition was not observed with other glycoforms, such as periodate-treated, sialidase-treated and sialidase/galactosidase-treated Tf, suggesting that the assay was glycoform-specific. SSA inhibition was also applicable for visualizing localization of α2,6-sialylated-Tf in a liver section. This is the first immunohistochemical demonstration of glycoform localization in a tissue section. SSA inhibition was utilized for establishing ELISA to quantify α2,6-sialylated carcinoembryonic antigen (CEA), a marker for various cancers. In addition, α2,6-sialylated-CEA was visualized in a colonic adenocarcinoma section by SSA inhibition. The method would further be applicable to a simple and rapid estimation of other α2,6-sialylated glycoproteins and have a potential aid to histopathological diagnosis.