Sambucus Sieboldiana Agglutinin Research Articles

In situ characterization of the agglutination of lectins via cross-linking of carbohydrates by time-resolved measurement of forward static light scattering.

We present real-time observations of a structurally variable process for cross-linking agglutination between multivalent lectins and glycoclusters using a small-angle forward static light scattering (F-SLS) technique. In this study, a cross-linking agglutination reaction was carried out using a tetravalent Neu5Acα2,6LacNAc-glycocluster and Sambucus sieboldiana agglutinin (SSA). The scattering intensity of time-resolved F-SLS increased with formation of the Neu5Acα2,6LacNAc-glycocluster-SSA cross-linked complex. Using this approach, fine sequential cross-linking agglutination between glycoclusters and lectins was observed in real-time. The rate of increase in the intensity of time-resolved F-SLS increased with the concentration of sialo-glycoclusters and SSA. Structural analysis based on the fractal dimension using time-resolved F-SLS patterns revealed that the density of the aggregates changed with progression of the cross-linking reaction until equilibrium was reached. This is the first report to evaluate the cross-linking agglutination reaction between glycoclusters and lectins and analysis of the subsequent structure of the obtained aggregates using time-resolved measurements of F-SLS.

Changes in Gastric Mucosal Glycosylation Before and After Helicobacter pylori Eradication Using Lectin Microarray Analysis.

Glycosylation is a common post-translational modification, and it has been reported that alterations in the glycosylation patterns on cells are related to cell proliferation, differentiation, tissue adhesion, and carcinogenesis. This study aimed to investigate the relationship between Helicobacter pylori infection and gastric mucosal glycosylation using a lectin microarray system. Gastric mucosal samples were obtained from 10 Helicobacter pylori-non-infected patients, 10 H. pylori-infected patients, and 10 after H. pylori-eradicated patients who underwent gastric mucosal biopsy by endoscopy in our institute. The gastric gland cells which were isolated from formalin-fixed, paraffin-embedded gastric mucosal biopsy samples using laser capture microdissection were used for lectin microarray to obtain lectin-glycan interaction values. Comparison of the lectin-glycan interaction values before and after eradication in the same patients showed significant increases for Ricinus communis agglutinin 120, Trichosanthes japonica agglutinin II, Euonymus europaeus lectin, jacalin, Amaranthus caudatus agglutinin, and Maclura pomifera agglutinin and significant decreases for Urtica dioica agglutinin, Lycopersicon esculentum lectin, Ulex europaeus agglutinin, Sambucus nigra agglutinin, Sambucus sieboldiana agglutinin, and Trichosanthes japonica agglutinin I. Furthermore, jacalin and MPA in the gastric antrum were significantly decreased with H. pylori infection compared with the without infection group and improved to the levels seen without infection as a result of eradication. Lycopersicon esculentum lectin, Sambucus nigra agglutinin, Sambucus sieboldiana agglutinin, and Trichosanthes japonica agglutinin I in the gastric body were significantly increased with H. pylori infection and improved to the level seen without infection as a result of eradication. H. pylori infection changes the lectin binding state which is related to various cancers on the gastric mucosal cell. Furthermore, those changes are reversible by H. pylori eradication.

Aqueous synthesis of sialylglycopeptide‐grafted glycopolymers with high affinity for the lectin and the influenza virus hemagglutinin

ABSTRACTGlycopolymers with pendant complex‐type sialyl N‐glycans containing heptapeptides, that is, sialylglycopeptides (SGPs), were synthesized using a water soluble polymer backbone bearing N‐hydroxysulfosuccinimidyl esters by post‐polymerization modification in water. Although SGP has three amino groups on the peptide chain, the substitution reaction occurs preferentially at the N‐terminus α‐amino group in the lysine residue onto the polymer side chain because the reactivity of such α‐amino group is higher than that of the ε‐amino group in the lysine residue under mild acidic aqueous condition. The resulting SGP‐grafted glycopolymers exhibited strong interaction with the lectin Sambucus sieboldiana agglutinin and the human influenza A virus hemagglutinin, with higher binding associate constant values than those of free saccharide according to quartz crystal microbalance analysis. © 2020 Wiley Periodicals, Inc. J. Polym. Sci. 2020, 58, 548–556

Simple and Rapid Detection of Glycoforms by "Lectin Inhibition" Assay.

Glycoforms are otherwise identical proteins with different glycosylation. A lectin, Sambucus sieboldiana agglutinin (SSA), specifically binds glycoforms having α2,6-sialyl residues. The binding is found to inhibit antigen-antibody reaction; e.g., SSA inhibits anti-transferrin antibody binding to α2,6-sialylated transferrin (Tf) (SSA inhibition). SSA inhibition is not observed with other Tf glycoforms, indicating that the inhibition is glycoform-specific. Here we describe the application of SSA inhibition to ELISA as a specific assay for quantifying α2,6-sialylated Tf.

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.

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.

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.

Two cases of placental mesenchymal dysplasia: Case reports

Two cases of placental mesenchymal dysplasia: Case reports

Lectin-dependent inhibition of antigen-antibody reaction: application for measuring 2,6-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.

Glycan profiling of endometrial cancers using lectin microarray

Cell surface glycans change during the process of malignant transformation. To characterize and distinguish endometrial cancer and endometrium, we performed glycan profiling using an emerging modern technology, lectin microarray analysis. The three cell lines, two from endometrial cancers [well-differentiated type (G1) and poorly differentiated type (G3)] and one from normal endometrium, were successfully categorized into three independent groups by 45 lectins. Furthermore, in cancer cells, a clear difference between G1 and G3 type was observed for the glycans recognized with six lectins, Ulex europaeus agglutinin I (UEA-I), Sambucus sieboldiana agglutinin (SSA), Sambucus nigra agglutinin (SNA), Trichosanthes japonica agglutinin I (TJA-I), Amaranthus caudatus agglutinin (ACA), and Bauhinia purpurea lectin (BPL). The lectin microarray analysis using G3 type tissues demonstrated that stage I and stage III or IV were distinguished depending on signal pattern of three lectins, Dolichos biflorus agglutinin (DBA), BPL, and ACA. In addition, the analysis of the glycans on the ovarian cancer cells showed that only anticancer drug-sensitive cell lines had almost no activities to specific three lectins. Glycan profiling by the lectin microarray may be used to assess the characteristics of tumors and potentially to predict the success of chemotherapy treatment.

Design and Synthesis of High-Avidity Tetravalent Glycoclusters as Probes for Sambucus sieboldiana Agglutinin and Characterization of their Binding Properties

We designed and synthesized three tetravalent sialo-glycoclusters that had different separations between the terminal sialic acids and the linking carboxy groups of the ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetate scaffold to serve as ligands for the sialic acid-binding lectin Sambucus sieboldiana agglutinin (SSA). The interaction between each glycocluster and SSA was characterized by hemagglutination inhibition, quantitative precipitation, and double-diffusion assays. For the precipitation assays, the precipitin curves indicated that the ligands and SSA bound in either a 1:1 or a 1:2 ratio, i.e., stoichiometrically. The strong interactions of these sialo-glycoclusters with SSA could be ascribed to a combination of multivalency and spacer effects. We also assessed the nature of the ligand-SSA complexes by isothermal titration calorimetry and dynamic light scattering. The results of those experiments indicated that formation of intermolecular complexes occurred at less than stoichiometric ratios of ligand to SSA concentrations and that, as the concentrations of the ligands increased, larger cross-linked aggregates formed. Large aggregates that were present concurrently with visible precipitation and with a particle size centered at ~600 to 800 nm were identified by dynamic light scattering.

Partial-filling affinity capillary electrophoresis of glycoprotein oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid

Partial-filling affinity capillary electrophoresis has been applied to the simultaneous analysis of interactions between glycoprotein oligosaccharides and certain plant lectins. A lectin solution and a mixture of glycoprotein-derived oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid were introduced to a neutrally coated capillary in this order, and separated by application of a negative voltage. Interaction of a lectin with each oligosaccharide in the mixture was observed as the specific retardation or dissipation of peaks, in addition to the size/charge separation of oligosaccharides by zone electrophoresis in the remainder (≈90%) of the capillary. The strength of the interaction with lectin was controlled by introducing an appropriate volume of lectin solution. Application of various specificities of lectins indicated characteristic migration profiles of the oligosaccharides. Moreover, sequential injection of four lectins ( Maachia amurensis mitogen, Sambucus sieboldiana agglutinin, Erythrina cristagalli agglutinin, Aleuria aurantia lectin) induced complete dissipation of complex-type oligosaccharides and enabled specific determination of the presence of high-mannose oligosaccharides without the interference or alteration of the electropherogram in porcine thyroglobulin. This method was also applied to determine the binding constants of ovalbumin-derived oligosaccharides to wheat germ agglutinin.

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.

Abstract 5568: Development of serum glycoproteomic profiling technology for the identification of pancreatic cancer biomarkers: simultaneous identification of glycosylation sites and site-specific quantification of glycan structure changes

Abstract Glycosylation is one of the most important and abundant post-translational modifications in nature. Glycoproteins play important roles during molecular and cellular recognition in development, growth, and cellular communication and in particular are involved in cancer progression. Glycoproteins have been used as therapeutic targets and biomarkers for cancer prognosis, diagnosis, and monitoring. As mass spectrometry-based systems biology begins to revolutionize our understanding of biomedical sciences, the ability to efficiently and comprehensively profile glycoproteins in biological samples of interest (body fluids, cell surface, etc.) is critical to many biological and clinical researchers. Here we report a new approach to identify carbohydrate-targeting serum biomarkers, termed isotopic glycosidase elution and labeling on lectin column chromatography (IGEL). It is based on glycan structure-specific enrichment of glycopeptides by lectin-column chromatography and site-directed tagging of N-glycosylation sites by 18O during the elution with N-glycosidase. The N-glycosidase elution in the presence of H218O results in the specific amino acid substitution, asparagine to 18O-asparatic acid residue, with 3-Da increase of the peptide molecular weight. The mass spectrometric identification of this 3-Da increase enables absolutely accurate determination of N-glycosylation sites in complicated samples like serum. Furthermore, the combination of IGEL with label-free quantification system Expressionist enabled us not only to identify N-glycosylation sites accurately but also to quantitatively compare glycan structures on each glycosylation site in a single LC/MS/MS analysis. We applied the IGEL method to the comparative analysis of 18 serum samples from 4 healthy controls, 4 chronic pancreatitis patients, 5 stage-I pancreatic cancer patients, and 5 stage-IV pancreatic cancer patients on the Sambucus sieboldiana agglutinin (SSA) lectin column recognizing alpha 2, 6-linked sialic acid residues. QSTAR-Elite tandem mass spectrometry analysis of SSA-IGEL purified serum samples determined 121 individual N-glycosylation sites captured by the SSA lectin. The further t-test and principal component analysis with Expressionist label-free quantification system revealed that the stage-I pancreatic cancer patients group was statistically distinguishable from normal and pancreatitis groups by the affinity changes of 12 glycosylation sites to SSA lectin. This fact indicated that these glycosylation sites should be potential carbohydrate-targeting biomarkers for pancreatic cancer. Thus our quantitative glycoproteomic technology could be extremely valuable for the identification of carbohydrate biomarkers from various body fluid specimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5568.

β-Galactoside α2,6-Sialyltransferase I Cleavage by BACE1 Enhances the Sialylation of Soluble Glycoproteins

BACE1 (beta-site amyloid precursor protein-cleaving enzyme-1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, amyloid beta-peptide, and has been implicated in triggering the pathogenesis of Alzheimer disease. We showed previously that BACE1 cleaves beta-galactoside alpha2,6-sialyltransferase I (ST6Gal I) to initiate its secretion, but it remained unclear how BACE1 affects the cellular level of alpha2,6-sialylation. Here, we found that BACE1 overexpression in Hep3B cells increased the sialylation of soluble secreted glycoproteins, but did not affect cell-surface sialylation. The sialylation of soluble glycoproteins was not increased by ST6Gal I overexpression alone, but was increased by co-overexpression of ST6Gal I and BACE1 or by expression of the soluble form of ST6Gal I, suggesting that soluble ST6Gal I produced by BACE1 plays, at least in part, a role in the sialylation of soluble glycoproteins. We also found that plasma glycoproteins from BACE1-deficient mice exhibited reduced levels of alpha2,6-sialylation compared with those from wild-type mice. We propose a novel regulatory mechanism in which cleavage and secretion of ST6Gal I enhance the sialylation of soluble glycoprotein substrates.

Elderberry Bark Lectins Evolved to Recognize Neu5Ac 2,6Gal/GalNAc Sequence from a Gal/GalNAc Binding Lectin Through the Substitution of Amino-Acid Residues Critical for the Binding to Sialic Acid

Bark lectins from the elderberry plants belonging to the genus Sambucus specifically bind to Neu5Acalpha2,6Gal/GalNAc sequence and have long been used for the analysis of sialoglycoconjugates that play important roles in many biological phenomena. However, molecular basis of such a unique carbohydrate binding specificity has not been understood. To answer these questions, we tried to identify the amino-acid residues in the Japanese elderberry bark lectin, Sambucus sieboldiana agglutinin that enabled the lectin to recognize sialic acid by using in silico docking simulation and site-directed mutagenesis. These studies showed that three amino-acid residues, S(197), A(233) and Q(234), in the C-terminal subdomain of SSA-B chain are critical for the binding to the sialic acid in Neu5Acalpha2,6Gal/GalNAc sequence. Replacement of one of these residues to the one in the corresponding position of ricin B-chain completely abolished the binding to a sialoglycoprotein, fetuin. Conserved presence of these amino acid residues in the corresponding sequences of two other elderberry lectins with similar binding specificity further supported the conclusion. These findings indicated that the replacement of the corresponding amino-acid residues in a putative Gal/GalNAc-specific ancestral lectin to these amino-acid residues generated the unique Neu5Acalpha2,6Gal/GalNAc-specific elderberry lectins in the course of molecular evolution.

Distribution of Sialoglycoconjugates in the Rat Cerebellum and Its Change with Aging

We examined the distribution of sialoglycoconjugates in the cerebellum of 9-week-old and 30-month-old rats using light microscopy and electron microscopy in combination with two lectins, Maackia amurensis lectin (MAL) for Sia(alpha)2-3Gal and Sambucus sieboldiana agglutinin (SSA) for Sia(alpha)2-6Gal. Each lectin showed characteristic staining patterns. In young adult rats, MAL stained a strongly granular layer, a weakly molecular layer, and the medullary lamina, while SSA more strongly stained the medullary lamina than the molecular and granular layers. After aging, different staining patterns were obtained. Intense SSA reactivity was observed in the granular layer and intense MAL reactivity was observed in the medullary lamina of the aged groups. The reactivity of Purkinje cells with MAL was downregulated in the aged rats. These results indicated that Sia(alpha)2-3Gal and Sia(alpha)2-6Gal were expressed in distinct regions of the rat cerebellum and that their expression patterns changed in the aged brain.

Alteration of sugar chains on α 1-acid glycoprotein secreted following cytokine stimulation of HuH-7 cells in vitro

In inflammation, hepatocytes secrete several proteins into serum, the so-called acute phase proteins. In addition to increased serum levels of α 1-acid glycoprotein (AGP), there are also changes in the composition of its sugar chains. To investigate the cytokine-stimulated alteration of sugar chains on AGP, we cultured the human hepatoma cell line HuH-7 cells in serum-free medium (IS-RPMI) with and without IL-1β and IL-6, and analyzed AGP secretion into the medium. AGP was increased during stimulation with both IL-1β and IL-6, although the effect of IL-1β was more pronounced. Lectin-binding assay for secreted AGP also indicated significant increases in the binding activities to Aleuria aurantia lectin (AAL), Sambucus sieboldiana agglutinin (SSA), and concanavalin-A (ConA). In particular, AAL binding activity increased with higher expression of the sialyl Lewis X (sLe X) antigen, NeuAc α2–3 Gal β1–4 (Fuc α1–3) GlcNAc-R. Thus, the increase in AGP fucosylation may be correlated with the increase of sLe X. The present results indicate that the serum-free culture of HuH-7 cells provides a useful model for investigating the secretion of proteins from hepatocytes, and the effects of cytokines on the changes in sugar chains of glycoproteins in vitro.

Up-regulation of the alpha2,6-sialyltransferase messenger ribonucleic acid increases glycoconjugates containing alpha2, 6-linked sialic acid residues in granulosa cells during follicular atresia of porcine ovaries.

The sugar chains in cellular glycoconjugates have many biological functions. Extensive morphological development and remodeling occur in the ovary of female animals. This caused us to study glycobiological characteristics of ovarian cells, particularly granulosa cells that undergo apoptosis during follicular atresia. The lectin Sambucus sieboldiana agglutinin (SSA) specific for Siaalpha2,6Gal/GalNAc showed positive staining for granulosa cells only in atretic follicles of porcine ovaries by lectin histochemistry. Lectin blot analysis for SSA demonstrated specific glycoproteins only in atretic follicles. Furthermore, we performed analysis of backbone structures of SSA-positive glycans carried by granulosa cell glycoproteins increased during atresia by glycosidase treatment. Most of these structures were Siaalpha2,6Galbeta1,4GlcNAc on complex-type N-glycans, suggesting that only ST6Gal I of four distinct alpha2,6-sialyltransferases catalyzes alpha2,6-sialic acid transfer in most of the increased glycoproteins of granulosa cells during follicular atresia. Reverse transcription-polymerase chain reaction analysis demonstrated that the expression of ST6Gal I mRNA was up-regulated in granulosa cells during atresia. These results suggested that the alteration of glycoconjugates by ST6Gal I in granulosa cells during atresia is involved in some processes of ovarian follicular atresia and granulosa cell apoptosis.

Identification of a major PAF acetylhydrolase in human serum/plasma as a 43 kDa glycoprotein containing about 9 kDa asparagine-conjugated sugar chain(s).

Platelet-activating factor (PAF) acetylhydrolase from human serum/plasma was identified on a polyvinylidene difluoride (PVDF) membrane by electroblotting proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme activity was detected in the 43 kDa region on the membrane as a decrease in the beta-radioluminescence of [3H]acetyl-PAF or by the convenient method for determining PAF acetylhydrolase activity (the TCA precipitation method). The enzyme activity on treatment with N-glycosidase F shifted to the 34 kDa region on the PVDF membrane. On the other hand, only one band was observed, corresponding to a molecular mass of 53 kDa, on analysis by SDS-PAGE with silver staining. Treatment of the 53 kDa protein with N-glycosidase F changed its molecular mass to 43 kDa (protein A). The NH2-terminal 32 amino acid sequence of protein A completely corresponds to that of the heterogenous enzyme with 54 amino acids deleted from the NH2 terminus reported by Tjoelker et al. (Nature 374, 549-553, 1995). Even after trypsin treatment of the N-glycosidase F-digested enzyme, its PAF-AH activity remained in the 34 kDa region, but the contaminating protein A disappeared, on the PVDF membrane. In addition, the majority of serum PAF-AH was retained on a Sambucus sieboldiana agglutinin (SSA)-agarose column and was eluted with the hapten sugar, lactose. These results indicate that PAF acetylhydrolase consisting of a 34 kDa protein and about 9 kDa asparagine-conjugated sugar chain(s) is a major enzyme in human serum/plasma.