Con A affinity glycoproteomics of normal human liver tissue

In order to establish the novel high throughput, high efficiency and low cost technological platform for the research of N-glycoproteomics, to resolve the significance of characteristic expression profile of glycoprotein and to find the proteins with biological functional importance, the glycoproteins with high-mannose core and the two antennary types were purified and enriched by the Con A affinity chromatography. Con A affinity protein expression profiles of normal human liver tissue were generated by using SDS-PAGE, two-dimensional electrophoresis (2-DE) followed by fast fluorescence staining based on multiplexed proteomics (MP) technology. 301 visible protein spots on the gel were detected and 85 of glycoproteins were further successfully identified via peptide mass fingerprinting (PMF) by a matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS) and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The glycosylation sites were predicted with NetNGlyc 1.0 and NetOGlyc 3.1 software, meanwhile they were classified according to the geneontology methods. The construction of Con A affinity glycoprotein database of normal human liver tissue would contribute to the subsequent research.     

Identification of a breast tumor-associated orosomucoid by concanavalin A affinity chromatography.

Con A-Sepharose affinity chromatography was utilized to examine the glycoproteins in phosphosaline extracts of normal and breast tumor tissues and breast patient sera. In extracts of normal breast tissue, normal sera and patient sera, all glycoproteins were eluted from the Con A-Sepharose with a linear gradient of 0.0-0.5 M alpha-methylmannose. Using breast tumor extracts, a glycoprotein peak which could not be eluted as with normal tissue extracts was observed. This tightly-binding peak could be eluted from the Con A-Sepharose with acetate buffer containing 1.0 M KCl. Polyacrylamide electrophoresis of this tightly-binding glycoprotein peak revealed one major glycoprotein and four minor glycoproteins. The major glycoprotein obtained from electrophoresis represented about 60% of the Con A-Sepharose tightly-binding protein and reacted with antiserum to human orosomucoid (alpha 1-acid glycoprotein). All glycoproteins isolated from tumor tissue extracts appeared to represent normal serum constituents as they were retained on an immunoadsorbent containing antibodies to normal serum proteins. The possible significance of the isolated tumor-associated orosomucoid is discussed.     

DSA affinity glycoproteome of human liver tissue

Due to the critical roles of glycoproteins in the activities of cells to tissues, mapping of liver glycoproteome may provide valuable basic information for finding disease marker proteins. In this study, Datura Stramonium Agglutinin (DSA) was chosen to enrich N-linked glycoproteins for its broader specificity with tri- or tetra-antennary complex type. DSA affinity glycoproteins’ profiles of human liver tissue were generated by two-dimensional electrophoresis (2-DE) followed by glycoprotein staining based on multiplexed proteomics (MP) technology. 64 ± 3 (n = 3) protein spots were detected and 41 of glycoproteins were identified via peptide mass fingerprinting (PMF) using MALDI-TOF-MS/MS and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The detailed carbohydrate moiety of some glycoproteins might be concluded according to the literatures. The construction of DSA affinity glycoprotein database would contribute to the subsequent research.     

Identification and characteristics of concanavalin A-binding neurospecific glycoproteins in human brain and brain tumors

Soluble and membrane-bound neurospecific Con A-binding glycoproteins from human brain and tumours were identified and characterized, using a procedure which included stepwise extraction with low and high ionic strength buffers, buffered. Triton X-100 and sodium deoxycholate followed by ConA-Sepharose column chromatography, SDS-PAAG electrophoresis and immunoblotting. Adsorbed antisera against different types of neurospecific glycoproteins were used. The bulk of neurospecific glycoproteins (11 and 13) were revealed in protein fractions extracted with low ionic strength buffers and Triton X-100. In astrocytomas and glyoblastomas, some neurospecific glycoproteins were absent. Some glycoproteins were found in tumours, but were absent in brain tissue. Soluble, 77 kD glycoprotein, 11 and 16 kD glycoproteins solubilized with high ionic strength buffers and intrinsic membrane-bound 51, 57, 61, 74 and 77 kD glycoproteins can be viewed as stable neurospecific markers in malignant brain tumours.     

Leaching of concanavalin A during affinity chromatographic isolation of cell surface glycoproteins from human fetal neurons and glial cells.

Preparation of surface glycoproteins from human fetal brain cells by affinity chromatography on Con A-Sepharose 4B was a problematic endeavor due to leaching of Con A from the matrix. Dissociation of Con A from the matrix took place irrespective of the presence of lipid and/or detergent and the buffer composition during chromatography and was apparently related to the nature of the protein under study. Pretreatment of Con A-Sepharose with 6 M guanidine or 8 M urea reduced Con A leaching. The Con A eluate also contained noncovalently associated glycolipid. Elution at 25 degrees C rendered fractions containing a higher degree of Con A and glycolipid contamination compared to the negligible contamination by these two components when elution was carried out at 4 degrees C. This phenomenon was attributed to the formation of heterogeneous mixed micelles of glycoprotein.     

Identification and analysis of altered alpha1,6-fucosylated glycoproteins associated with hepatocellular carcinoma metastasis

Tumor metastasis might be associated with the expression levels of cellular glycoproteins and the alteration of their glycan parts. In order to screen the aberrantly alpha1,6-fucosylated glycoproteins related to hepatocellular carcinoma (HCC) metastasis, a high-throughput glycomic approach which consisted of 2-DE, electronic transfer of proteins, lectin affinity blot and precipitation, and MALDI-TOF-MS/MS, was established. Lens culinaris agglutinin (LCA) affinity glycoprotein profiles of higher and lower metastatic HCC cell lines were compared and analyzed. Seven out of 34 identified glycoproteins were differentially displayed; they were cytokeratin 8 (CK8), annexin I, annexin II, heterogeneous nuclear ribonucleoprotein A/B, PDZ and LIM domain 1, RNA-binding motif protein 4, and poly(rC)-binding protein 1. On comparison with Hep3B, CK8 showed a higher affinity to Ricinus communis agglutinin 1 (RCA-I) and LCA, and annexin I presented a higher affinity to LCA and Con A by the lectin-binding assay. Furthermore, the up-regulation of CK8, annexin I, and annexin II were found by Western blot and immunofluorescence analysis in higher metastatic HCC cell lines. This implied that the alteration of CK8, annexin I, and annexin II both in their expression levels and their glycan parts might be related to metastatic ability, and play a critical role in the process of HCC metastasis.     

Expression of membrane glycoproteins in normal keratinocytes and squamous carcinoma cell lines

Con A acceptor glycoproteins were analyzed by 2D-PAGE and 125I-Con A overlay in three squamous carcinoma cell lines and compared with those in the simian virus (SV40)-transformed keratinocyte cell line SVK-14 and in normal keratinocytes. The majority of the glycoproteins identified by this technique were expressed at similar levels in all of the cells examined, independent of the culture conditions used. A cell surface glycoprotein gp34 (MW 34 kDa, pI 5.1) was increased in the tumor cells compared with normal keratinocytes and expression varied with the culture density. Another glycoprotein, gp21 (MW 21 kDa, pI 6.3), was found to be increased in expression in normal keratinocytes and stratified hyperconfluent cultures of squamous carcinoma cell lines. This paper describes the potential of this technique to identify membrane glycoproteins which may be expressed as a function of proliferation or differentiation.     

Isolement et caractérisation partielle des glycoprotéines de paroi des charophytes: Chara aspera et Nitella translucens

From purified cell walls and pectic, hemicellulosic and cellulosic fractions of Chara aspera and Nitella translucens, glycoproteins were isolated through affinity chromatography on concanvalin A (Con A)-Sepharose column. The chemical composition of protein fractions is qualitatively the same in both algae. The polysaccharidic moieties vary from one glycoprotein to another. None of the glycoproteins present contains cystine or hydroxyproline.     

Comparative glycoproteomics based on lectins affinity capture of N-linked glycoproteins from human Chang liver cells and MHCC97-H cells

We present here an effective technique for the large-scale separation and identification of N-linked glycoproteins from Chang liver cells, the human normal liver cells. To enrich N-linked glycoproteins from the whole cells, a procedure containing the lysis of human liver cells, the solubilization of total proteins, lectin affinity chromatography including Concanavalin A and wheat germ agglutinin was established. Furthermore, captured N-linked glycoproteins were separated by 2-DE, and identified by MS and database searching. Finally, we found 63 N-glycoproteins in Chang liver cells. In addition, using the above method, we identified 7 remarkably up-regulated glycoproteins from MHCC97-H cells, highly metastatic liver cancer cells, compared to Chang liver cells. These up-regulated glycoproteins were associated with liver cancer and might be used as biomarkers for tumor diagnosis. Results showed that we established a high-throughput proteomic analysis for separating N-linked glycoproteins from human liver cells. This strategy greatly improved the glycoprotein analysis method associated with proteome-wide glycosylation changes related to liver cancer. Our work was part of the HUPO Human Liver Proteome Project (HLPP) studies and was supported by CHINA HUPO.     

Approaches to the study of N-linked glycoproteins in human plasma using lectin affinity chromatography and nano-HPLC coupled to electrospray linear ion trap--Fourier transform mass spectrometry

In this publication, we will describe the combination of lectin affinity chromatography with nano high performance liquid chromatography (HPLC) coupled to a linear ion trap Fourier transform mass spectrometer (capillary LC-LTQ/FTMS) to characterize N-linked glycosylation structures in human plasma proteins. We used a well-characterized glycoprotein, tissue plasminogen activator (rt-PA), which is present at low levels in blood, as a standard to determine the dynamic range of this approach. N-linked glycopeptides derived from rt-PA could be characterized at a ratio of 1:200 in human plasma (rtPA: total plasma protein, w/w) by accurate mass measurement in the FTMS and fragmentation (MS(n)) in the linear ion trap. We demonstrated that this platform has the potential to characterize the general N-linked glycosylation structures of abundant glycoproteins present in human plasma without the requirement for antibody-based purification, or additional carbohydrate analytical protocols. This conclusion was supported by the determination of carbohydrate structures for three glycoproteins, IgG, haptoglobin, and alpha-1-acid glycoprotein, at their natural levels in a human plasma sample, but only after the lectin enrichment step.     

Identification of liver plasma membrane glycoproteins which bind to 125I-labelled concanavalin A following electrophoresis in sodium dodecyl sulfate.

Following electrophoresis of ovalbumin in sodium dodecyl sulfate (SDS) this glycoprotein bound 125I-labelled concanavalin A (Con A). The reaction was specific and proportional to the amount of glycoprotein present on the gel. This technique was used to study the Con-A-binding glycoproteins of liver cell surfaces. Mouse liver plasma membranes were purified and subfractionated to yield two fractions corresponding to the bile canalicular surface and the surface between adjacent hepatocytes (Evans, W.H. (1970) Biochem. J. 116, 833-842). Both fractions bound 125I-labelled Con A, the former binding two to three times more lectin than the latter. Following SDS gel electrophoresis individual membrane glycoproteins reacted with 125I-labelled Con A. Both membrane subfractions yielded qualitatively similar Con A binding profiles, seven binding proteins being present in each. The results are consistent with a generally uniform distribution of glycoproteins over the hepatocyte surface. The reaction of lectins with glycoproteins following SDS gel electrophoresis should find general application in the study of membrane composition.     

Specific isolation of surface glycoproteins from intact cells by biotinylated concanavalin A and immobilized streptavidin

An indirect affinity chromatography procedure utilizing biotinylated lectins and designed for the specific isolation of surface glycoproteins is described. The method is illustrated with intact acute leukemic lymphoblastic cells (ALL cells) with biotin-epsilon-aminocaproyl-concanavalin A (biocap-Con A) and streptavidin-Sepharose 4B. Biocap-Con A, containing on average 27 biotin residues per tetrameric lectin molecule, is used to isolate Con A-binding glycoproteins from the surface of [35S]methionine-radiolabeled intact cells. The biocap-Con A/glycoprotein complexes, after solubilization in detergent, are retrieved on immobilized streptavidin. The surface glycoproteins isolated from intact ALL cells by this method are subjected to two-dimensional gel electrophoresis and detected by autoradiography. More than fifty Con A-binding glycoproteins can be separated from the ALL cells. These glycoproteins retrievable from the cell surface were compared to those retrieved by the indirect affinity chromatography procedure from isolated plasma membrane fractions. Certain groups of glycoproteins present in the fraction isolated from intact cells were not detected in that from the plasma membrane preparations. The advantage of using the biocap-con A/streptavidin system with intact cells rather than isolated plasma membranes for the detection of surface glycoproteins is discussed.     

Partial purification and characterization of Schistosoma mansoni soluble egg antigen with Con A-Sepharose chromatography

A crude preparation of Schistosoma mansoni soluble egg antigen (SEA) was subjected to affinity chromatography with concanavalin A (Con A) bound to Sepharose 4B. The resulting Con A fractions (bound and unbound) were characterized with sodium dodecyl sulfate (SDS) gel electrophoresis, immunoelectrophoresis, immunodiffusion, and lymphocyte blastogenesis techniques. In the fraction that did not bind to Con A there were at least two distinct antigens, and there were also at least two distinct antigens in the fractions that did bind to Con A. With SDS polyacrylamide gel electrophoresis, at least 20 distinct protein bands (Coomassie blue staining) and three glycoprotein bands (PAS reactive) were present in the unbound fractions from Con A chromatography. The bound fractions separated into at least six distinct glycoproteins with SDS electrophoresis. Although both the bound and unbound fractions contained precipitating antigens, only the bound fractions were capable of eliciting lymphocyte blastogenic responses.     

二维电泳和质谱技术鉴定肝癌血清差异表达的N-连接糖蛋白

缺乏有效的早期诊断方法是导致肝细胞癌(hepatocellular carcinoma, HCC)预后极差的主要原因之一.蛋白质异常糖基化与恶性肿瘤细胞侵袭、转移等生物学过程关系密切,人体内至少有50%的蛋白质发生了糖基化修饰.本实验采用IgY12去除血清高丰度蛋白、多植物凝集素亲和层析技术分别从20例肝癌和年龄、性别匹配的20例非癌慢性肝病患者血清中纯化N 连接糖蛋白、二维电泳分析差异表达的蛋白质斑点,质谱检测、生物信息学等技术鉴定了18个差异表达的糖蛋白和/或其异质体（12种高表达和6种低表达）.ExPASy数据库比对结果表明,本实验鉴定的糖蛋白质分子含有至少1个已报道的N 糖基化位点.这些差异表达的糖蛋白属于急性期反应蛋白,分别具有蛋白酶抑制、生物转运、凝血和纤溶等功能,表明肝癌的发生发展过程中机体产生的急性期反应物可能是潜在的肝癌血清标志物.     

Relationship of the glycan structure of glycoproteins to the desialylation process by rat liver endothelium

To investigate the variations in desialylation of glycoproteins by liver endothelium, we compared endothelial desialylation for 3 glycoproteins, human ceruloplasmin, human and rat transferrin. Radiolabeled glycoproteins were chased through purified rat liver endothelium and then fractionated by lectin affinity chromatography. Endothelium processed glycoproteins were fractionated by RCA120 chromatography into sialylated and desialylated components. The latter was then studied by Con A chromatography. Desialylation occurred only when the molecule contained at least a single triantennary chain of glycan. Desialylation was minimal in the case of human transferrin which contains mostly biantennary branching pattern. Thus, it appears that a single triantennary glycan chain is necessary and sufficient to trigger desialylation of glycoproteins by liver endothelium and this process is an all-or-none phenomenon.     

岩藻糖糖链与肝癌细胞的迁移作用

通过凝集素印迹转移电泳和亲和层析技术,对岩藻糖糖基化蛋白在肝癌细胞中的作用进行了研究.在化学诱发的大鼠肝癌过程中, 分子质量在23 ku到40 ku范围内与荆豆凝集素(UEA)及扁豆凝集素(LCA)结合的岩藻糖糖基化蛋白显著减少, 诱癌至17～20周这些条带重新恢复,而分子质量为80 ku的条带却在诱癌过程中逐周增加.比较高、低转移性肝癌细胞的岩藻糖糖基化蛋白, 发现高转移性肝癌细胞具有多种增强的条带.利用橘果粉胞凝集素(AAL)和LCA亲和层析柱分离了这些岩藻糖基化糖蛋白, 并用这些糖蛋白直接作用于肝癌细胞,发现AAL-糖蛋白具有显著抑制肝癌细胞迁移的作用,迁移细胞数从对照的(100±4.9)%下降到(48.1±2.5)% (P＜0.01), LCA-糖蛋白也有类似作用.用胰酶和木瓜蛋白酶水解蛋白质部分后,形成的糖肽抑制肝癌细胞迁移的作用并不改变,甚至增强.此外直接用肝癌转移灶的组织测定了岩藻糖转移酶活性,发现α1,6岩藻糖基转移酶活性显著比正常肝组织高,而α1,3岩藻糖基转移酶活性没有显著的改变.用系列凝集素分析发现这些糖链主要能结合伴刀豆凝集素A, 也能结合E-型及L-型植物凝集素, 显示这种糖蛋白的糖链可能含有较多的高甘露糖型.这些结果提示糖链在诱癌过程中结构有了改变,使之在肝癌细胞的迁移和转移中起重要作用.     

Chronic ethanol administration alters hepatic surface membranes as evidenced by decreased concanavalin A binding

The effects of chronic ethanol administration on the hepatic surface membrane were examined. The binding of the lectin, concanavalin A (Con A), to isolated hepatocytes was used to ascertain changes in the hepatic plasma membrane, especially in regard to glycoprotein composition, due to chronic ethanol feeding. Hepatocytes, isolated from rats fed ethanol for 5 to 7 weeks, had a decreased ability to bind Con A when compared to hepatocytes from either the pair-fed controls or ad libitum chow-fed rats. Since decreased Con A binding was more apparent at high Con A concentrations, reduced lectin binding likely reflected changes in the composition of surface membrane glycoproteins in the livers of the ethanol-fed rats. When ethanol (50 mM) was added to the incubation medium containing hepatocytes from ethanol-fed rats, pair-fed controls, or chow-fed rats, no effects on Con A binding were observed. These results indicate that chronic ethanol administration induces changes in the oligosaccharide chains of plasma membrane glycoproteins in the liver. Such alterations may play a role in the pathogenesis of alcoholic liver disease.     

Alteration by concanavalin A of the slow dissociable component in the human growth hormone-receptor interaction

In mice liver plasma membranes (PM), the binding affinity of receptors for [125I] human growth hormone (hGH) was dependent on the association time: after 18 hours, a high affinity receptor form with KA = 6.8 X 10(9) M-1 accumulated and, as compared to after 1 hour, an increase up to 88%, in a slow dissociating component was observed. Preincubation of PM with concanavalin A (Con A) or other lectins from Lens culinaris (LCA), Ricinus communis (RCA I), Wheat germ agglutinin (WGA) specifically inhibited the binding of hGH to receptors by 54, 28, 50 and 25%, respectively. Furthermore, PM pretreatment with Con A concomitantly increased the rate of dissociation of the hormone-receptor (H-R) complex to 92 or 65% after association for 1 or 18 hours. These Con A-induced alterations resulted from a reduced fraction of the slow dissociable component together with an increased rate constant. The treatment of PM with Con A subsequent to incubation with the hormone did not decrease hormone binding but caused the conversion of the class of hGH receptors exhibiting fast dissociation kinetics towards a form exhibiting slow ones. These data strongly suggest a role for glycoproteins of the N-acetyllactosaminic type in the affinity state of liver membrane hGH receptors.     

酶标白桂木凝集素糖蛋白结合特性的分析

采用辣根过氧化物酶 ( HRP)标记白桂木凝集素 ( AHL) ,应用酶联夹心法及糖竞争抑制实验 ,研究 AHL的糖蛋白结合特性 .研究表明 ,AHL能与两种不同类型的糖蛋白结合 ,一类以胃蛋白酶为代表 ,AHL能以高亲和力与胃蛋白酶结合 .其次能与β-乳球蛋白、牛血清清蛋白结合 ,但结合力依次递减 .AHL也能与透明质酸以较高亲和力相结合 .AHL与胃蛋白酶、β-乳球蛋白、牛血清清蛋白、透明质酸的结合受 Me- Gal的强烈竞争抑制 ,亦受 Me- Man\D- Gal\Raf的抑制 .另一类为Con A,AHL与 Con A的结合受 Me- Man的强烈竞争抑制 ,并受 Me- Glc\D- Man\D- Fru\D- Glc的较强抑制 .各种糖的封闭性抑制实验结果与竞争性抑制实验相似 .提示 AHL上存在 O-糖苷键结合位点 .     

Separation of Herpes Simplex Virus-Induced Antigens by Concanavalin A Affinity Chromatography

Biologically active herpes simplex virus (HSV)-induced antigens were selectively removed from extracts of infected BHK cells by affinity chromatography by utilizing an insoluble form of concanavalin A (Con A). Soluble extracts of (3)H-glucosamine-labeled, HSV-infected cells were absorbed to a Con A column. Bound material was eluted with alpha-methyl-d-mannoside (alphaMM) and NaCl. The specific activity of the eluted glycoproteins increased by 10-fold. Two broad groups of viral-induced antigens were isolated from Con A. Group I includes two antigens which bind to Con A by a specific mechanism because the antigens are dissociated by alphaMM. Group II contains three antigens which bind to Con A but apparently by a nonspecific or electrolytic mechanism. One antigen in group I was identified as the glycoprotein antigen, CP-1, described previously.