RESUMO
In human tumors, glycoproteins often exhibit abnormal glycosylation patterns, e.g. certain Lewis structures, TF antigen, Tn antigen and/or their sialylated forms, creating additional binding sites for glycoreceptors. In the present study, we have analyzed the carbohydrate specificity of the C-type lectin CLEC10A using glycan profiling by enzyme-linked immunosorbent assay (ELISA). In addition to the known ligands, we show binding to two tumor-associated antigens, namely Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn, with an affinity of CLEC10A in the micromolar range. Detailed analyses of the glycan-lectin interactions were carried out by surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR. CLEC10A binds Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn with dissociation constants of 297 and 80 µM, respectively, as determined by SPR. Comparison of the STD nuclear magnetic resonance (NMR) binding epitopes of Tn and Neu5Acα2,6-Tn revealed a constant binding mode of the N-acetylgalactosamine moiety. This finding is in good agreement with binding studies of CLEC10A transfectomas, which show a well-defined interaction of transmembrane CLEC10A with 6-sialylated-Tn structures. Since both Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn together with the previously known Tn antigen are expressed in human tumors such as mammary carcinoma, the interaction with CLEC10A expressed by macrophages and dendritic cells could be of major functional significance in tumor progression.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Lectinas Tipo C/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Ácidos Neuramínicos/metabolismo , Animais , Antígenos Glicosídicos Associados a Tumores/química , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Ligação ProteicaRESUMO
Specialized protein domains bind to posttranslational modifications (PTMs) of proteins, such as phosphorylation or glycosylation. When such PTM-binding protein domains are used as analytical tools, the functional states of cells and tissues can be determined with high precision. Here, we describe the use of recombinant CLEC10A (CD301), a human glycoreceptor of the C-type lectin family, for the detection of ligands in sections from formalin-fixed, paraffin-embedded normal and cancerous mammary tissues. A construct, in which part of the carbohydrate recognition domain (CRD) was deleted, was used as a negative control. In comparison to normal mammary glands, a pronounced staining of tumor tissues was observed. Because the construct with the truncated CRD did not show any tissue staining, the binding of the wild-type glycoreceptor can be attributed to its carbohydrate recognition domain. To distinguish our novel approach from immunohistochemistry, we propose the designation "protein domain histochemistry" (PDH).
Assuntos
Neoplasias da Mama/patologia , Mama/patologia , Histocitoquímica/métodos , Lectinas Tipo C/análise , Lectinas Tipo C/metabolismo , Neoplasias da Mama/diagnóstico , Clonagem Molecular , Feminino , Células HEK293 , Humanos , Inclusão em Parafina/métodos , Polissacarídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Fixação de Tecidos/métodosRESUMO
Drastic enrichment of potential disease-specific glycoprotein markers in human plasma can be achieved by the combination of affinity- and immuno-depletion. In the affinity-fractionation step all glycoproteins carrying a certain glycostructure are isolated by lectin affinity chromatography, thus depleting other components. Against the respective glycoprotein fraction isolated from the plasma of healthy individuals antibodies are raised in llamas. The llama heavy chain antibodies (which are particularly stable) directed at the isolated plasma glycoprotein fraction are immobilized and the immunoaffinity column thus obtained is used to deplete the respective glycoprotein fraction of patient plasma samples. Depletion of proteins normally found in human plasma by 99.8-99.9% can be achieved, resulting in a 800-1000-fold enrichment of potential disease-specific proteins in the flow-through of the immunoaffinity column.
Assuntos
Biomarcadores/sangue , Cromatografia de Afinidade/métodos , Glicoproteínas/sangue , Imunoensaio/métodos , Animais , Anticorpos Imobilizados/química , Antígenos/administração & dosagem , Antígenos/química , Antígenos/imunologia , Proteínas Sanguíneas/química , Camelídeos Americanos/imunologia , Cromatografia de Afinidade/instrumentação , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoensaio/instrumentação , Cadeias Pesadas de Imunoglobulinas/química , Lectinas/química , VacinaçãoRESUMO
Protein biomarker discovery in the low concentration range of human body fluids requires the enrichment of the proteins of interest. Here we report on a tandem affinity strategy: In the first step, we isolated a human plasma glyco-subproteome of healthy individuals by wheat germ agglutinin (WGA) lectin affinity chromatography. In the second step, the proteins of this subproteome were used to raise antibodies in llama (Lama glama). The heavy-chain fraction of the llama antibodies was used to deplete from the WGA lectin binding fraction all proteins normally found in human plasma. In this way, we selectively enriched the glycoprotein, CEA, a known cancer marker which had been spiked into normal plasma. As a proof of concept, we applied this method to the analysis of plasma sample from colon cancer patients. We could demonstrate the selective enrichment of CEA by a factor of 600-800.