RESUMO
A new platform for N-glycoprotein analysis from serum that combines matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) workflows with antibody slide arrays is described. Antibody panel based (APB) N-glycan imaging allows for the specific capture of N-glycoproteins by antibodies on glass slides and N-glycan analysis in a protein-specific and multiplexed manner. Development of this technique has focused on characterizing two abundant and well-studied human serum glycoproteins, alpha-1-antitrypsin and immunoglobulin G. Using purified standard solutions and 1 µL samples of human serum, both glycoproteins can be immunocaptured and followed by enzymatic release of N-glycans. N-Glycans are detected with a MALDI FT-ICR mass spectrometer in a concentration-dependent manner while maintaining specificity of capture. Importantly, the N-glycans detected via slide-based antibody capture were identical to that of direct analysis of the spotted standards. As a proof of concept, this workflow was applied to patient serum samples from individuals with liver cirrhosis to accurately detect a characteristic increase in an IgG N-glycan. This novel approach to protein-specific N-glycan analysis from an antibody panel can be further expanded to include any glycoprotein for which a validated antibody exists. Additionally, this platform can be adapted for analysis of any biofluid or biological sample that can be analyzed by antibody arrays.
Assuntos
Biomarcadores/metabolismo , Glicômica/métodos , Glicoproteínas/metabolismo , Cirrose Hepática/diagnóstico , Imagem Óptica/métodos , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Estudos de Casos e Controles , Glicoproteínas/química , Glicosilação , Humanos , Cirrose Hepática/metabolismo , Polissacarídeos/químicaRESUMO
(1) Glycoproteins account for ~80% of proteins located at the cell surface and in the extracellular matrix. A growing body of evidence indicates that α-L-fucose protein modifications contribute to breast cancer progression and metastatic disease. (2) Using a combination of techniques, including matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) based in cell and on tissue imaging and glycan sequencing using exoglycosidase analysis coupled to hydrophilic interaction ultra-high performance liquid chromatography (HILIC UPLC), we establish that a core-fucosylated tetra-antennary glycan containing a single N-acetyllactosamine (F(6)A4G4Lac1) is associated with poor clinical outcomes in breast cancer, including lymph node metastasis, recurrent disease, and reduced survival. (3) This study is the first to identify a single N-glycan, F(6)A4G4Lac1, as having a correlation with poor clinical outcomes in breast cancer.
Assuntos
Amino Açúcares/análise , Neoplasias da Mama/patologia , Fucose/análise , Polissacarídeos/análise , Animais , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Feminino , Glicoproteínas/química , Glicosilação , Humanos , Linfonodos/patologia , Camundongos Endogâmicos BALB C , Metástase Neoplásica/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Análise de SobrevidaRESUMO
Hepatocellular carcinoma (HCC) accounts for >700,000 deaths worldwide, largely related to poor rates of diagnosis. Our previous work identified glycoproteins with increased levels of fucosylation in HCC. Plate-based assays to measure this change were compromised by increased levels of heterophilic antibodies with glycan lacking terminal galactose residues, which allowed for increased binding to the lectins used in these assays. To address this issue, we developed a multi-step protein A/G incubation and filtration method to remove the contaminating signal. However, this method was time consuming and expensive so alternative methods were desired. Herein, we describe a simple method relying on PEG precipitation that allows for the removal of IgG and IgM but retention of glycoproteins of interest. This method was tested on three sample sets, two internal and one external. PEG depletion of heterophilic IgG and IgM reduced in the coefficient of variation as observed with the protein A/G filtration method from 26.82% to 7.50% and allowed for the measurement of fucosylated protein. This method allowed for the measurement of fucosylated kininogen, which could serve as a biomarker of HCC. In conclusion, a new and simple method for the depletion of heterophilic IgG and IgM was developed and allowed for the analysis of fucosylated kininogen in patients with liver disease.
Assuntos
Anticorpos Heterófilos/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Cininogênios/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Adulto , Idoso , Anticorpos Heterófilos/química , Biomarcadores Tumorais/química , Feminino , Glicosilação , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulina M/química , Imunoglobulina M/metabolismo , Cininogênios/química , Lectinas , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/química , Polietilenoglicóis/químicaRESUMO
Hepatocellular carcinoma (HCC) remains as the fifth most common cancer in the world and accounts for more than 700,000 deaths annually. Changes in serum glycosylation have long been associated with this cancer but the source of that material is unknown and direct glycan analysis of HCC tissues has been limited. Our laboratory previously developed a method of in situ tissue based N-linked glycan imaging that bypasses the need for microdissection and solubilization of tissue prior to analysis. We used this methodology in the analysis of 138 HCC tissue samples and compared the N-linked glycans in cancer tissue with either adjacent untransformed or tissue from patients with liver cirrhosis but no cancer. Ten glycans were found significantly elevated in HCC tissues as compared to cirrhotic or adjacent tissue. These glycans fell into two major classes, those with increased levels of fucosylation and those with increased levels of branching with or without any fucose modifications. In addition, increased levels of fucosylated glycoforms were associated with a reduction in survival time. This work supports the hypothesis that the increased levels of fucosylated N-linked glycans in HCC serum are produced directly from the cancer tissue.
Assuntos
Carcinoma Hepatocelular/metabolismo , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Polissacarídeos/metabolismo , Adulto , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/patologia , Feminino , Fucose/metabolismo , Glicosilação , Histocitoquímica/métodos , Humanos , Estimativa de Kaplan-Meier , Fígado/patologia , Cirrose Hepática/sangue , Cirrose Hepática/patologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Polissacarídeos/química , Análise Serial de Tecidos/métodosRESUMO
Background: Hepatocellular carcinoma (HCC) has the greatest increase in mortality among all solids tumors in the United States related to low rates of early tumor detection. Development of noninvasive biomarkers for the early detection of HCC may reduce HCC-related mortality.Methods: We have developed an algorithm that combines routinely observed clinical values into a single equation that in a study of >3,000 patients from 5 independent sites improved detection of HCC as compared with the currently used biomarker, alpha-fetoprotein (AFP), by 4% to 20%. However, this algorithm had limited benefit in those with AFP <20 ng/mL. To that end, we have developed a secondary algorithm that incorporates a marker, fucosylated kininogen, to improve the detection of HCC, especially in those with AFP <20 ng/mL and early-stage disease.Results: The ability to detect early-stage AFP-negative (AFP <20 ng/mL) HCC increased from 0% (AFP alone) to 89% (for the new algorithm). Glycan analysis revealed that kininogen has several glycan modifications that have been associated with HCC, but often not with specific proteins, including increased levels of core and outer-arm fucosylation and increased branching.Conclusions: An algorithm combining fucosylated kininogen, AFP, and clinical characteristics is highly accurate for early HCC detection.Impact: Our biomarker algorithm could significantly improve early HCC detection and curative treatment eligibility in patients with cirrhosis. Cancer Epidemiol Biomarkers Prev; 26(5); 795-803. ©2017 AACR.
Assuntos
Algoritmos , Biomarcadores Tumorais/análise , Carcinoma Hepatocelular/diagnóstico , Detecção Precoce de Câncer/métodos , Cininogênios/análise , Neoplasias Hepáticas/diagnóstico , Idoso , Carcinoma Hepatocelular/metabolismo , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Estados Unidos , alfa-Fetoproteínas/análiseRESUMO
The Aleuria aurantia lectin (AAL) derived from orange peel fungus contains five fucose-binding sites that recognizes fucose bound in α-1,2, α-1,3, α-1,4, and α-1,6 linkages to N-acetylglucosamine and galactose. Recently, we have created several recombinant AAL (rAAL) proteins that had altered binding affinity to fucose linkages. In this report, we further characterize the binding specificity of one of the mutated lectins, N224Q lectin. This lectin was characterized by lectin Western blotting, surface plasmon resonance, and glycan microarray and shown to have increased binding to fucosylated glycan. Subsequently, we used this lectin to identify secreted fucosylated glycoproteins from a fetal hepatic cell line. Proteomic analysis revealed several glycoproteins secreted by the fetal cell line that were bound by N224Q lectin. These findings were confirmed by subsequent proteomic analysis of human serum from control patients or patients with hepatocellular carcinoma. These represent candidate oncofetal markers for liver cancer.
Assuntos
Carcinoma Hepatocelular/metabolismo , Fucose/metabolismo , Glicoproteínas/metabolismo , Lectinas/metabolismo , Neoplasias Hepáticas/metabolismo , Polissacarídeos/metabolismo , Ascomicetos/química , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/diagnóstico , Linhagem Celular , Células Cultivadas , Fucose/análise , Glicoproteínas/análise , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Lectinas/química , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/diagnóstico , Polissacarídeos/química , Ligação Proteica , Proteômica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Alterations in N-linked glycosylation have long been associated with cancer but for the most part, the reasons why have remained poorly understood. Here we show that increased core fucosylation is associated with de-differentiation of primary hepatocytes and with the appearance of markers indicative of a transition of cells from an epithelial to a mesenchymal state. This increase in core fucosylation was associated with increased levels of two enzymes involved in α-1,6 linked fucosylation, GDP-mannose 4, 6-dehydratase (Gmds) and to a lesser extent fucosyltransferase 8 (Fut8). In addition, the activation of cancer-associated cellular signaling pathways in primary rat hepatocytes can increase core fucosylation and induce additional glycoform alterations on hepatocyte proteins. Specifically, we show that increased levels of protein sialylation and α-1,6-linked core fucosylation are observed following activation of the ß-catenin pathway. Activation of the Akt signaling pathway or induction of hypoxia also results in increased levels of fucosylation and sialylation. We believe that this knowledge will help in the better understanding of the genetic factors associated with altered glycosylation and may allow for the development of more clinically relevant biomarkers.
Assuntos
Carcinoma Hepatocelular/patologia , Desdiferenciação Celular/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Fucosiltransferases/genética , Hidroliases/metabolismo , Neoplasias Hepáticas/patologia , beta Catenina/metabolismo , Animais , Biomarcadores/metabolismo , Carcinoma Hepatocelular/diagnóstico , Células Cultivadas , Fucosiltransferases/metabolismo , Glicosilação , Hepatócitos/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/diagnóstico , Células-Tronco Mesenquimais/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais/fisiologia , Ativação Transcricional , Regulação para CimaRESUMO
Liver disease, in the form of hepatocellular carcinoma (HCC) accounts for > 700,000 deaths worldwide. A major reason for this is late diagnosis of HCC. The currently used biomarker, serum alpha-fetoprotein (AFP) is elevated in 40-60% of those with HCC and other markers that can either compliment or replace AFP are desired. Our previous work has identified a number of proteins that contain altered glycans in HCC. Specifically, these altered glycans were increased levels of core and outer arm fucosylation. To determine the clinical usefulness of those identified glycoproteins, a plate based assay was developed that allowed for the detection of fucosylated glycoforms. While this method was applicable to a number of independent patient sets, it was unable to specifically detect fucosylated glycoforms in many patient samples. That is, some material was present in serum that led to non-specific signal in the lectin- fluorescence -linked immunosorbent assay (lectin-FLISA). To address this issue, a systematic process was undertaken to identify the material. This material was found to be increased levels of lectin reactive IgM. Removal of both IgG and IgM using a multi-step protein A/G incubation and filtration step removed the contaminating signal and allowed for the analysis of specific protein glycoforms. This assay was subsequently used on two sample sets, one that was shown previously to be unable to be tested via a lectin FLISA and in a larger independent sample set. The clinical usefulness of this assay in the early detection of HCC is discussed.
Assuntos
Carcinoma Hepatocelular/sangue , Imunoglobulina M/sangue , Técnicas de Imunoadsorção , Lectinas/análise , Neoplasias Hepáticas/sangue , alfa-Fetoproteínas/análise , Biomarcadores Tumorais/sangue , Glicosilação , Humanos , Imunoglobulina G/análise , Imunoglobulina G/sangue , Imunoglobulina M/análiseRESUMO
Liver cancer is the fifth most common cancer, but the second leading cause of cancer death, in the world, with more than 700,000 fatalities annually. The major etiology of liver cancer is infection with an hepatotropic virus such as hepatitis B virus or hepatitis C virus infection. While chronic viral infection remains the main cause of liver disease and risk of hepatocellular carcinoma (HCC), rates of nonviral-associated HCC are occurring at an alarmingly increasing rate. Like many cancers, survival rates are closely associated with time of detection. If HCC is caught early, survival rates can be as high as 50%. Regrettably, most cases of HCC are caught late where survival rates can be as low as 2-7%. Thus, there has been great interest in discovering serum biomarkers that could be used to identify those with HCC. To this end, many groups have examined the N-linked glycans to identify changes that occur with HCC. As the liver secretes the vast majority of proteins into the serum, this has often been a starting point for study. In serum, alterations in core fucosylation, outer-arm fucosylation, increased sialylation, and glycan branching have been observed in patients with HCC. Similar findings have been found directly in HCC tissue suggesting that these glycan changes may play a role in tumor formation and development.