Tyramide signal amplification for antibody-overlay lectin microarray: a strategy to improve the sensitivity of targeted glycan profiling

Antibody-overlay lectin microarray (ALM) has been used for targeted glycan profiling to identify disease-related protein glycoforms. In this context, high sensitivity is desired because it allows for the identification of disease-related glycoforms that are often present at low concentrations. We describe a new tyramide signal amplification (TSA) for the antibody-overlay lectin microarray procedure for sensitive profiling of glycosylation patterns. We demonstrate that TSA increased the sensitivity of the microarray over 100 times for glycan profiling using the model protein prostate specific antigen (PSA). The glycan profile of PSA enriched from LNCAP cells, obtained at a subnanogram level with the aid of TSA, was consistent with the previous reports. We also established the glycan profile of prostate specific membrane antigen (PSMA) using the TSA and ALM. Thus, the TSA for antibody-overlay lectin microarray is a sensitive, rapid, comprehensive, and high-throughput method for targeted glycan profiling and can potentially be used for the identification of disease-related protein glycoforms.     

Optimization of evanescent-field fluorescence-assisted lectin microarray for high-sensitivity detection of monovalent oligosaccharides and glycoproteins

Lectin microarray is an emerging technique, which will accelerate glycan profiling and discovery of glycan-related biomarkers. One of the most important stages in realizing the potential of the technique is to achieve sufficiently high sensitivity to detect even the low concentrations of some target glycoproteins which occur in sera or tissues. Previously, we developed a lectin microarray based on an evanescent-field fluorescence-assisted detection principle that allows rapid profiling of glycoproteins. Here, we report optimization of procedures for lectin spotting and immobilization to improve the sensitivity and reproducibility of the lectin microarray. The improved microarray allows high-sensitivity detection of even monovalent oligosaccharides that generally have a low affinity with lectins (K(d)>10(-6) M). The LOD observed for RCA120, a representative plant lectin, with asialofetuin, and an asialo-biantennary N-glycan probe were determined to be 100 pg/mL and 100 pM, respectively. With the improved lectin microarray system, closely related structural isomers, i.e., Le(a) and Le(x), were clearly differentiated by the difference in signal patterns on relevant multiple lectins, even though specific lectins to detect these glycan structures were not available. The result proved a previously proposed concept of lectin-based glycan profiling.     

Vitronectin (Vn) glycosylation patterned by lectin affinity assays—A potent glycoproteomic tool to discriminate plasma Vn from cancer ascites Vn

Changes in glycosylation have been associated with human cancer, but their complexity poses an analytical challenge. Ovarian cancer is a major cause of death in women because of an often late diagnosis. At least one‐third of patients presents ascites fluid at diagnosis, and almost all have ascites at recurrence. Vitronectin (Vn) is a multifunctional glycoprotein that is suggested to be implicated in ovarian cancer metastasis and is found within ascites. The present study evaluated the potential of using lectin affinity for characterizing the glycosylation pattern of Vn. Human Vn was purified from 1 sample of ovarian cancer ascites or a pool of plasma samples. Consistent findings were observed with both dot blot and lectin array assays. Based on a panel of 40 lectins, the lectin array revealed discriminant patterns of lectin binding to Vn glycans. Interestingly, almost all the highlighted interactions were found to be higher with Vn from ascites relative to the plasma counterpart. Also, the lectin array was able to discriminate profiles of lectin interactions (ConA, SNA‐I, PHA‐E, PHA‐L) between Vn samples that were not evident using dot blot, indicating its high sensitivity. The model of ConA binding during thermal unfolding of Vn confirmed the higher accessibility of mannosylated glycans in Vn from ascites as monitored by turbidimetry. Thus, this study demonstrated the usefulness of lectins and the lectin array as a glycoproteomic tool for high throughput and sensitive analysis of glycosylation patterns. Our data provide novel insights concerning Vn glycosylation patterns in clinical specimens, paving the way for further investigations regarding their functional impact and clinical interest.     

The use of lectin microarray for assessing glycosylation of therapeutic proteins

Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. Lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins.     

Glycome profiling by lectin microarray reveals dynamic glycan alterations during epidermal stem cell aging

Aging in the epidermis is marked by a gradual decline in barrier function, impaired wound healing, hair loss, and an increased risk of cancer. This could be due to age‐related changes in the properties of epidermal stem cells and defective interactions with their microenvironment. Currently, no biochemical tools are available to detect and evaluate the aging of epidermal stem cells. The cellular glycosylation is involved in cell–cell communications and cell–matrix adhesions in various physiological and pathological conditions. Here, we explored the changes of glycans in epidermal stem cells as a potential biomarker of aging. Using lectin microarray, we performed a comprehensive glycan profiling of freshly isolated epidermal stem cells from young and old mouse skin. Epidermal stem cells exhibited a significant difference in glycan profiles between young and old mice. In particular, the binding of a mannose‐binder rHeltuba was decreased in old epidermal stem cells, whereas that of an α2‐3Sia‐binder rGal8N increased. These glycan changes were accompanied by upregulation of sialyltransferase, St3gal2 and St6gal1 and mannosidase Man1a genes in old epidermal stem cells. The modification of cell surface glycans by overexpressing these glycogenes leads to a defect in the regenerative ability of epidermal stem cells in culture. Hence, our study suggests the age‐related global alterations in cellular glycosylation patterns and its potential contribution to the stem cell function. These glycan modifications detected by lectins may serve as molecular markers for aging, and further functional studies will lead us to a better understanding of the process of skin aging.     

Lectin microarray profiling of metastatic breast cancers

Altered protein glycosylation compared with the disease-free state is a universal feature of cancer cells. It has long been established that distinct glycan structures are associated with specific forms of cancer, but far less is known about the complete array of glycans associated with certain tumors. The cancer glycome has great potential as a source of biomarkers, but progress in this field has been hindered by a lack of available techniques for the elucidation of disease-associated glycosylation. In the present study, lectin microarrays consisting of 45 lectins with different binding preferences covering N- and O-linked glycans were coupled with evanescent-field activated fluorescent detection in the glycomic analysis of primary breast tumors and the serum and urine of patients with metastatic breast cancer. A single 50 μm section of a primary breast tumor or <1 μL of breast cancer patient serum or urine was sufficient to detect glycosylation alterations associated with metastatic breast cancer, as inferred from lectin-binding patterns. The high-throughput, sensitive and relatively simple nature of the simultaneous analysis of N- and O-linked glycosylation following minimal sample preparation and without the need for protein deglycosylation makes the lectin microarray analysis described a valuable tool for discovery phase glycomic profiling.     

应用凝集素芯片分析癌细胞膜表面糖链表达

细胞膜表面糖复合物的糖链结构与肿瘤细胞增殖、侵染、转移等发展过程密切相关.凝集素芯片技术的出现实现了对癌症的糖组进行快速、高通量的检测.通过模式细胞系PANC-1证明了构建的凝集素芯片体系的准确性、重复性、特异性,应用这一芯片体系初步检测了几种癌细胞系(HT-29、SGC-7901、BEL-7402、H460)的膜表面糖链表达.这几种癌细胞系表面都有唾液酸、乙酰葡萄糖/葡萄糖、乙酰半乳糖/半乳糖、甘露糖等糖链.根据实验结果,推测它们的细胞膜表面α1-6岩藻糖链表达水平可能较高,而α1-3岩藻糖链表达水平较低;这些聚糖可能是癌症潜在的标志物.凝集素芯片有助于推动癌细胞膜表面糖链的快速分析和筛选出癌症相关的糖链标志物.     

Lectin microarrays identify cell-specific and functionally significant cell surface glycan markers

Glycosylation is among the most complex posttranslational modifications with an extremely high level of diversity that has made it refractory to high-throughput analyses. Despite its resistance to high-throughput techniques, glycosylation is important in many critical cellular processes that necessitate a productive approach to their analysis. To facilitate studies in glycosylation, we developed a high-throughput lectin microarray for defining mammalian cell surface glycan signatures. Using the lectin microarray we established a binary analysis of cell binding and hierarchical organization of 24 mammalian cell lines. The array was also used to document changes in cell surface glycosylation during cell development and differentiation of primary murine immune system cells. To establish the biological and clinical importance of glycan signatures, the lectin microarray was applied in two systems. First, we analyzed the cell surface glycan signatures and were able to predict mannose-dependent tropism using a model pathogen. Second, we used the glycan signatures to identify novel lectin biomarkers for cancer stem-like cells in a murine model. Thus, lectin microarrays are an effective tool for analyzing diverse cell processes including cell development and differentiation, cell-cell communication, pathogen-host recognition, and cell surface biomarker identification.     

An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feeding

Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub‐array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N‐glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon‐γ (IFN‐γ). Galactose (±uridine), glucosamine (±uridine), and N‐acetylmannosamine (ManNAc) (±cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN‐γ sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP‐Hex (～20‐fold), UDP‐HexNAc (6‐ to 15‐fold) and CMP‐sialic acid (30‐ to 120‐fold), respectively. Up‐regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP‐HexNAc and CMP‐sialic acid by another two‐ to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN‐γ sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine‐ and cytidine‐activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality. Biotechnol. Bioeng. 2010;107: 321–336. © 2010 Wiley Periodicals, Inc.     

Piezoelectric printing and probing of Lectin NanoProbeArrays for glycosylation analysis

Glycans have great potential as disease biomarkers and therapeutic targets. However, the major challenge for glycan biomarker identification from clinical samples is the low abundance of key glycosylated proteins. To demonstrate the potential for glycan analysis with nanoliter amounts of glycoprotein, we have developed a new technology (Lectin NanoProbeArray) based on piezoelectric liquid dispensing for non-contact printing and probing of a lectin array. Instead of flooding the glycoprotein probe on the lectin array surface, as in conventional microarray screening, a piezoelectric printer is used to dispense nanoliters of fluorescently labeled glycoprotein probe over the lectin spots on the array. As a proof-of-concept, the ability of Lectin NanoProbeArrays to precisely identify and reliably distinguish between the closely related glycoforms of fetuin is illustrated here. Sensitivity levels comparable to lectin arrays that use evanescent-field scanners was achieved along with several orders of magnitude reduction in the amount of probe required for glycosylation analysis.     

Differential carbohydrate binding and cell surface glycosylation of human cancer cell lines

Currently there is only a modest level knowledge of the glycosylation status of immortalised cell lines that are commonly used in cancer biology as well as their binding affinities to different glycan structures. Through use of glycan and lectin microarray technology, this study has endeavoured to define the different bindings of cell surface carbohydrate structures to glycan-binding lectins. The screening of breast cancer MDA-MB435 cells, cervical cancer HeLa cells and colon cancer Caco-2, HCT116 and HCT116-FM6 cells was conducted to determine their differential bindings to a variety of glycan and lectin structures printed on the array slides. An inverse relationship between the number of glycan structures recognised and the variety of cell surface glycosylation was observed. Of the cell lines tested, it was found that four bound to sialylated structures in initial screening. Secondary screening in the presence of a neuraminidase inhibitor (4-deoxy-4-guanidino-Neu5Ac2en) significantly reduced sialic acid binding. The array technology has proven to be useful in determining the glycosylation signatures of various cell-lines as well as their glycan binding preferences. The findings of this study provide the groundwork for further investigation into the numerous glycan-lectin interactions that are exhibited by immortalised cell lines.     

Impact of cultivation conditions on N‐glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture

Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor‐binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N‐glycosylation occupancy as well as glycan composition). In this study, capillary gel electrophoresis with laser‐induced fluorescence detection (CGE‐LIF) based glycoanalysis (N‐glycan fingerprinting) was used to determine the impact of cultivation conditions on the HA N‐glycosylation pattern of Madin–Darby canine kidney (MDCK) cell‐derived influenza virus A PR/8/34 (H1N1). We found that adaptation of adherent cells to serum‐free growth has only a minor impact on the HA N‐glycosylation pattern. Only relative abundances of N‐glycan structures are affected. In contrast, host cell adaptation to serum‐free suspension growth resulted in significant changes in the HA N‐glycosylation pattern regarding the presence of specific N‐glycans as well as their abundance. Further controls such as different suppliers for influenza virus A PR/8/34 (H1N1) seed strains, different cultivation scales and vessels in standard or high cell density mode, different virus production media varying in either composition or trypsin activity, different temperatures during virus replication and finally, the impact of β‐propiolactone inactivation resulted—at best—only in minor changes in the relative N‐glycan structure abundances of the HA N‐glycosylation pattern. Surprisingly, these results demonstrate a rather stable HA N‐glycosylation pattern despite various (significant) changes in upstream processing. Only the adaptation of the production host cell line to serum‐free suspension growth significantly influenced HA N‐glycosylation regarding both, the type of attached glycan structures as well as their abundances. Biotechnol. Bioeng. 2013; 110: 1691–1703. © 2013 Wiley Periodicals, Inc.     

Enrichment and identification of glycoproteins in human saliva using lectin magnetic bead arrays

Aberrant glycosylation of proteins is a hallmark of tumorigenesis and could provide diagnostic value in cancer detection. Human saliva is an ideal source of glycoproteins due to the relatively high proportion of glycosylated proteins in the salivary proteome. Moreover, saliva collection is noninvasive and technically straightforward, and the sample collection and storage is relatively easy. Although differential glycosylation of proteins can be indicative of disease states, identification of differential glycosylation from clinical samples is not trivial. To facilitate salivary glycoprotein biomarker discovery, we optimized a method for differential glycoprotein enrichment from human saliva based on lectin magnetic bead arrays (saLeMBA). Selected lectins from distinct reactivity groups were used in the saLeMBA platform to enrich salivary glycoproteins from healthy volunteer saliva. The technical reproducibility of saLeMBA was analyzed with liquid chromatography–tandem mass spectrometry (LC–MS/MS) to identify the glycosylated proteins enriched by each lectin. Our saLeMBA platform enabled robust glycoprotein enrichment in a glycoprotein- and lectin-specific manner consistent with known protein-specific glycan profiles. We demonstrated that saLeMBA is a reliable method to enrich and detect glycoproteins present in human saliva.     

N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

Studies of protein N‐glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N‐glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N‐glycan patterns as documented using mass spectrometry and glycan‐recognising antibodies, indicating successful identification of null mutations in the target glyco‐genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3‐fucosyltransferase (Lj3fuct) mutant completely lacked α1,3‐core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N‐glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N‐acetylglucosaminyltransferase I, and α1,3‐fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N‐glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N‐glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian‐like N‐glycosylation features.     

Focused differential glycan analysis with the platform antibody-assisted lectin profiling for glycan-related biomarker verification

Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery.     

The transmembrane domain of N –acetylglucosaminyltransferase I is the key determinant for its Golgi subcompartmentation

Golgi‐resident type–II membrane proteins are asymmetrically distributed across the Golgi stack. The intrinsic features of the protein that determine its subcompartment‐specific concentration are still largely unknown. Here, we used a series of chimeric proteins to investigate the contribution of the cytoplasmic, transmembrane and stem region of Nicotiana benthamiana N–acetylglucosaminyltransferase I (GnTI) for its cis/medial‐Golgi localization and for protein–protein interaction in the Golgi. The individual GnTI protein domains were replaced with those from the well‐known trans‐Golgi enzyme α2,6–sialyltransferase (ST) and transiently expressed in Nicotiana benthamiana. Using co‐localization analysis and N–glycan profiling, we show that the transmembrane domain of GnTI is the major determinant for its cis/medial‐Golgi localization. By contrast, the stem region of GnTI contributes predominately to homomeric and heteromeric protein complex formation. Importantly, in transgenic Arabidopsis thaliana, a chimeric GnTI variant with altered sub‐Golgi localization was not able to complement the GnTI‐dependent glycosylation defect. Our results suggest that sequence‐specific features in the transmembrane domain of GnTI account for its steady‐state distribution in the cis/medial‐Golgi in plants, which is a prerequisite for efficient N–glycan processing in vivo.     

综述: 卵巢癌生物标志物的糖链谱研究进展

糖类抗原125(CA125)被认为是卵巢癌诊断的“金标准”,但在临床应用中普遍存在着特异性不高的问题．肿瘤形成和发展过程中常伴有糖基化修饰异常和糖链结构的改变,不同的肿瘤具有特异的异常糖链结构．近年来,借助凝集素芯片、多重质谱分析等糖蛋白组学和糖组学研究技术,发现不同来源CA125的O-糖链和N-糖链结构存在着明显的微观不均一性,以这些特征性糖链结构为标志物,可以显著提高CA125对卵巢癌的诊断特异性．在过去的10年,研究者们除对CA125糖链结构和糖基化模式做了深入的研究外,还利用糖组的研究方法,直接对来自卵巢癌患者血液、体液(腹水、囊泡液等)中糖蛋白的糖链做了精细的结构解析,结果显示,可有效鉴别卵巢癌患者和健康志愿者的特异性N-糖链结构,有可能成为灵敏度高和特异性好的卵巢癌生物标志物．卵巢癌生物标志物研究发展的总趋势是从传统的对蛋白质的定性和定量研究,逐步转向于对标志物糖基化修饰和特异性糖链结构的鉴定以及定量分析．本文从糖组学的视角,对卵巢癌标志物糖组学的研究现状及发展趋势进行了综述和展望．     

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control

The glycoside hydrolase family 31 (GH31) α‐glucosidases play vital roles in catabolic and regulated degradation, including the α‐subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N‐glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β‐subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα‐binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme.