Machine Learning Reveals Serum Glycopatterns as Potential Biomarkers for the Diagnosis of Nonalcoholic Fatty Liver Disease (NAFLD).

Nonalcoholic fatty liver disease (NAFLD) has emerged as the predominant chronic liver condition globally, and underdiagnosis is common, particularly in mild cases, attributed to the asymptomatic nature and traditional ultrasonography's limited sensitivity to detect early-stage steatosis. Consequently, patients may experience progressive liver pathology. The objective of this research is to ascertain the efficacy of serum glycan glycopatterns as a potential diagnostic biomarker, with a particular focus on the disease's early stages. We collected a total of 170 serum samples from volunteers with mild-NAFLD (Mild), severe-NAFLD (Severe), and non-NAFLD (None). Examination via lectin microarrays has uncovered pronounced disparities in serum glycopatterns identified by 19 distinct lectins. Following this, we employed four distinct machine learning algorithms to categorize the None, Mild, and Severe groups, drawing on the alterations observed in serum glycopatterns. The gradient boosting decision tree (GBDT) algorithm outperformed other models in diagnostic accuracy within the validation set, achieving an accuracy rate of 95% in differentiating the None group from the Mild group. Our research indicates that employing lectin microarrays to identify alterations in serum glycopatterns, when integrated with advanced machine learning algorithms, could constitute a promising approach for the diagnosis of NAFLD, with a special emphasis on its early detection.

Comprehensive analysis of glycosphingolipid glycans by lectin microarrays and MALDI-TOF mass spectrometry.

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates present on the cell membrane; they play significant roles in many bioprocesses such as cell adhesion, embryonic development, signal transduction and carcinogenesis. Analyzing such amphiphilic molecules is a major challenge in the field of glycosphingolipidomics. We provide a step-by-step protocol that uses a lectin microarray to analyze GSL glycans from cultured cells. The procedure describes (i) extraction of GSLs from cell pellets, (ii) N-monodeacylation using sphingolipid ceramide N-deacylase digestion to form lyso-GSLs, (iii) fluorescence labeling at the newly exposed amine group, (iv) preparation of a lectin microarray, (v) GSL-glycan analysis by a lectin microarray, (vi) complementary mass spectrometry analysis and (vii) data acquisition and analysis. This method is high-throughput, low cost and easy to conduct, and it provides detailed information about glycan linkages. This protocol takes ~10 d.

Abnormal Galactosylated-Glycans recognized by Bandeiraea Simplicifolia Lectin I in saliva of patients with breast Cancer.

Currently, the definitive diagnosis in breast cancer requires biopsy and histopathology, such the most effective markers are tissue-based. However, the advantages of saliva in collection and storage make it possible for assessing human pathology and contributing to the development of cancer-related biomarkers for clinical application. The present study validated alteration of salivary protein glycopatterns recognized by Bandeiraea simplicifolia lectin I (BS-I) in the saliva of patients with breast diseases using saliva microarrays, and the N/O-glycan profiles of their salivary glycoproteins isolated by the BS-I-magnetic particle conjugates from 259 female subjects (66 healthy volunteers (HV), 65 benign breast cyst or tumor patients (BB), 66 patients with breast cancer in stage I (BC-I) and 62 patients with breast cancer in stage II (BC-II)) were analyzed by MALDI-TOF/TOF-MS. The results showed that the expression level of galactosylated glycans recognized by BS-I was significantly increased in patients with breast cancer compared with HV (p < 0.05). Totally, there were 11/10, 10/19, 7/24 and 7/9 galactosylated N-/O-linked glycans were identified and annotated from the pooled salivary samples of HV, BB, BC-I and BC-II, respectively. One galactosylated N-glycan peak (m/z 2773.977), and 4 galactosylated O-glycan peaks (m/z 868.295, 882.243, 884.270 and 1030.348) were found only in BC-I. These findings could provide pivotal information on galactosylated N/O-linked glycans related to breast cancer, and promote the study of biomarkers for early-stage breast cancer based on precise alterations of galactosylated N/O-glycans in saliva.

Alterations in serum protein glycopatterns related to small cell lung cancer, adenocarcinoma and squamous carcinoma of the lung.

Background: The main reason why lung cancer has maintained a high rate of morbidity and mortality is that its early diagnosis is difficult. No current lung cancer screening is recommended by any major medical organization due to the lack of sensitive and specific screening technologies. Thus, this study aimed to systematically investigate the correlation between the alterations in serum glycosylation and three main types of lung cancers (SCLC, ADC and SqCC). Materials and methods: We investigated the protein glycopatterns in sera from 333 subjects (65 healthy volunteers, 38 benign lung disease patients, 49 small cell lung cancer patients, and 181 NSCLC patients) using a lectin microarray. A serum microarray was produced to evaluate and verify the terminal carbohydrate moieties of the glycoproteins in individual serum samples from 30 cases simultaneously. Results: There were 16 lectins (e.g., RCA120, BS-I, and UEA-I), 24 lectins (e.g., HHL, PTL-I, and MAL-II), and 18 lectins (e.g., GSL-I, LEL, and ACA) that exhibited significant differences in serum protein glycopatterns in the patients with SCLC, ADC and SqCC compared with the controls (HV and BPD). There were 6 lectins (e.g., EEL, NPA, and LEL) that exhibited significantly increased NFIs in ADC and SqCC compared with SCLC. Also, there were 5 lectins (e.g., Jacalin, BS-I, and UEA-I) that exhibited significantly decreased NFIs in ADC compared with SCLC and SqCC. Conclusions: This study can facilitate the discovery of potential biomarkers for the differential diagnosis of lung cancer based on the precise alteration in serum protein glycopatterns.

Integrated Glycome Strategy for Characterization of Aberrant LacNAc Contained N-Glycans Associated With Gastric Carcinoma.

Aberrant glycosylation is not only a feature of malignant cell transformation, but also plays an important role in metastasis. In the present study, an integrated strategy combining the lectin microarrays and lectin cytochemistry was employed to investigate and verify the altered glycopatterns in gastric cancer (GC) cell lines as well as resected tumor specimens from matched tissue sets of 46 GC patients. Subsequently, lectin-mediated affinity capture glycoproteins, and MALDI-TOF/TOF-MS were employed to further acquire precise structural information of the altered glycans. According to the results, the glycopatterns recognized by 10 (e.g., ACA, MAL-I, and ConA) and 3 lectins (PNA, MAL-I, and VVA) showed significantly variations in GC cells and tissue compared to their corresponding controls, respectively. Notably, the relative abundance of Galß-1,4GlcNAc (LacNAc) recognized by MAL-I exhibited a significant increase in GC cells (p < 0.001) and tissue from patients at stage II and III (p < 0.05), and a significant increase in lymph node positive tumor cases, compared with lymph node negative tumor cases (p < 0.05). More LacNAc contained N-glycans were characterized in tumor sample with advanced stage compared to corresponding control. Moreover, there were 10 neo-LacNAc-contained N-glycans (e.g., m/z 1625.605, 1803.652, and 1914.671) only presented in GC tissue with advanced stage. Among these, six N-glycans were modified with sialic acid or fucose based on LacNAc to form sialylated N-glycans or lewis antigens, respectively. Our results revealed that the aberrant expression of LacNAc is a characteristic of GC, and LacNAc may serve as a scaffold to be further modified with sialic acid or fucose. Our findings provided useful information for us to understand the development of GC.

Analysis of Glycosphingolipid Glycans by Lectin Microarrays.

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates of cell membranes. Identification of unknown GSL-glycan structures is still a major challenge. To address this challenge, we developed a novel strategy for analysis of GSL-glycans from cultured cells based on a lectin microarray that can directly detect and reveal glycopatterns of GSL extracts without the need for glycan release. There were six steps to perform the analysis of GSL-glycans: (i) extraction of GSLs from cell pellets, (ii) quantification of GSL-glycans using orcinol-sulfuric acid reaction, (iii) preparation of lyso-GSLs by using sphingolipid ceramide N-deacylase, (iv) fluorescence labeling of lyso-GSLs, (v) detection by a lectin microarray, (vi) data acquisition and analysis. Simultaneously, a supplementary verification analysis for GSL-glycans was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Optimized experimental conditions, which consisted of the blocking buffer, incubation buffer, and appropriate GSL concentration, were investigated by analyzing the glycopatterns of a standard ganglioside (GM1a) via lectin microarray. The analysis of GSL-glycans from human hepatocarcinoma cell lines (MHCC97L, MHCC97H, and HCCLM3) showed that there were 27 lectins (e.g., WFA, MAL-II, and LTL) to give significantly different signals compared with a normal human liver cell line (HL-7702), indicating up- and/or down-regulations of corresponding glycopatterns such as α1-2 fucosylation and α2-3 sialylation, and changes of certain glycostructures such as Galß1-3GalNAcß1-4(NeuAcα2-3)Galß1-4Glc:Cer and GalNAcα1-3(Fucα1-2)Galß1-3GlcNAcß1-3Galß1-4Glc:Cer. The lectin microarray analysis of lyso-GSLs labeled by fluorescence has proven to be credible, which can provide the glycopatterns and detailed linkage information on GSL-glycans.

Identification of abnormal fucosylated-glycans recognized by LTL in saliva of HBV-induced chronic hepatitis, cirrhosis, and hepatocellular carcinoma.

The hepatitis B virus (HBV)-induced chronic liver diseases are serious health threats worldwide. There is evidence to display the alterations of salivary N-linked glycans related to the development of HBV-infected liver diseases. Here, we further investigated the alterations of fucosylated N/O-glycans recognized by LTL in saliva from 120 subjects (30 healthy volunteers (HV), 30 patients with hepatitis B (HB), 30 patients with hepatic cirrhosis (HC), and 30 patients with hepatocellular carcinoma (HCC)) using salivary microarrys and MALDI-TOF/TOF-MS. The results showed that the expression level of fucosylated glycans recognized by LTL was significantly increased in HCC compared with other subjects (P < 0.0001). Besides, the fucosylated glycoproteins were isolated from pooled saliva of HV, HB, HC, and HCC by LTL-magnetic particle conjugates. Then, N/O- glycans were released from the isolated glycoproteins with PNGase F and NaClO, and were identified by MALDI-TOF-MS, respectively. Totally, there were 21/20, 25/18, 29/19, and 28/24 N/O-glycan peaks that were identified and annotated with proposed structures in saliva of HV, HB, HC, and HCC. Among the total, there were 8 N-glycan peaks (e.g., m/z 1905.634, 2158.777 and 2905.036) and 15 O-glycan peaks (e.g., 1177.407, 1308.444 and 1322.444) that only presented in patients with HBV-induced liver diseases. One N-glycan peak (m/z 2205.766) was unique in HC, and 9 O-glycan peaks (e.g., m/z 1157.420, 1163.417 and 1193.402) were unique in HCC. This study could facilitate the discovery of biomarkers for HC and HCC based on precise alterations of fucosylated N/O-glycans in saliva.

Increased expression of core-fucosylated glycans in human lung squamous cell carcinoma.

Lung cancer is the most frequent cancer and the leading cause of cancer around the world. As one of the major types of lung cancer, lung squamous cell carcinoma (LUSC) is closely associated with smoking and shows poor sensitivity to therapy and prognosis. Although alteration of glycopatterns are reliable indicators of cancer, little is known about the alterations of protein glycosylation related to LUSC. In this study, we compared the differential expression levels of glycopatterns in seven pairs of LUSC tissues and normal pericarcinomatous tissues (PCTs) using lectin microarrays. Fluorescence-based lectin histochemistry and lectin blotting were utilized to validate and assess the expression and distribution of certain glycans in LUSC tissues and PCTs. And we further analyzed their total N-linked glycans using MALDI-TOF/TOF-MS to provide more information about the aberrant glycopatterns. The results showed that the expression level of the core fucosylation recognized by Pisum sativum agglutinin (PSA) and Lens culinaris agglutinin (LCA) was significantly increased in LUSC tissues compared with PCTs. There were 10 and 15 fucosylated N-linked glycans that were detected in PCTs and LUSC tissues respectively, 10 fucosylated N-glycans were common, while five fucosylated N-glycans were unique to LUSC tissues. And the abundance of the fucosylated N-glycans was increased from 40.9% (PCTs) to 48.3% (LUSC). These finding is helpful to elucidate the molecular mechanisms underlying the lung diseases and develop new treatment strategies.

Characterization of proteins with Siaα2-3/6Gal-linked glycans from bovine milk and role of their glycans against influenza A virus.

The bovine milk proteins have a wide range of functions, but the role of the attached glycans in their biological functions has not been fully understood yet. Here, the glycopatterns of whole bovine milk proteins were analyzed using lectin microarrays. Then, the proteins with Siaα2-3/6Gal-linked glycans were isolated and characterized. The roles of Siaα2-3/6Gal-linked glycans of the isolated proteins were assessed by inhibiting viral activity against influenza A viruses (IAV). In total, there were 69 sialylated proteins to be identified and annotated. The sialylated proteins have the ability to inhibit the attachment of IAV mimics to MDCK cells; however, the role of inhibition was abolished when the sialic acid moieties were destroyed. The results demonstrate that the sialic acid moieties of proteins could serve as competitive substrates to disturb the viral attachment to cell surface receptors. Our findings will help to assess the potential application of sialylated glycoproteins in bovine milk against IAV.

Identification of N- and O-linked glycans recognized by AAL in saliva of patients with atrophic gastritis and gastric cancer.

BACKGROUND AND AIM: Gastric cancer (GC) is a common and fatal malignancy with a worldwide occurrence. There still lacks effective biomarkers for precisely evaluating GC. Saliva is a biological fluid with enormous diagnostic potentials which emerged many advantages. We aimed to discover the novel biomarkers for accurately distinguishing early GC based on saliva glycopatterns. METHODS: We used Aleuria Aurantia Lectin (AAL)-magnetic particle conjugates to isolate fucosylated glycoproteins in the pooled saliva of healthy volunteers (HV, n= 51) and patients with atrophic gastritis (AG, n= 51) or GC (n= 51), following to release the N- and O-linked glycans from the isolated proteins with PNGase F and NaClO, and further identified the released glycans by MALDI-TOF/TOF-MS, respectively. RESULTS: A total of 9/9, 8/11, and 9/9 fucosylated N-/O-linked glycans were annotated in the isolated salivary proteins from HV, AG, and GC, respectively. Among these, six fucosylated N-linked glycansand four O-linked glycans exhibited significantly increased expression levels in GC, while five fucosylated N-linked glycans and ten fucosylated O-linked glycans exhibited significantly decreased expression levels in GC. The proportion of fucosylated N-linked glycans was decreased in GC (41.66%) compared with AG (43.63%) and HV (52.57%), as well as the fucosylated O-linked glycans was apparently decreased in GC (19.58%) compared with AG (25.43%) and HV (55.54%). CONCLUSIONS: This study could provide pivotal information to distinguish among HV, AG, and GC, and facilitate the discovery of biomarkers for GC diagnosis based on precise alterations of N- and O-linked glycans in saliva.

Comparative Analysis for Glycopatterns and Complex-Type N-Glycans of Glycoprotein in Sera from Chronic Hepatitis B- and C-Infected Patients.

Background: Chronic infection with HBV (CHB) or HCV (CHC) is the most common chronic viral hepatitis that can lead to cirrhosis and hepatocellular carcinoma in humans, their infections have distinct pathogenic processes, however, little is known about the difference of glycoprotein glycopatterns in serum between hepatitis B virus (HBV)- and hepatitis C virus (HCV)-infected patients. Methods: A method combining the lectin microarrays, letin-mediated affinity capture glycoproteins, and MALDI-TOF/TOF-MS was employed to analyze serum protein glycopatterns and identify the glycan structures from patients with CHB (n = 54) or CHC(n = 47), and healthy volunteers (HV, n = 35). Lectin blotting was further utilized to validate and assess the expression levels of their serum glycopatterns. Finally, the differences of the glycoprotein glycopatterns were systematically compared between CHB and CHC patients. Conclusions: As a result, there were 11 lectins (e.g., HHL, GSL-II, and EEL) exhibited significantly increased expression levels, and three lectins (LCA, VVA, and ACA) exhibited significantly decreased expression levels of serum protein glycopatterns only in the CHB patients. However, DBA exhibited significantly decreased expression levels, and two lectins (WGA and SNA) exhibited significantly increased expression levels of serum glycopatterns only in the CHC patients. Furthermore, LEL and MAL-I showed a coincidentally increasing trend in both CHC and CHB patients compared with the HV. The individual analysis demonstrated that eight lectins (MPL, GSL-I, PTL-II, UEA-I, WGA, LEL, VVA, and MAL-I) exhibited a high degree of consistency with the pooled serum samples of HV, CHB, and CHC patients. Besides, a complex-type N-glycans binder PHA-E+L exhibited significantly decreased NFIs in the CHB compared with HV and CHC subjects (p < 0.01). The MALDI-TOF/TOF-MS results of N-linked glycans from the serum glycoproteins isolated by PHA-E+L-magnetic particle conjugates showed that there was an overlap of 23 N-glycan peaks (e.g., m/z 1419.743, 1663.734, and 1743.581) between CHB, and CHC patients, 5 glycan peaks (e.g., m/z 1850.878, 1866.661, and 2037.750) were presented in virus-infected hepatitis patients compared with HV, 3 glycan peaks (1460.659, 2069.740, and 2174.772) were observed only in CHC patients. Our data provide useful information to find new biomarkers for distinguishing CHB and CHC patients based on the precision alteration of their serum glycopatterns.

Salivary glycopatterns as potential biomarkers for diagnosis of gastric cancer.

Gastric cancer (GC) is still an extremely severe health issue with high mortality due to the lacking of effective biomarkers. In this study, we aimed to investigate the alterations of salivary protein glycosylation related to GC and assess the possibility of salivary glycopatterns as potential biomarkers for the diagnosis of GC. Firstly, 94 patients with GC (n = 64) and atrophic gastritis (AG) (n = 30), as well as 30 age- and sex-matched healthy volunteers (HV) were enrolled in the test group to probe the difference of salivary glycopatterns using lectin microarrays, the results were validated by saliva microarrays and lectin blotting analysis. Then, the diagnostic model of GC (Model GC) and AG (Model AG) were constructed based on 15 candidate lectins which exhibited significant alterations of salivary glycopattern by logistic stepwise regression. Finally, two diagnostic models were assessed in the validation group including HV (n = 30) and patients with GC (n = 23) and AG (n = 24) and achieved high diagnostic power (Model GC (AUC: 0.89, sensitivity: 0.96 and specificity: 0.80), Model AG (AUC: 0.83, sensitivity: 0.92 and specificity: 0.72)). This study provides pivotal information to distinguish HV, AG and GC based on precise alterations in salivary glycopatterns, which have great potential to be biomarkers for diagnosis of GC.