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1.
Fungal Genet Biol ; : 103285, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31648060

RESUMO

Protein O-mannosyltransferases (PMTs) have been identified in fungi but not in plants and nematodes, which makes PMTs become attractive targets for developing a new strategy against phytopathogens. Three PMTs have been identified in Fusarium oxysporum, a fungal pathogen that causes vascular wilt in a broad range of economical crops. By deletion or suppression of the pmt genes, we showed that all mutants displayed retarded growth, reduced conidiation, cell wall defects, ER stress and attenuated virulence in F. oxysporum f.sp. cucumerinum. In addition, the Δpmt1 exhibited reduced thermotolerance, while the Δpmt4 and the pmt2 conditional mutant exhibited abnormal polarized growth. Comparative glycoproteome analysis of these pmt mutants revealed that PMTs preferentially modified random coils with flanking regions rich in Ser, Thr, Ala, Glu, Asp and Lys at the stem region of membrane proteins, the N-terminal region close to signal peptide of secreted proteins, or surface of soluble proteins. PMT1 specifically acted on nuclear proteins and proteins that are responsible for protein folding, which might contribute to thermotolerance. PMT4 specifically acted on the membrane and soluble proteins in secretory pathways, especially the GPI anchoring pathway, which might contribute to synthesis and transportation of GPI anchored proteins and thus polarized growth. PMT2 was responsible for modification of proteins that are required for protein folding and cell wall synthesis, which might make PMT2 essential. Our results gave an insight to understanding of the roles of each O-mannosyltransferase in F. oxysporum f.sp. cucumerinum and provide a new perspective to prevent Fusarium wilt.

2.
J Proteome Res ; 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31647243

RESUMO

The major protein in Chinese yam (Dioscorea opposita Thunb.) glycoprotein, 30CYGP, exhibits strong immunomodulatory activities. Research has identified the sequence and structure of 30CYGP; however, 30CYGP N-glycoform composition and immunoactivity remain unknown. We isolated and purified 30CYGP from Chinese yam and used that material to release the N-glycans contained within. The N-glycans were labeled with 1-phenyl-3-methyl-5-pyrazolone and analyzed via ESI-MS and online LC-MS. Additionally, the immunoactivities of 30CYGP and de-glycosylated 30CYGP in the RAW264.7 cell line were investigated. Six 30CYGP N-glycans were observed in total, in which three were modified with xylose (XM: 40%) and three with xylose and fucose (XFM: 60%). Furthermore, de-glycosylated 30CYGP had significantly weaker immunoactivity than 30CYGP. This study demonstrated that novel N-glycoforms may enhance 30CYGP immunoactivity. Further research on the role of varied glycosylation patterns in immunoactivity is needed.

3.
J Agric Food Chem ; 67(38): 10702-10712, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31490688

RESUMO

Human milk oligosaccharides are complex carbohydrates with multibiofunctional health benefits to newborns. Human milk free oligosaccharides (HMOs) are well characterized. However, changes in the N/O-glycome during lactation are poorly reported. Herein, we qualitatively and quantitatively investigated N/O-glycome profiles and their alteration in human milk at different lactation stages. N-Glycans were mainly fucosylated and nonsialylated, nonfucosylated throughout lactation. O-Glycans mainly consisted of sialylated and nonsialylated, nonfucosylated in colostrum and transitional milk, and fucosylated and nonfucosylated, nonsialylated in mature milk. Fucosylated and sialylated N-glycans gradually decreased and increased, respectively, as lactation progressed; O-glycans showed the reverse. Interestingly, changes in HMO abundance decreased during lactation, complementing HMG N/O-glycome changes. In conclusion, temporal HMG glycosylation changes provide the groundwork for developing infant formula that is closer to breast milk at different lactation stages.


Assuntos
Glicoproteínas/química , Lactação , Leite Humano/química , Adulto , Colostro/química , Feminino , Glicoproteínas/metabolismo , Glicosilação , Humanos , Espectrometria de Massas , Leite Humano/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo
4.
Anal Biochem ; 582: 113355, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276651

RESUMO

Quantitative analysis of glycosphingolipids (GSLs) has been hindered by the lack of chromogenic groups for spectral detection or active functional groups for specific derivatization. In this study, a highly sensitive method based on ozonolysis-induced release and isotopic Girard's reagent P labeling of GSL glycans coupled with mass spectrometric detection for the quantification of animal tissue GSLs is developed. First, different approaches for the release of glycans from GSLs were compared with each other and the ozonolysis-based method was found to have the highest glycan yield under relative mild reaction conditions. Then a relative quantification method of ozonolysis-released GSL glycans based on stable isotope labeling using nondeuterated (d0-) and 2,3,4,5,6-pentadeuterated (d5-) Girard's reagent P (GP) was established, and its good linearity, accuracy and reproducibility were statistically verified. Finally, the new method was successfully applied to revealing the difference between porcine brain and liver as well as between the brain of normal and aging rats in GSL glycome by online hydrophilic interaction liquid chromatography coupling with ultraviolet detection and tandem mass spectrometry (HILIC-UV-MS/MS). This novel method is versatile and sensitive, enabling accurate quantitative analysis of tissue GSLs and showing great significance for glycomic studies.

5.
J Agric Food Chem ; 67(32): 8958-8966, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31334644

RESUMO

The functional role of human milk oligosaccharides (HMOs) is closely associated with their type, composition, and structure. However, a detailed analysis of HMOs is difficult because neutral oligosaccharides (NHMOs) are mixed with sialylated oligosaccharides (SHMOs) in milk. Here, NHMOs were separated from SHMOs by DEAE-52 anion chromatography, and lactose was removed by graphite carbon solid-phase extraction. Lactose-free NHMOs were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) based on Girard's reagent P on-target derivatization (GPOD), and SHMOs were analyzed by MALDI-TOF-MS following selective sialic acid derivatization and GPOD. Sixty-four oligosaccharides were detected: 36 NHMOs, of which 28 were fucosylated, and 28 SHMOs, of which 8 with α-2,3-linked monosialic acid, 2 with α-2,3-linked disialic acid, 10 with α-2,6-linked monosialic acid, 2 with α-2,6-linked disialic acid, and 5 with both α-2,3- and α-2,6-linked disialic acid. These findings provide the groundwork for further characterization of the structure and activity of HMOs.


Assuntos
Betaína/análogos & derivados , Leite Humano/química , Oligossacarídeos/química , Betaína/química , Feminino , Humanos , Ácido N-Acetilneuramínico/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
6.
Food Funct ; 10(7): 4231-4241, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31259337

RESUMO

The fruits of Lycium barbarum are considered medicinal foods with high nutritional value and bioactivity. In this study, we aimed to evaluate the effect of a crude L. barbarum polysaccharide (LBP) and two derived fractions, LBP-1 and LBP-2, on the lifespan of Drosophila melanogaster (fruit fly). The average lifespan of fruit flies was extended by supplementing their diet with either of the three LBP preparations. In vivo analysis of antioxidant activities detected increased superoxide dismutase (SOD) and catalase (CAT) activities and decreased malondialdehyde (MDA) levels. Dietary LBP supplements significantly reduced the mortality rate of fruit flies induced by paraquat and hydrogen peroxide. Importantly, the strongest anti-aging activity was exhibited by the LBP-2 fraction, containing arabinogalactan with a molecular weight of 9 × 104 Da. Further studies showed that the anti-aging activity of LBP was, at least in part, mediated by an age-related signaling pathway (MAPK, TOR, S6K) and the expression of longevity genes (Hep, MTH, and Rpn11).

7.
Carbohydr Polym ; 222: 115009, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31320075

RESUMO

We obtained four soluble acid xylan fractions AGP-III-A, AGP-III-B, AGP-III-C and AGP-III-D from the insoluble Artemisia sphaerocephala Krasch gum (ASKG) polysaccharide by weak alkali treatment combined with H2O2-Vc oxidative degradation. Activity studies showed that the degradation components could reduce the cell viability of several cancer cell lines in a concentration-dependent manner, especially 4-O-Methylglucuronoxylan AGP-III-C with specific molecular weight and branching degree significantly reduced cancer cells viability and induced HepG2 apoptosis, also caused mitochondrial membrane dysfunction upregulated ROS levels, and induced G0/G1 arrest in HepG2 cells by cell cycle assay. Further, AGP-III-C mediates apoptosis in HepG2 cells by upregulating MAPK phosphorylation. The structure of AGP-III-C was characterized by uronic acid reduction, permethylation with GC-MS, and 2D-NMR analysis. The structure of AGP-III-C had a linear (1→4)-linked ß-Xylf residue backbone with one branched 4-O-Me-α-GlcAp attached to the main chain by a (1→2)-glycosidic bond at every two ß-(1→4)-Xylf units.

8.
Anal Chem ; 91(16): 10492-10500, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31329418

RESUMO

Changes of α-2,3-/α-2,6-linked sialic acids (SAs) in sialylglycans have been found to be closely related with some diseases. However, accurate quantification of sialylglycans at the isomeric level remains challenging due to their instability, structural complexity, and low mass spectrometry (MS) detection sensitivity. Herein, we propose an analytical strategy named "glycoqueuing", which allows sequential chromatographic elution and high-sensitivity MS quantification of various sialylglycan isomers based on isotopic labeling followed by analysis via online reversed-phase high performance liquid chromatography coupling with MS (RP-HPLC-MS). The new method was validated by detailed structural identification and quantification of fetal bovine serum (FBS) N-linked sialylglycan isomers, during which many branching isomers were successfully differentiated, and 28 sialylglycan compositions with Neu5Gc residues were analyzed. The method was successfully applied to isomer-specific, quantitative comparison of sialylated N-glycans between bovine and rabbit immunoglobulin G (IgG) and the search for serum sialylated N-glycan biomarker candidates of hepatocellular carcinoma, during which a 55% increase of α-2,6-sialylated fucosylated N-glycans was revealed, demonstrating the great applicability and potential clinical usage of the method.

9.
MBio ; 10(2)2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940702

RESUMO

The maternal milk glycobiome is crucial for shaping the gut microbiota of infants. Although high core fucosylation catalyzed by fucosyltransferase 8 (Fut8) is a general feature of human milk glycoproteins, its role in the formation of a healthy microbiota has not been evaluated. In this study, we found that the core-fucosylated N-glycans in milk of Chinese mothers selectively promoted the colonization of specific gut microbial groups, such as Bifidobacterium spp. and Lactobacillus spp. in their breast-fed infants during lactation. Compared with Fut8+/+ (WT) mouse-fed neonates, the offspring fed by Fut8 +/- maternal mice had a distinct gut microbial profile, which was featured by a significant reduction of Lactobacillus spp., Bacteroides spp., and Bifidobacterium spp. and increased abundance of members of the Lachnospiraceae NK4A136 group and Akkermansia spp. Moreover, these offspring mice showed a lower proportion of splenic CD19+ CD69+ B lymphocytes and attenuated humoral immune responses upon ovalbumin (OVA) immunization. In vitro studies demonstrated that the chemically synthesized core-fucosylated oligosaccharides possessed the ability to promote the growth of tested Bifidobacterium and Lactobacillus strains in minimal medium. The resulting L-fucose metabolites, lactate and 1,2-propanediol, could promote the activation of B cells via the B cell receptor (BCR)-mediated signaling pathway.IMPORTANCE This study provides novel evidence for the critical role of maternal milk protein glycosylation in shaping early-life gut microbiota and promoting B cell activation of neonates. The special core-fucosylated oligosaccharides might be promising prebiotics for the personalized nutrition of infants.


Assuntos
Linfócitos B/imunologia , Bifidobacterium/metabolismo , Fucose/metabolismo , Trato Gastrointestinal/imunologia , Lactobacillus/metabolismo , Leite Humano/química , Polissacarídeos/metabolismo , Animais , China , Feminino , Trato Gastrointestinal/microbiologia , Humanos , Lactente , Ativação Linfocitária , Camundongos , Polissacarídeos/química
10.
J Cell Biochem ; 120(8): 13372-13381, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30920024

RESUMO

Herein, we found that serum chemokine ligand 14 (CXCL14) was significantly enhanced in patients with idiopathic pulmonary fibrosis (IPF). In our current study, mouse L929 fibroblasts were stimulated with lipopolysaccharide (LPS) (100 ng/mL). Cell proliferation, the levels of matrix metalloproteinase 2 (MMP2) and MMP9, as well as extracellular matrix (ECM) content were assessed to evaluate the fibrogenesis of L929 cells. Proliferating cell nuclear antigen and cell viability were assessed to evaluate cell proliferation. Hydroxyproline (Hyp), collagen I/III, connective tissue growth factor (CTGF), and phosphorylated Smad2/3 (p-Smad2/3) were assessed to evaluate ECM secretion and deposition. α-Smooth muscle actin (α-SMA) was used to measure the occurrence of differentiation from fibroblast toward myofibroblast. Our data suggested that knockdown of CXCL14 prevented LPS-induced fibrogenesis of L929 cells through inhibiting cell proliferation and decreasing the expression of MMP2/9, Hyp, collagen I/III, CTGF, p-Smad2/3, and α-SMA. Notably, upregulation of protein phosphatase magnesium-dependent 1A (PPM1A) was involved in this process. On the contrary, recombinant CXCL14 protein led to an opposite effect. We first suggested that overexpression of PPM1A ameliorated LPS-induced fibrogenesis. Furthermore, we substantiated that knockdown of CXCL14 exerted an antifibrotic effect in IPF in vitro probably via the upregulation of PPM1A. Besides, evidently enhanced CXCL14, yet reduced PPM1A, was found in bleomycin-induced rat pulmonary fibrosis, confirming the roles of CXCL14 and its potential association with PPM1A in IPF in vivo. In conclusion, CXCL14 could be considered as a therapeutic target for preventing fibrogenesis of mouse L929 fibroblasts.

11.
Int J Biol Macromol ; 131: 744-751, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30904534

RESUMO

Schisandra sphenanthera and Schisandra chinensis are widely consumed either as food or for medicinal purposes. Nevertheless, no detailed comparative assessments of their physicochemical properties and biological activity have been reported. In this paper, using hot-water extraction, alcohol precipitation, and deproteinization, we obtained polysaccharidic extracts from Schisandra sphenanthera and Schisandra chinensis (denoted as SSP and SCP, respectively) and investigated their antioxidant and immunological activities. The extracts were different from each other with regard to sugar, protein, and uronic acid contents. Both extracts were mainly composed of arabinose, glucose, and galactose, but their contents varied greatly; SSP had more galacturonic acid. Compared with SCP, SSP had stronger free radical scavenging ability, protective effects on biomolecules, cellular antioxidant activity, owing to its higher protein (35.35 ±â€¯1.73%) and uronic acid (12.81 ±â€¯1.15%) contents. With respect to cell viability, neutral red phagocytosis, NO production, and acid phosphatase activity, SCP had stronger effects than SSP; this was largely due to its high levels of mannose, galactose, arabinose, and glucose. These results provide evidence to support the use Schisandra-derived polysaccharides for several purposes, including clinical, agricultural, and industrial applications.


Assuntos
Antioxidantes/química , Antioxidantes/farmacologia , Fenômenos Químicos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Polissacarídeos/química , Polissacarídeos/farmacologia , Schisandra/química , Animais , Antioxidantes/isolamento & purificação , Biomarcadores , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/isolamento & purificação , Depuradores de Radicais Livres/farmacologia , Imunomodulação/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Extração Líquido-Líquido , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Monossacarídeos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/isolamento & purificação , Polissacarídeos/isolamento & purificação , Análise Espectral
12.
Anal Chim Acta ; 1048: 105-114, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30598139

RESUMO

Sensitive glycomics analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is of great importance but significantly hampered by their low ionization efficiency and labile sialic acid moieties. Chemical derivatization offers a viable way to improve both the ionization efficiency and analytical sensitivity of the glycans in MS analysis by altering their hydrophobicity or charge property. Here we employed Girard's reagent T (GT) for on-target derivatization (GTOD) of reducing glycan under mild acid condition to form stable hydrazones at room temperature, allowing rapid and sensitive identification of neutral and sialylated glycans in positive-ion mode as only permanently positive charged molecular ions without multiple ion adducts by MALDI-TOF-MS. The MS signal intensities of lactose, sialylated N-glycans derived from bovine fetuin and neutral N-glycans derived from RNaseB and ovalbumin were boosted by 7.44, 9.13, 12.96 and 13.47 folds on average (n = 3), respectively. More importantly, after GTOD strategy, unwanted desialylation of sialylated glycans during MS was suppressed. The detection limit of the assay is desirable since the nanogram of N-glycans derived from 0.16 µg ovalbumin could be detected. The assay demonstrated good stability (RSD≤2.95%, within 10 days), reliable reproducibility (RSD = 2.96%, n = 7) and a desirable linear dynamic range from 78 nmol/mL to 10 µmol/mL. The strategy has been successfully applied to MS analysis of reducing glycans from human milks, neutral and sialylated O-, N-glycans from glycoproteins, and reducing glycans derived from glycosphingolipids, presenting neater [M]+ signals which allow detection of more low-abundance glycans and assignation of Neu5Ac vs. Neu5Gc or fucose vs. hexose in glycans due to the absence of the ambiguous interpretation from multiple peaks (ion adducts [M+Na]+ and [M+K]+). Moreover, the GTOD assay prevents desialylation during MALDI-TOF-MS profiling and enables distinct linkage-specific characterization of terminal sialic acids of N-glycans derived from human serum protein when combines with an esterification.


Assuntos
Betaína/análogos & derivados , Glicômica/métodos , Oligossacarídeos/química , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Betaína/química , Proteínas Sanguíneas/química , Feminino , Glicoproteínas/química , Glicoesfingolipídeos/química , Humanos , Proteínas do Leite/química , Leite Humano/química , Reprodutibilidade dos Testes , Ácidos Siálicos/química
13.
Biochemistry ; 58(8): 1120-1130, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30661358

RESUMO

The glycan moiety of glycoproteins plays key roles in various biological processes. However, there are few versatile methods for releasing, separating, and recovering monomeric reducing N-glycans for further functional analysis. In this study, we developed a new method to achieve the release, separation, and recovery of monomeric reducing N-glycans using enzyme E (Pronase E) combined with 9-chloromethyl chloroformate (Fmoc-Cl) and glycosylasparaginase (GA). Ovalbumin, ribonuclease B, ginkgo, and pine nut glycoproteins were used as materials and sequentially enzymatically hydrolyzed with Pronase E, derivatized with Fmoc-Cl, and enzymatically hydrolyzed with GA. The products produced by this method were then detected by electrospray ionization mass spectrometry, high-performance liquid chromatography (HPLC), and online hydrophilic interaction chromatography (HILIC-MS) separation. The results showed that all N-glycans with essentially one amino acid obtained with Pronase E were labeled with Fmoc-Cl and could be efficiently separated and detected via HPLC and HILIC-MS. Finally, the isolated Asn-glycan derivatives were digested with GA, enabling the recovery of all monomeric reducing N-glycans modified by core α-1,3 fucose. This method was simple, inexpensive, and broadly applicable and could therefore be quite important for analysis of the structure-function relationships of glycans.

14.
Bioconjug Chem ; 29(11): 3847-3855, 2018 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-30380836

RESUMO

The advancement of glycoscience is critically dependent on the access to a large number of glycans for their functional study. Naturally occurring glycans are considered a viable source for diverse and biologically relevant glycan libraries. A mixture of free reducing glycans released from natural sources can be fluorescently tagged and separated by chromatography to produce a natural glycan library. Anthranilic acid (AA) has been widely used to fluorescently tag reducing glycans for HPLC or LC/MS analysis. However, AA conjugated glycans are not efficiently immobilized on microarray slides due to the lack of a primary alkylamine functional group. In this study, we have developed simple and efficient chemistry for bioconjugation and further functionalization of glycan-AA conjugates. This new approach enables quick preparation of glycan microarrays and neoglycoproteins from glycan-AA conjugates, which can be separated by weak anion exchange (WAX) and C18 reversed-phase HPLC.

15.
Glycoconj J ; 35(4): 411-420, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30196374

RESUMO

Despite the great significance of release and analysis of glycans from glycoproteins, the existing N-glycan release methods are undermined by some limitations and deficiencies. The traditional enzymatic protocols feature high N-glycan release specificity but are generally costly and inefficient for some types of N-glycans. The existing chemical methods require harsh reaction conditions or are accompanied by the remarkable formation of by-products. Herein, we describe a versatile chemical method for the release and analysis of N-glycans from glycoproteins. This method differs from the existing methods as only aqueous ammonia is used to catalyze the N-glycan release reactions. Optimization of reaction conditions was performed using RNase B as a model glycoprotein and the obtained results indicated a highest N-glycan yield in ammonia at 60 °C for 16 h. Comparison of this method with traditional enzymatic protocols and recently reported NaClO methods confirmed the good reliability and efficiency of the novel approach. We also successfully applied this method to some complex biological samples, such as Ginkgo seed protein, fetal bovine serum (FBS) and hen egg white, and demonstrated its great compatibility with various neutral N-glycans, core α-1,3-fucosylated N-glycans and sialylated N-glycans. This method is very simple and cost-effective, enabling convenient analysis and large-scale preparation of released reducing N-glycans from various biological samples for structural and functional glycomics studies.

16.
Oncol Lett ; 16(2): 2490-2494, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30013642

RESUMO

The aim of this study was to investigate the expression of FGF2 and FGFR2 in patients with idiopathic pulmonary fibrosis (IPF) and lung cancer (LC) as well as their clinical significance. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting were used to detect FGF2 and FGFR2 expression in LC and adjacent normal tissues of LC patients and lavage fluid of idiopathic pulmonary fibers patients and normal controls (confirmed by bronchoalveolar lavage examination). The expression levels of FGF2 mRNA and protein in the non-small cell LC tissues were significantly higher than those in the adjacent normal tissues (P<0.001). The expression level of FGF2 protein in lavage fluid of patients with IPF was higher than that of the control group (P<0.001). The expression level of FGFR2 mRNA in the non-small cell LC tissues was significantly higher than that in the adjacent normal tissues (P<0.001). The expression level of FGFR2 protein in the non-small cell LC tissues was higher than that in the adjacent normal lung tissues (P<0.001). The expression levels of FGF2 mRNA and FGFR2 mRNA in cancer tissues were not significantly correlated with age, sex and history of smoking (P>0.05), but were significantly correlated with lymph node metastasis, tumor differentiation and TNM staging. FGF2 and FGFR2 proteins were highly expressed in cancer tissues of LC patients and lavage fluid of patients with IPF. The expression of FGF2 mRNA and FGFR2 mRNA was correlated with lymph node metastasis and TNM stage. The high expression levels of FGF2 mRNA and FGFR2 mRNA were associated with tumor metastasis and poor prognosis of LC patients.

17.
J Proteomics ; 187: 47-58, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-29885470

RESUMO

Glycoproteins play pivotal roles in a series of biological processes and their glycosylation patterns need to be structurally and functionally characterized. However, the lack of versatile methods to release N-glycans as functionalized forms has been undermining glycomics studies. Here a novel method is developed for dissociation of N-linked glycans from glycoproteins for analysis by MS and online LC/MS. This new method employs aqueous ammonia solution containing NaBH3CN as the reaction medium to release glycans from glycoproteins as 1-amino-alditol forms. The released glycans are conveniently labeled with 9-fluorenylmethyloxycarbonyl (Fmoc) and analyzed by ESI-MS and online LC/MS. Using the method, the neutral and acidic N-glycans were successfully released without peeling degradation of the core α-1,3-fucosylated structure or detectable de-N-acetylation, revealing its general applicability to various types of N-glycans. The Fmoc-derivatized N-glycans derived from chicken ovalbumin, Fagopyrum esculentum Moench Pollen and FBS were successfully analyzed by online LC/MS to distinguish isomers. The 1-amino-alditols were also permethylated to form quaternary ammonium cations at the reducing end, which enhance the MS sensitivity and are compatible with sequential multi-stage mass spectrometry (MSn) fragmentation for glycan sequencing. The Fmoc-labeled N-glycans were further permethylated to produce methylated carbamates for determination of branches and linkages by sequential MSn fragmentation. SIGNIFICANCE OF THE STUDY: N-Glycosylation represents one of the most common post-translational modification forms and plays pivotal roles in the structural and functional regulation of proteins in various biological activities, relating closely to human health and diseases. As a type of informational molecule, the N-glycans of glycoproteins participate directly in the molecular interactions between glycan epitopes and their corresponding protein receptors. Detailed structural and functional characterization of different types of N-glycans is essential for understanding the functional mechanisms of many biological activities and the pathologies of many diseases. Here we describe a simple, versatile method to indistinguishably release all types of N-glycans as functionalized forms without remarkable side reactions, enabling convenient, rapid analysis and preparation of released N-glycans from various complex biological samples. It is very valuable for studies on the complicated structure-function relationship of N-glycans, as well as for the search of N-glycan biomarkers of some major diseases and N-glycan related targets of some drugs.

18.
J Proteome Res ; 17(7): 2345-2357, 2018 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-29775069

RESUMO

Most glycoproteins and biological protein samples undergo both O- and N-glycosylation, making characterization of their structures very complicated and time-consuming. Nevertheless, to fully understand the biological functions of glycosylation, both the glycosylation forms need to be analyzed. Herein we report a versatile, convenient one-pot method in which O- and N-glycans are simultaneously released from glycoproteins and chromogenically labeled in situ and thus available for further characterization. In this procedure, glycoproteins are incubated with 1-phenyl-3-methyl-5-pyrazolone (PMP) in aqueous ammonium hydroxide, making O-glycans released from protein backbones by ß-elimination and N-glycans liberated by alkaline hydrolysis. The released glycans are promptly derivatized with PMP in situ by Knoevenagel condensation and Michael addition, with peeling degradation almost completely prevented. The recovered mixture of O- and N-glycans as bis-PMP derivatives features strong ultraviolet (UV) absorbing ability and hydrophobicity, allowing for high-resolution chromatographic separation and high-sensitivity spectrometric detection. Using this technique, O- and N-glycans were simultaneously prepared from some model glycoproteins and complex biological samples, without significant peeling, desialylation, deacetylation, desulfation or other side-reactions, and then comprehensively analyzed by online HILIC-UV-ESI-MS/MS and RP-HPLC-UV-ESI-MS/MS, with which some novel O- and N-glycan structures were first found. This method provides a simple, versatile strategy for high-throughput glycomics analysis.

19.
Sci Rep ; 8(1): 4688, 2018 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-29549280

RESUMO

Milk oligosaccharides (MOs) are complex carbohydrates with multifunctional health benefits for the neonate. Poor reproductive performance in primiparous gilts limits their productivity. Changes in the structure and abundance of porcine MO (PMOs) through lactation with parity remains unknown and may explain superior new-born growth in litters from multiparous sows relative to gilts. We report 55 PMOs structures, of which 25 are new (17 sialylated and 8 neutral). Their incidence in gilt and sow colostrum was almost identical (53 vs. 54), but not in transitional milk (48 vs. 53) nor mature milk (41 vs. 47). These PMOs including neutral-, sialyl- and fucosyl- MOs in colostrum were more abundant in the gilt than the sow, but always decreased during lactation. Structural diversity decreased, although fucosylated MO were conserved. In conclusion, high diversity and levels of MO in porcine milk is parity dependent. Given the similarity between porcine and human MO profiles, our findings may help define key roles for MOs as potential dietary additives to improve growth of neonates from first pregnancies in both human and sows.

20.
Anal Biochem ; 549: 1-11, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29524379

RESUMO

Sensitive analysis of glycans by liquid chromatography/mass spectrometry is significantly hampered by the lack of chromogenic or fluorescent groups on the glycan structures, as well as, their poor ionization properties. In the present, a heterobifunctional chromogenic reagent 3-amino-1-phenyl-2-pyrazoline-5-ketone (PAP) bearing amino and active methylene groups, which readily reacts with reducing glycans, was used for detection of the pre-column-labeled glycans via high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). The PAP derivatives with active methylene and amino groups were obtained via reductive amination in acidic medium and condensation of an active PAP methylene group with the reducing end of glycans in alkaline medium, respectively, and the PAP derivatives could be further functionalized, e.g., via glycan microarray preparation. The conditions for the two reaction modes were optimized, the HPLC separation method of PAP derivatives was investigated, and the PAP derivatives of some glycans derived from biological samples were obtained and analyzed by ESI-MS and LC-MS. Using this new reagent, reducing glycans can be selectively derivatized by different reaction mechanisms, having great importance for functional glycomics studies.

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