Centella asiatica (L.) Urban (Apiaceae), a small annual plant that grows in India, Sri Lanka, Malaysia, and other parts of Asia, is well-known as a medicinal herb with a long history of therapeutic uses. The bioactive compounds present in C. asiatica leaves include ursane-type triterpene sapogenins and saponins-asiatic acid, madecassic acid, asiaticoside, and madecassoside. Various bioactivities have been shown for these compounds, although most of the steps in the biosynthesis of triterpene saponins, including glycosylation, remain uncharacterized at the molecular level. This chapter describes an approach that integrates partial enzyme purification, proteomics methods, and transcriptomics, with the aim of reducing the number of cDNA candidates encoding for a glucosyltransferase involved in saponin biosynthesis and facilitating the elucidation of the pathway in this medicinal plant.
The aim of this research is to investigate whether edible bird's nest (EBN) attenuates cortical and hippocampal neurodegeneration in ovariectomized rats. Ovariectomized rats were randomly divided into seven experimental groups (n = 6): the ovariectomy (OVX) group had their ovaries surgically removed; the sham group underwent surgical procedure similar to OVX group, but ovaries were left intact; estrogen group had OVX and received estrogen therapy (0.2 mg kg(-1) per day); EBN treatment groups received 6%, 3%, and 1.5% EBN, respectively. Control group was not ovariectomized. After 12 weeks of intervention, biochemical assays were performed for markers of neurodegeneration, and messenger ribonucleic acid (mRNA) levels of oxidative stress-related genes in the hippocampus and frontal cortex of the brain were analysed. Caspase 3 (cysteine-aspartic proteases 3) protein levels in the hippocampus and frontal cortex were also determined using western blotting. The results show that EBNs significantly decreased estrogen deficiency-associated serum elevation of advanced glycation end-products (AGEs), and they changed redox status as evidenced by oxidative damage (malondialdehyde content) and enzymatic antioxidant defense (superoxide dismutase and catalase) markers. Furthermore, genes associated with neurodegeneration and apoptosis were downregulated in the hippocampus and frontal cortex by EBN supplementation. Taken together, the results suggest that EBN has potential for neuroprotection against estrogen deficiency-associated senescence, at least in part via modification of the redox system and attenuation of AGEs.
Matched MeSH terms: Glycosylation End Products, Advanced/metabolism
OBJECTIVE: We previously reported ethnic disparity in adverse outcomes among Asians with type 2 diabetes (T2DM) in Singapore. Central arterial stiffness can aggravate systemic vasculopathy by propagating elevated systolic and pulse pressures forward, thereby accentuating global vascular injury. We aim to study ethnic disparity in central arterial stiffness and its determinants in a multi-ethnic T2DM Asian cohort.
METHODS: Arterial stiffness was estimated by carotid-femoral pulse wave velocity (PWV) and augmentation index (AI) using applanation tonometry method in Chinese (N = 1045), Malays (N = 458) and Indians (N = 468). Linear regression model was used to evaluate predictors of PWV and AI.
RESULTS: PWV was higher in Malays (10.1 ± 3.0 m/s) than Chinese (9.7 ± 2.8 m/s) and Indians (9.6 ± 3.1 m/s) (P = 0.018). AI was higher in Indians (28.1 ± 10.8%) than Malays (25.9 ± 10.1%) and Chinese (26.1 ± 10.7%) (P < 0.001). Malays remain associated with higher PWV (β = 0.299, P = 0.048) post-adjustment for age, gender, duration of diabetes, hemoglobin A1c, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), soluble receptor for advanced glycation end-products, urinary albumin-to-creatinine ratio, and insulin usage, which were all independent predictors of PWV. Indians remain associated with higher AI (β = 2.776, P < 0.001) post-adjustment for age, gender, BMI, SBP, DBP, and height, which were independent predictors of AI. These variables explained 27.7% and 33.4% of the variance in PWV and AI respectively.
CONCLUSIONS: Malays and Indians with T2DM have higher central arterial stiffness, which may explain their higher risk for adverse outcomes. Modifying traditional major vascular risk factors may partially alleviate their excess cardiovascular risk through modulating arterial stiffness.
KEYWORDS: Arterial stiffness; Augmentation index; Pulse wave velocity; Type 2 diabetes
Matched MeSH terms: Glycosylation End Products, Advanced/blood
The nucleotide sequence of the haemagglutinin-neuraminidase (HN) glycoprotein gene of Newcastle disease virus (NDV) variant strain V4(UPM) was determined by direct genomic RNA sequencing and confirmed by cycle sequencing. The gene comprises 1996 nucleotides encoding a 615 amino acid protein of size 67.4 kDa. The nucleotide and amino acid sequences of this strain were compared with those of the parent strain V4(QUE). There are 16 nucleotide substitutions on V4(UPM), eight of which are silent mutations and another eliminated a potential Asn-linked glycosylation site in V4(UPM). In addition, an Arg (403) residue was shown to be absent in the variant strain. This deletion is thought to be significant because of its location in a highly conserved region of the HN protein.
Most studies have focused on the role of the cellular prion protein (PrP(C)) in neurodegenerative diseases, whereas the function of this ubiquitous protein outside the nervous system remains elusive. Therefore, the anti-apoptotic property of PrP(C) in oral squamous cell carcinoma (HSC-2) and colon adenocarcinoma (LS 174T) was evaluated in this study, by stable shRNA knockdown and overexpression, respectively. PrP(C) confers resistance against oxidative stress-apoptosis as indicated by MTT assay, Annexin V-FITC/PI and DCFH-DA staining, but this property is abolished upon N-glycosylation inhibition by tunicamycin. Our results indicate that the inhibition of glycosylation in cancer cells overexpressing PrP(C) could represent a potential therapeutic target.
The prevalence of oral squamous cell carcinoma (OSCC) is high in South and Southeast Asia regions. Most OSCC patients are detected at advanced stages low 5-year survival rates. Aberrant expression of glycosylated proteins was found to be associated with malignant transformation and cancer progression. Hence, identification of cancer-associated glycoproteins could be used as potential biomarkers that are beneficial for diagnosis or clinical management of patients. This study aims to identify the differentially expressed glycoproteins using lectin-based glycoproteomics approaches. Serum samples of 40 patients with OSCC, 10 patients with oral potentially malignant disorder (OPMD), and 10 healthy individuals as control group were subjected to two-dimensional gel electrophoresis (2-DE) coupled with lectin Concanavalin A and Jacalin that specifically bind to N- and O-glycosylated proteins, respectively. Five differentially expressed N- and O-glycoproteins with various potential glycosylation sites were identified, namely N-glycosylated α1-antitrypsin (AAT), α2-HS-glycoprotein (AHSG), apolipoprotein A-I (APOA1), and haptoglobin (HP); as well as O-glycosylated AHSG and clusterin (CLU). Among them, AAT and APOA1 were further validated using enzyme-linked immunosorbent assay (ELISA) (n = 120). It was found that AAT and APOA1 are significantly upregulated in OSCC and these glycoproteins are independent risk factors of OSCC. The clinical utility of AAT and APOA1 as potential biomarkers of OSCC is needed for further evaluation.
We successfully developed an in-house, competitive enzyme immunoassay to measure advanced glycosylation end-products (AGE) in serum. The assay involved coating microtitre wells with AGE-BSA at 8 micrograms/ml for 4 hours, followed by overnight incubation of 20 microliters sample (prediluted at 1:6) with 80 microliters antiserum (1:8000). HRP-labelled goat anti-rabbit was used as the second antibody and 3,5',5,5'-tetramethylbenzidine dihydrochloride as the substrate. Incubation was carried out at 4 degrees C. As suggested in an earlier study, we standardised the AGE units against normal human serum (NHS). Thus, one AGE unit was defined as the inhibition that resulted when the 1:6 diluted NHS was assayed. Mean (+/- SD) AGE level in normal subjects (n = 37) was significantly lower than in diabetes subjects with microalbuminuria (n = 57) (6.0 +/- 0.7 versus 10.2 +/- 4.7 units/ml, p = 0.0001). With the availability of in-house assay and by standardising the AGE unit with the other laboratories, more studies could be undertaken and results compared, and possibly, further elucidate the roles of AGE in the pathogenesis of diabetic complications.
Matched MeSH terms: Glycosylation End Products, Advanced/blood*; Glycosylation End Products, Advanced/immunology
There are few reports of congenital disorders of glycosylation (CDGs) in the Asian population, although they have been reported worldwide. We identified a Malaysian infant female at 2 days of life with CDG type Ia. The diagnosis was suspected on the basis of inverted nipples and abnormal fat distribution. She had cerebellar hypoplasia and developed coagulopathy, hypothyroidism and severe pericardial effusion and died at 7 months of life. The diagnosis was supported by abnormal serum transferrin isoform pattern that showed elevated levels of the disialotransferrin isoform and trace levels of the asialotransferrin isoform. Enzyme testing of peripheral leukocytes showed decreased level of phosphomannomutase (PMM) activity (0.6 nmol/min per mg protein, normal range 1.6-6.2) and a normal level of phosphomannose isomerase activity (19 nmol/min per mg protein, normal range 12-25), indicating a diagnosis of CDG type Ia. Mutation study of the PMM2 gene showed the patient was heterozygous for both the common p.R141H (c.422T>A) mutation and a novel sequence change in exon 7, c.618C>A. The latter change is predicted to result in the replacement of the highly conserved phenylalanine residue at position 206 with a leucine residue (p.F206L) and occurs in the same codon as the previously reported p.F206S mutation. Analysis of 100 control chromosomes has shown that the p.F206L sequence change is not present, making it highly likely that this change is functionally important. To the best of our knowledge, this is the first report of CDG in the Malay population. Prenatal diagnosis was successfully performed in a subsequent pregnancy for this family.
Matched MeSH terms: Congenital Disorders of Glycosylation/diagnosis; Congenital Disorders of Glycosylation/enzymology*; Congenital Disorders of Glycosylation/genetics*
The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in vivo.
Breast cancer is one of the major issues in the field of oncology, reported with a higher prevalence rate in women worldwide. In attempt to reveal the potential biomarkers for breast cancer, the findings of differentially glycosylated haptoglobin and osteonectin in previous study have drawn our attention towards glycoproteins of secretome from the MCF-7 cancer cell line. In the present study, further analyses were performed on the medium of MCF-7 cells by subjecting it to two-dimensional analyses followed by image analysis in contrast to the medium of human mammary epithelial cells (HMEpC) as a negative control. Carboxypeptidase A4 (CPA4), alpha-1-antitrypsin (AAT), haptoglobin (HP), and HSC70 were detected in the medium of MCF-7, while only CPA4 and osteonectin (ON) were detected in HMEpC medium. In addition, CPA4 was detected as upregulated in the MCF-7 medium. Further analysis by lectin showed that CPA4, AAT, HP, and HSC70 were secreted as N-glycan in the medium of MCF-7, with HP also showing differentially N-glycosylated isoforms. For the HMEpC, only CPA4 was detected as N-glycan. No O-glycan was detected in the medium of HMEpC but MCF-7 expressed O-glycosylated CPA4 and HSC70. All these revealed that glycoproteins could be used as glycan-based biomarkers for the prognosis of breast cancer.
4-Methylbenzimidazole 1-28 novel derivatives were synthesized and evaluated for their antiglycation and antioxidant activities. Compounds 1-7 and 11 showed excellent activities ranged 140-280 μM, better than standard drug rutin (294.46 ± 1.50 μM). Compound 1-28 were also evaluated for DPPH activities. Compounds 1-8 showed excellent activities, ranging 12-29 μM, better than standard drug n-propylgallate (IC50 = 30.30 ± 0.40 μM). For superoxide anion scavenging activity, compounds 1-7 showed better activity than standard n-propylgallate (IC50 = 106.34 ± 1.6 μM), ranged 82-104 μM. These compounds were found to be nontoxic to THP-1 cells.
A series of 4-methoxybenzoylhydrazones 1-30 was synthesized and the structures of the synthetic derivatives elucidated by spectroscopic methods. The compounds showed a varying degree of antiglycation activity, with IC50 values ranging between 216.52 and 748.71 µM, when compared to a rutin standard (IC50=294.46±1.50 µM). Compounds 1 (IC50=216.52±4.2 µM), 3 (IC50=289.58±2.64 µM), 6 (IC50=227.75±0.53 µM), 7 (IC50=242.53±6.1) and 11 (IC50=287.79±1.59) all showed more activity that the standard, and these compounds have the potential to serve as possible leads for drugs to inhibit protein glycation in diabetic patients. A preliminary SAR study was performed.
BACKGROUND:
Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53-55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53-55 kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information.
OBJECTIVE:
We sought to clone the transcript encoding the Hev b 4 major protein, and characterize the native protein and its recombinant form in relation to IgE binding.
METHODS:
The 5'/3' rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients.
RESULTS:
The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue.
CONCLUSION:
The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular weight of the protein and the value calculated from its cDNA.
The valorization of organosolv pretreatment (OP) is a required approach to the industrialization of the current enzyme-mediated lignocellulosic biorefinery. Recent literature has demonstrated that the solvolysis happening in the OP can modify the soluble components into value-added active compounds, namely organosolv modified lignin (OML) and organosolv modified sugars (OMSs), in addition to protecting them against excessive degradation. Among them, the OML is coincidental with the "lignin-first" strategy that should render a highly reactive lignin enriched with β-O-4 linkages and less condensed structure by organosolv grafting, which is desirable for the transformation into phenolic compounds. The OMSs are valuable glycosidic compounds mainly synthesized by trans-glycosylation, which can find potential applications in cosmetics, foods, and healthcare. Therefore, a state-of-the-art OP holds a big promise of lowering the process cost by the valorization of these active compounds. Recent advances in organosolv modified components are reviewed, and perspectives are made for addressing future challenges.
Intravenous immunoglobulin (IVIG) is the first line treatment for Guillain-Barré syndrome and multifocal motor neuropathy, which are caused by anti-ganglioside antibody-mediated complement-dependent cytotoxicity. IVIG has many potential mechanisms of action, and sialylation of the IgG Fc portion reportedly has an anti-inflammatory effect in antibody-dependent cell-mediated cytotoxicity models. We investigated the effects of different IVIG glycoforms on the inhibition of antibody-mediated complement-dependent cytotoxicity. Deglycosylated, degalactosylated, galactosylated and sialylated IgG were prepared from IVIG following treatment with glycosidases and glycosyltransferases. Sera from patients with Guillain-Barré syndrome, Miller Fisher syndrome and multifocal motor neuropathy associated with anti-ganglioside antibodies were used. Inhibition of complement deposition subsequent to IgG or IgM autoantibody binding to ganglioside, GM1 or GQ1b was assessed on microtiter plates. Sialylated and galactosylated IVIGs more effectively inhibited C3 deposition than original IVIG or enzyme-treated IVIGs (agalactosylated and deglycosylated IVIGs). Therefore, sialylated and galactosylated IVIGs may be more effective than conventional IVIG in the treatment of complement-dependent autoimmune diseases.
A unique taste-modifying activity that converts the sense of sourness to the sense of sweetness occurs in the fruit of the plant Curculigo latifolia, intrinsic to West Malaysia. The active component, known as curculin, is a protein consisting of two identical subunits. We have found a new taste-modifying protein, named neoculin, of the same origin. Both chemical analysis and cDNA cloning characterized neoculin as a heterodimeric protein consisting of an acidic, glycosylated subunit of 113 amino acid residues and a basic subunit that is the monomeric curculin itself.
Foods rich in carbohydrates or fats undergo the Maillard reaction during frying, which promotes the color, flavor and sensory characteristics formation. In the meanwhile, Maillard reaction intermediates and advanced glycation end products (AGEs) have a negative impact on food sensory quality and gut homeostasis. This negative effect can be influenced by food composition and other processing factors. Whole grain products are rich in polyphenols, which can capture carbonyl compounds in Maillard reaction, and reduce the production of AGEs during frying. This review summarizes the Maillard reaction production intermediates and AGEs formation mechanism in fried food and analyzes the factors affecting the sensory formation of food. In the meanwhile, the effects of Maillard reaction intermediates and AGEs on gut homeostasis were summarized. Overall, the innovative processing methods about the Maillard reaction are summarized to optimize the sensory properties of fried foods while minimizing the formation of AGEs.
Matched MeSH terms: Glycosylation End Products, Advanced*
The retinal pigment epithelium (RPE) plays a central role in neuroretinal homoeostasis throughout life. Altered proteolysis and inflammatory processes involving RPE contribute to the pathophysiology of age-related macular degeneration (AMD), but the link between these remains elusive. We report for the first time the effect of advanced glycation end products (AGE)-known to accumulate on the ageing RPE's underlying Bruch's membrane in situ-on both key lysosomal cathepsins and NF-κB signalling in RPE. Cathepsin L activity and NF-κB effector levels decreased significantly following 2-week AGE exposure. Chemical cathepsin L inhibition also decreased total p65 protein levels, indicating that AGE-related change of NF-κB effectors in RPE cells may be modulated by cathepsin L. However, upon TNFα stimulation, AGE-exposed cells had significantly higher ratio of phospho-p65(Ser536)/total p65 compared to non-AGEd controls, with an even higher fold increase than in the presence of cathepsin L inhibition alone. Increased proportion of active p65 indicates an AGE-related activation of NF-κB signalling in a higher proportion of cells and/or an enhanced response to TNFα. Thus, NF-κB signalling modulation in the AGEd environment, partially regulated via cathepsin L, is employed by RPE cells as a protective (para-inflammatory) mechanism but renders them more responsive to pro-inflammatory stimuli.
Matched MeSH terms: Glycosylation End Products, Advanced/metabolism*
Aiming for new glycolipids with enhanced chemical stability and close structural similarity to natural cell membrane lipids for the development of a drug delivery system, we have synthesized double amide analogs of glyco-glycerolipids. The synthesis applied a Staudinger reaction based coupling of a 1,3-diazide with fatty acid chlorides. While the concept furnished the desired glucosides in reasonable yields, the corresponding lactosides formed a tetrahydropyrimidine based 1:1 coupling product instead. This unexpected coupling result likely originates from steric hindrance at the iminophosphorane intermediate and provides an interesting core structure for potentially bioactive surfactants. The assembly behavior of both glycolipid types was investigated by optical polarizing microscopy, DSC and surface tension studies.