Radioimmunochemical evidence for a role of carbohydrate in antigenic determinant(s) on human liver alpha-L-fucosidase.
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ABSTRACT:
SUBMITTER: Alhadeff JA
PROVIDER: S-EPMC1144299 | biostudies-other | 1984 Oct
REPOSITORIES: biostudies-other
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Project description:The subunit composition of human liver alpha-L-fucosidase was studied by using three different procedures for treating and electrophoresing the purified enzyme. Although the presence on only one subunit was found by conventional procedures using sodium dodecyl sulphate and urea, two non-identical subunits with mol. wts. of 59 000 and 54 000 were found when the alpha-L-fucosidase was reduced and S-carboxymethylated with iodoacetate followed by electrophoresis on polyacrylamide gels containing 0.1% sodium dodecyl sulphate and 8.0 M-urea.
| S-EPMC1186428 | biostudies-other
Project description:The carbohydrate component of Glycoprotein 2 (12% carbohydrate) from rye-grass (Lolium perenne) pollen has saccharide sequences that contribute to its antigenicity. Radioimmunoassay inhibition tests show that the antiserum to this glycoprotein cross-reacts with a number of other plant glycoproteins. In contrast, antiserum to another glycoprotein from rye-grass pollen, Glycoprotein 1 (5% carbohydrate), does not cross-react with any of the test glycoconjugates. Treatment of glycoproteins with sodium metaperiodate (0.02 M, 4 degree C, 6 h, in the dark) causes the loss of their ability to cross-react antigenically with Glycoprotein 2, and a loss of capacity to bind 125I-labelled concanavalin A. The cross-reactivity of this plant glycoprotein with other glycoconjugates imposes limitations on the interpretation of ultrastructural studies aimed at localizing a particular glycoprotein to a cellular site by using fluorescent or ferritin-labelled antisera. A radioimmunoassay inhibition technique for quantitative determination of the amounts of antigens in plants is also described.
| S-EPMC1163184 | biostudies-other
Project description:In previous studies we described a decreased alpha-L-fucosidase activity in colorectal tumors, appearing as a prognostic factor of tumoral recurrence. The aim of this work was to extend the knowledge about tissue alpha-L-fucosidase in colorectal cancer by quantifying the expression of its encoding gene FUCA1 in tumors and healthy mucosa. FUCA1 mRNA levels were measured by RT-qPCR in paired tumor and normal mucosa tissues from 31 patients. For the accuracy of the RT-qPCR results, five candidate reference genes were validated in those samples. In addition, activity and expression of alpha-L-fucosidase in selected matched tumor and healthy mucosa samples were analyzed. According to geNorm and NormFinder algorithms, RPLP0 and HPRT1 were the best reference genes in colorectal tissues. These genes were used for normalization of FUCA1 expression levels. A significant decrease of more than 60% in normalized FUCA1 expression was detected in tumors compared to normal mucosa (p = 0.002). Moreover, a gradual decrease in FUCA1 expression was observed with progression of disease from earlier to advanced stages. These findings were confirmed by Western blot analysis of alpha-L-fucosidase expression. Our results demonstrated diminished FUCA1 mRNA levels in tumors, suggesting that expression of tissue alpha-L-fucosidase could be regulated at transcriptional level in colorectal cancer.
| S-EPMC3759947 | biostudies-literature
Project description:BackgroundThis study investigated the prognostic and immunological significance of alpha-L-fucosidase 2 (FUCA2) in hepatocellular cancer (HCC).MethodsThe expression of FUCA2 and its clinical and prognostic values were explored across several databases, namely the University of Alabama Cancer Database, The Cancer Genome Atlas, Gene Expression Profiling Interactive Analysis, and the Human Protein Atlas. The prognostic relevance of FUCA2 was investigated using Kaplan-Meier curves, nomograms, and Cox analysis. The "limma" package in R was used to identify differentially expressed genes between high and low FUCA2 expression. A protein interaction network was established using the Search Tool for the Retrieval of Interacting Genes (STRING), whereas hub genes and clustering modules were identified using Cytoscape. "clusterProfiler", an R package, was used to examine the potential function of FUCA2. Using gene set enrichment analysis, signaling pathways associated with FUCA2 expression were identified. Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT), Tumor Immune Estimation Resource (TIMER) 2.0, and Tumor and Immune System Interaction Database (TISIDB) were used to examine immune infiltration and FUCA2 in HCC.ResultsMany datasets indicated that FUCA2 expression is higher in HCC, and that this is related to age and overall survival (OS). With the cutoff value of 50% as the dividing threshold, the patients were divided into a high-FUCA2 expression group (n=167) and a low-FUCA2 expression group (n=168). High levels of FUCA2 expression coincided with decreased OS. FUCA2 expression in HCC was associated with immune infiltrates. The functional mechanisms of FUCA2 depend on signal release, extracellular matrix collagen, and neuroactive ligands and receptors.ConclusionsIn HCC, increased FUCA2 expression is associated with a poor prognosis and immune infiltration. FUCA2 may serve as an immunological and predictive biomarker for HCC.
| S-EPMC10007884 | biostudies-literature
Project description:?-L-Fucosidases are enzymes involved in metabolism of ?-L-fucosylated molecules, compounds with a fundamental role in different life essential processes including immune response, fertilization and development, but also in some serious pathological events. According to the CAZy database, these enzymes belong to families 29 and 95. Some of them are also reported to be able to catalyze transglycosylation reactions, during which ?-L-fucosylated molecules, representing compounds of interest especially for pharmaceutical industry, are formed.Activity-based screening of a genomic library was used to isolate the gene encoding a novel ?-L-fucosidase. The enzyme was expressed in E.coli and affinity chromatography was used for purification of His-tagged ?-L-fucosidase. Standard activity assay was used for enzyme characterization. Thin layer chromatography and mass spectrometry were used for transglycosylation reactions evaluation.Using a genomic library of Paenibacillus thiaminolyticus, constructed in E.coli DH5? cells, nucleotide sequence of a new ?-L-fucosidase isoenzyme was determined and submitted to the EMBL database (HE654122). However, no similarity with enzymes from CAZy database families 29 and 95 was detected. This enzyme was produced in form of histidine-tagged protein in E.coli BL21 (DE3) cells and purified by metaloaffinity chromatography. Hydrolytic and transglycosylation abilities of ?-L-fucosidase iso2 were tested using different acceptor molecules.In this study, new enzyme ?-L-fucosidase iso2 originating from Paenibacillus thiaminolyticus was described and prepared in recombinant form and its hydrolytic and transglycosylation properties were characterized. As a very low amino acid sequence similarity with known ?-L-fucosidases was found, following study could be important for different biochemical disciplines involving molecular modelling.
| S-EPMC4445282 | biostudies-literature
Project description:Previous studies have documented the presence of a novel alpha-L-fucosidase in mouse liver that contains unique basic isoelectric forms and that is antigenically similar to, but not identical with, human liver alpha-L-fucosidase [Laury-Kleintop, Damjanov & Alhadeff (1985) Biochem. J. 230, 75-82]. In the present investigation, mouse liver alpha-L-fucosidase was purified approx. 26,500-fold in 10% overall yield by antibody-affinity chromatography with the IgG fraction of goat anti-(human alpha-L-fucosidase) antibody coupled to Sepharose 4B. Native polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis indicated that the mouse fucosidase is highly purified if not homogeneous. Isoelectric focusing demonstrated that all enzymic forms found in crude mouse liver supernatant fluids were purified by the antibody-affinity procedure.
| S-EPMC1148162 | biostudies-other
Project description:Nonalcoholic fatty liver disease (NAFLD) associated with type 2 diabetes (T2D) easily progresses toward severe forms of nonalcoholic steatohepatitis (NASH) and fibrosis, and the underlying mechanism is under active investigation. Here, we established the role of transcription factor 7-like 2 (TCF7L2), the most significant T2D susceptibility gene, in NAFLD development and progression. Hepatic TCF7L2 expression was decreased in liver biopsies of patients with NAFLD. Based on the major risk factors for NAFLD development, liver-specific TCF7L2 knockout mice were subjected to a high-fat diet providing fatty acids (FAs) and refeeding/high-carbohydrate diet stimulating de novo lipogenesis. Hepatic TCF7L2 deficiency significantly increased the lipid synthetic pathways and hepatic TG accumulation by preferentially metabolizing carbohydrates than FAs. Mechanistically, TCF7L2 regulated miRNAs targeting SREBF1c and enhanced proteasome-mediated MLXIPL (ChREBP) degradation. Our findings will deepen the pathophysiological mechanism of NAFLD associated with dietary carbohydrate and diabetes, and will provide a potential target for treatment of NAFLD.
2023-01-09 | GSE162449 | GEO
Project description:Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed "recoding". In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for -1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed -1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discussed.
| S-EPMC8037382 | biostudies-literature
Project description:Western-blot analysis [with lectins, polyclonal antibodies (pAbs) and four monoclonal antibodies (mAbs)] was employed to investigate the structural relationship between the separated isoforms and subunits of purified human liver alpha-L-fucosidase. SDS/PAGE and Western-blot analysis indicated the presence of two protein bands of 51 kDa and 56 kDa that were recognized by the pAbs. Polyacrylamide-gel isoelectric focusing (PAG-IEF) followed by blotting indicated that the pAbs and mAbs recognized at least five fucosidase isoforms (pI values 3.6-6.0). Lectin blotting indicated an enrichment of sialic acid residues in the more acidic isoforms. Western-blot analysis indicated that four mAbs recognized the 51 kDa subunit and at least two mAbs recognized the 56 kDa subunit. The subunit composition of the isoforms (separated by PAG-IEF) of human liver alpha-L-fucosidase was investigated by SDS/PAGE. One or two closely spaced bands were found for each isoform with a trend of increasing relative amounts of the high-molecular-mass band in the more acidic isoforms relative to the more neutral isoforms. Neuraminidase treatment of alpha-L-fucosidase resulted in a decrease in the amount of the high-molecular-mass subunit and an increase in the amount of the low-molecular-mass subunit, suggesting that these subunits are related at least in part by sialic acid residues. In addition, blotting with lectins indicated the presence of sialic acid residues only in the high-molecular-mass subunit. N-Glycanase treatment led to the disappearance of the glycosylated 56 kDa and 51 kDa protein bands and the appearance of non-glycosylated protein bands at 48 kDa and 45 kDa. The overall results indicate that (1) N-glycosylation contributes to, but does not account completely for, structural differences in the fucosidase subunits and (2) the more acidic isoforms of fucosidase contain enriched relative amounts of the sialylated high-molecular-mass subunit.
| S-EPMC1130862 | biostudies-other
Project description:cDNA clones for alpha-L-fucosidase were isolated from a rat liver lambda gt11 expression library by using both monospecific polyclonal antibodies against the affinity-purified enzyme and biotinylated rat liver fucosidase cDNA sequences as probes. The largest clone, lambda FC9, contained a 1522 bp full-length cDNA insert (FC9) that encoded the 434-amino acid-residue subunit (Mr 50439) of rat liver alpha-L-fucosidase. A putative signal peptide 28 amino acid residues in length preceded the sequence for the mature protein. In addition, FC9 specified for 11 nucleotide residues of 5' untranslated sequence, 78 nucleotide residues of 3' untranslated sequence and a poly(A) tail. The deduced amino acid sequence from FC9 in conjunction with the experimentally determined N-terminus of the mature enzyme suggested that rat liver fucosidase did not contain a pro-segment. However, there was the possibility of limited N-terminal processing (one to five amino acid residues) having occurred after removal of the predicted signal peptide. Amino acid sequences deduced from FC9 were co-linear with amino acid sequences measured at the N-terminus of purified fucosidase and on two of its CNBr-cleavage peptides. An unusual aspect of rat liver alpha-L-fucosidase protein structure obtained from the FC9 data was its high content of tryptophan (6%). The coding sequence from FC9 showed 82% sequence identity with that from a previously reported incomplete human fucosidase sequence [O'Brien, Willems, Fukushima, de Wet, Darby, DiCioccio, Fowler & Shows, (1987) Enzyme 38, 45-53].
| S-EPMC1133642 | biostudies-other