Project description:Human senescence-associated β-galactosidase (SA-β-gal), the most widely used biomarker of aging, is a valuable tool for assessing the extent of cell 'healthy aging' and potentially predicting the health life span of an individual. Human SA-β-gal is an endogenous lysosomal enzyme expressed from GLB1, the catalytic domain of which is very different from that of E. coli β-gal, a bacterial enzyme encoded by lacZ. However, existing chemical probes for this marker still lack the ability to distinguish human SA-β-gal from β-gal of other species, such as bacterial β-gal, which can yield false positive signals. Here, we show a molecular design strategy to construct fluorescent probes with the above ability with the aid of structure-based steric hindrance adjustment catering to different enzyme pockets. The resulting probes normally work as traditional SA-β-gal probes, but they are unique in their powerful ability to distinguish human SA-β-gal from E. coli β-gal, thus achieving species-selective visualization of human SA-β-gal for the first time. NIR-emitting fluorescent probe KSL11 as their representative further displays excellent species-selective recognition performance in biological systems, which has been herein verified by testing in senescent cells, in lacZ-transfected cells and in E. coli-β-gal-contaminated tissue sections of mice. Because of our probes, it was also discovered that SA-β-gal content in mice increased gradually with age and SA-β-gal accumulated most in the kidneys among the main organs of naturally aging mice, suggesting that the kidneys are the organs with the most severe aging during natural aging.
Project description:T cells are known as the most potent killer cells of the immune system, designed by nature to prevent unwanted challenges. The first class of therapeutic products harnessing the power of T cells for target-specific treatment of oncological diseases was bispecific antibodies. The first T-cell engaging bispecific antibodies that obtained approval were catumaxomab and blinatumomab1,2. Eight years later, the first chimeric antigen receptor (CAR)-T cells received regulatory approval3. CAR-T cells are the cellular interpretation of T-cell engaging therapies and have shown remarkable clinical results. CAR-T cells belong to the regulatory group of advanced therapy medicinal products (ATMPs). Due to the cell-/gene-based complex nature, ATMPs are far more challenging to develop than other, more defined, medicinal products. Despite very encouraging clinical results, there have been many set-backs in the development of ATMPs during the past 20 years. Therefore, the approval of the first two CAR-Ts KYMRIAH and YESCARTA is highly encouraging for the field. In this article we review the current landscape of CAR-Ts as a special class of ATMPs. This comprises the pathway to approval including the use of dedicated regulatory tools and challenges that were faced during the procedure. Furthermore, we highlight important future trends in the field.
Project description:A gene encoding a third alpha-galactosidase (AglB) from Aspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other alpha-galactosidases revealed that it belongs to a subfamily of alpha-galactosidases that also includes A. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic alpha-galactosidases. The expression of aglA, aglB, aglC, and lacA, the latter of which encodes an A. niger beta-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins.
Project description:Binding of a single-chain Fv antibody to Escherichia coli β-galactosidase (β-gal) is known to stabilize the enzyme and activate several inactive point mutants, historically called antibody-mediated enzyme formation mutants. To understand the nature of this activation, we have determined by electron cryo-microscopy the structure of the complex between β-gal and the antibody scFv13R4. Our structure localizes the scFv13R4 binding site to the crevice between domains 1 and 3 in each β-gal subunit. The mutations that scFv13R4 counteracts are located between the antibody binding site and the active site of β-gal, at one end of the TIM-barrel that forms domain 3 where the substrate lactose is hydrolyzed. The mode of binding suggests how scFv stabilizes both the active site of β-gal and the tetrameric state.
Project description:Beta (β)-galactosidase is a lysosomal enzyme that removes terminal galactose residues from glycolipids and glycoproteins. It is upregulated in, and used as a marker for, senescent cells. Microglia are brain macrophages implicated in neurodegeneration, and can upregulate β-galactosidase when senescent. We find that inflammatory activation of microglia induced by lipopolysaccharide results in translocation of β-galactosidase to the cell surface and release into the medium. Similarly, microglia in aged mouse brains appear to have more β-galactosidase on their surface. Addition of β-galactosidase to neuronal-glial cultures causes microglial activation and neuronal loss mediated by microglia. Inhibition of β-galactosidase in neuronal-glial cultures reduces inflammation and neuronal loss induced by lipopolysaccharide. Thus, activated microglia release β-galactosidase that promotes microglial-mediated neurodegeneration which is prevented by inhibition of β-galactosidase.
Project description:Microbial β-galactosidase enzymes are widely used as biocatalysts in industry to produce prebiotic galactooligosaccharides (GOS) from lactose. GOS mixtures are used as beneficial additives in infant formula to mimic the prebiotic effects of human milk oligosaccharides (hMOS). The structural variety in GOS mixtures is significantly lower than in hMOS. Since this structural complexity is considered as the basis for the multiple biological functions of hMOS, it is important to broaden the variety of GOS structures. In this study, residue R484 near +1 subsite of the C-terminally truncated β-galactosidase from Bacillus circulans (BgaD-D) was subjected to site saturation mutagenesis. Especially the R484S and R484H mutant enzymes displayed significantly altered enzyme specificity, leading to a new type of GOS mixture with altered structures and linkage types. The GOS mixtures produced by these mutant enzymes contained 14 structures that were not present in the wild-type enzyme GOS mixture; 10 of these are completely new structures. The GOS produced by these mutant enzymes contained a combination of (β1 → 3) and (β1 → 4) linkages, while the wild-type enzyme has a clear preference toward (β1 → 4) linkages. The yield of the trisaccharide β-d-Galp-(1 → 3)-β-d-Galp-(1 → 4)-d-Glcp produced by mutants R484S and R484H increased 50 times compared to that of the wild-type enzyme. These results indicate that residue R484 is crucial for the linkage specificity of BgaD-D. This is the first study showing that β-galactosidase enzyme engineering results in an altered GOS linkage specificity and product mixture. The more diverse GOS mixtures produced by these engineered enzymes may find industrial applications.
Project description:The beta-galactosidase gene of Streptococcus pneumoniae, bgaA, encodes a putative 2,235-amino-acid protein with the two amino acid motifs characteristic of the glycosyl hydrolase family of proteins. In addition, an N-terminal signal sequence and a C-terminal LPXTG motif typical of surface-associated proteins of gram-positive bacteria are present. Trypsin treatment of cells resulted in solubilization of the enzyme, documenting that it is associated with the cell envelope. In order to obtain defined mutants suitable for lacZ reporter experiments, the bgaA gene was disrupted, resulting in a complete absence of endogenous beta-galactosidase activity. The results are consistent with beta-galactosidase being a surface protein that seems not to be involved in lactose metabolism but that may play a role during pathogenesis.
Project description:GM1 gangliosidosis and Morquio B disease are autosomal recessive diseases caused by the defect in the lysosomal β-galactosidase (β-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding and prevent endoplasmic reticulum-associated degradation and promote trafficking to the lysosome. In this report, we describe the enzymological properties of purified recombinant human β-Gal(WT) and two representative mutations in GM1 gangliosidosis Japanese patients, β-Gal(R201C) and β-Gal(I51T). We have also evaluated the PC effect of two competitive inhibitors of β-Gal. Moreover, we provide a detailed atomic view of the recognition mechanism of these compounds in comparison with two structurally related analogues. All compounds bind to the active site of β-Gal with the sugar-mimicking moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity, and the PC potential are strongly affected by the mono- or bicyclic structure of the core as well as the orientation, nature, and length of the exocyclic substituent. These results provide understanding on the mechanism of action of β-Gal selective chaperoning by newly developed PC compounds.
Project description:Stevia rebaudiana Bertoni is a plant cultivated worldwide due to its use as a sweetener. The sweet taste of stevia is attributed to its numerous steviol glycosides, however, their use is still limited, due to their bitter aftertaste. The transglycosylation of steviol glycosides, aiming at the improvement of their taste, has been reported for many enzymes, however, glycosyl hydrolases are not extensively studied in this respect. In the present study, a β-glucosidase, MtBgl3a, and a β-galactosidase, TtbGal1, have been applied in the transglycosylation of two steviol glycosides, stevioside and rebaudioside A. The maximum conversion yields were 34.6 and 33.1% for stevioside, while 25.6 and 37.6% were obtained for rebaudioside A conversion by MtBgl3a and TtbGal1, respectively. Low-cost industrial byproducts were employed as sugar donors, such as cellulose hydrolyzate and acid whey for TtbGal1- and MtBgl3a- mediated bioconversion, respectively. LC-HRMS analysis identified the formation of mono- and di- glycosylated products from stevioside and rebaudioside A. Overall, the results of the present work indicate that both biocatalysts can be exploited for the design of a cost-effective process for the modification of steviol glycosides.