Structural and functional insights into the enzymatic activities of lipases from Burkholderia stagnalis and Burkholderia plantarii.

Lipases with high interesterification activity are important enzymes for industrial use. The lipase from Burkholderia stagnalis (BsL) exhibits higher interesterification activity than that from Burkholderia plantarii (BpL) despite their significant sequence similarity. In this study, we determined the crystal structure of BsL at 1.40 Å resolution. Utilizing structural insights, we have successfully augmented the interesterification activity of BpL by over twofold. This enhancement was achieved by substituting threonine with serine at position 289 through forming an expansive space in the substrate-binding site. Additionally, we discuss the activity mechanism based on the kinetic parameters. Our study sheds light on the structural determinants of the interesterification activity of lipase.

Comparison of enzymatic activities of lipases from Burkholderia plantarii and Burkholderia cepacia.

Lipases (EC 3.1.1.3) are enzymes used in the oils and fats industries to modify the physicochemical properties of triacylglycerol (TAG). Lipase-catalyzed interesterification at high temperatures is an effective method for modifying the physicochemical properties of TAG. The lipase from Burkholderia plantarii (BpL) exhibits excellent catalytic activity for non-regiospecific interesterification at high temperatures, with depressed lipase hydrolytic activity. The detailed catalytic mechanism for reactions involving catalytic residues has not been elucidated because of the lack of a conventional method for estimating interesterification activity. We used our original water-in-oil emulsion system to estimate the interesterification activity of lipases. BpL showed 10% hydrolytic and 140% interesterification activities compared to the lipase from Burkholderia cepacia, which has a high sequence homology with BpL. By comparing the sequence and crystal structure data of the lipases, we clarified that two amino acids near the active center are one of the factors controlling the hydrolytic and interesterification activities of the enzyme.

Palatability of Aripiprazole Gummies Prepared from Commercially Available Products: Pharmaceutical Formulation for Improving Patient Adherence.

Good adherence to medication is critical for successfully treating psychiatric disorders. Tailor-made pharmaceutical formulations can provide a suitable dosage form to meet the specific needs of individual patients who exhibit poor adherence to industrially manufactured products. Herein, we prepared aripiprazole (ARP) gummies (ARP-Gs) using a commercially available ARP formulation. We aimed to clarify the palatability of ARP-Gs by performing a gustatory sensation test in healthy volunteers. We performed two types of organoleptic masking of ARP-Gs, cocoa- and fruit-flavoured gummies (6.0 mg of ARP/3.5 g of gummy), and conducted two different gustatory sensation tests for each ARP-G. Ten young, healthy volunteers (mean ± standard deviation, 23.7 ± 1.2 years) were enrolled in each trial. The overall palatability of ARP-Gs was evaluated using the 100-mm visual analogue scale (VAS). Receiver operating characteristic (ROC) curve analysis was performed between VAS scores of total ARP-G palatability and acceptability assessed using a 5-point rating scale. Among cocoa-flavoured ARP-Gs, those combining aspartame, cocoa powder, and banana flavour (ABC-ARP-G) exhibited the highest VAS scores for total palatability. Similarly, the VAS scores of grapefruit-flavoured ARP-Gs (GF-ARP-G) showed the highest values considering all fruit-flavoured ARP-Gs. The VAS scores for ABC-ARP-G and GF-ARP-G greatly exceeded the cut-off values of acceptability calculated using the ROC curve. We developed two types of ARP-Gs with organoleptic masking as tailor-made pharmaceutical formulations. ABC-ARP-Gs and GF-ARP-Gs could be acceptable in patients. ARP-Gs could be an alternative to currently available pharmaceutical formulations to enhance their adherence and meet the specific needs of individual patients.

Twin studies on the effect of genetic factors on serum agalactosyl immunoglobulin G levels.

The level of immunoglobulin G (IgG) lacking the terminal galactose, referred to as agalactosyl IgG, was found to be increased in chronic inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease (IBD), particularly in Crohn's disease, which is suggested to have a genetic component. This oligosaccharide modification of IgG is mainly regulated by the expression of glyco-genes; however, the association between genetic factors and changes in the IgG glycosylation has not been fully elucidated. The aim of the present study was to assess the role of genetics in this process by comparing the serum agalactosyl IgG levels between members of monozygotic and dizygotic twin pairs who underwent medical check-ups at the same time. The serum agalactosyl IgG level was assayed using high-performance liquid chromatography. Hematological and biochemical markers, including γ-glutamyltranspeptidase (γGTP), alanine aminotransferase (ALT) and white blood cell (WBC) count, were also measured. Although the serum γGTP levels (and, to a lesser extent, ALT and WBC levels) exhibited a correlation within monozygotic twin pairs, agalactosyl IgG levels were not found to be correlated between members of either type of twin pairs. Thus, the role of genetic factors in determining serum agalactosyl IgG levels may be less significant compared to the effect of environmental factors or the onset of inflammatory disease.

Overexpression of α1,6-fucosyltransferase in hepatoma enhances expression of Golgi phosphoprotein 2 in a fucosylation-independent manner.

Golgi phosphoprotein 2 (GP73) is a type II Golgi protein, which was found on examination of the fucosylated proteome as a potential tumor marker for hepatocellular carcinoma (HCC). The serum levels of both total and fucosylated GP73 were increased in the sera of patients with HCC. Fucosylation is one of the most important oligosaccharide modifications involved in cancer and is catalyzed by α1,6-fucosyltransferase (Fut8). In the present study, we investigated the effect of Fut8 overexpression on GP73 production in the human hepatoma cell line Hep3B. The Fut8 expression vector was transfected into Hep3B cells and the expression of GP73 was investigated by Western blotting and real-time PCR. Overexpression of Fut8 dramatically enhanced the expression of GP73 at the transcriptional level. Surprisingly, this effect was not dependent on cellular fucosylation. Overexpression of a mutant Fut8, which was unable to be localized to the Golgi, did not induce GP73 production, suggesting that the localization of Fut8 in the Golgi apparatus was important for the increase in GP73 expression. This is the first demonstration of GP73 regulation through overexpression of a glycosyltransferase, which may lead to Golgi stress.

Clinical application of a lectin-antibody ELISA to measure fucosylated haptoglobin in sera of patients with pancreatic cancer.

BACKGROUND: Recent advanced techniques in glycobiology have produced a number of tumor marker candidates. As a result from the glycomic approach, we found that fucosylated haptoglobin in sera was a possible tumor marker for pancreatic cancer (PC). Although Aleuria aurantia lectin (AAL) blotting can detect fucosylated haptoglobin, it is difficult to quantify fucosylated haptoglobin precisely. To overcome this problem, we developed a fucosylated haptoglobin detection kit as a sandwich enzyme-linked immune sorbent assay (ELISA) using AAL and the Fab portion of anti-haptoglobin antibody. In the present study, we investigated the clinical application of this lectin-antibody ELISA kit to measure fucosylated haptoglobin in PC. METHODS: We measured fucosylated haptoglobin in patients with PC with a lectin-antibody ELISA kit. The fucosylated haptoglobin measured with this assay was compared with lectin blotting data, and the discrepancy was analyzed by immunoprecipitation methods. The concentration of fucosylated haptoglobin was investigated with respect to the clinical stage of PC. We also measured fucosylated haptoglobin, using 397 cases of several types of cancers including PC, benign diseases, and normal controls. RESULTS: The sensitivity and specificity for the differential diagnosis of PC from normal controls was 50% and 91%, respectively. The results from lectin-antibody ELISA were significantly correlated with data from previous AAL blotting studies. Positive rates of fucosylated haptoglobin with this method in patients with PC were significantly higher in cases of stage IV compared with other clinical stages. Fucosylated haptoglobin was increased in several types of cancers, in which fucosylated haptoglobin was reported to increase. CONCLUSIONS: While certain cases showed a discrepancy in fucosylated haptoglobin concentrations between the lectin-antibody ELISA and conventional lectin blotting, this novel type of lectin-antibody ELISA might be useful for a tumor marker for PC.

Identification of an inducible factor secreted by pancreatic cancer cell lines that stimulates the production of fucosylated haptoglobin in hepatoma cells.

Fucosylation is one of the most important oligosaccharide modifications and is involved in cancer and inflammation. Recently, fucosylated haptoglobin was identified as a possible tumor marker for pancreatic cancer. The molecular mechanism underlying increases in fucosylated haptoglobin in sera of patients with pancreatic cancer seems to be complicated. Our previous study [N. Okuyama, Y. Ide, M. Nakano, T. Nakagawa, K. Yamanaka, K. Moriwaki, K. Murata, H. Ohigashi, S. Yokoyama, H. Eguchi, O. Ishikawa, T. Ito, M. Kato, A. Kasahara, S. Kawano, J. Gu, N. Taniguchi, E. Miyoshi, Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation, Int. J. Cancer 118 (11) (2006) 2803-2808] demonstrated that pancreatic cancer cells secrete a factor, which induces the production of haptoglobin in hepatoma cells. In the present study, we found that interleukin 6 (IL6) expressed in pancreatic cancer is a factor that induces the haptoglobin production, using a neutralizing antibody for IL6. Real-time PCR analyses revealed the up-regulation of fucosylation regulatory genes after IL6 treatment, resulting increases in fucosylated haptoglobin being revealed by a lectin ELISA. This pathway could be one of the possible mechanisms underlying increases in haptoglobin in sera of patients with pancreatic cancer.