The toxicology mechanism of endophytic fungus and swainsonine in locoweed.

Locoweed is a perennial herbaceous plant included in Astragalus spp. and Oxytropis spp. that contains the toxic indolizidine alkaloid swainsonine. The livestock that consume locoweed can suffer from a type of toxicity called locoism. There are aliphaticnitro compounds, selenium, selenium compounds, and alkaloids in locoweed. The toxic component in locoweed has been identified as swainsonine, an indolizidine alkaloid. Swainsonine inhibits lysosomal a-mannosidase and mannosidase II, resulting in altered oligosaccharide degradation and incomplete glycoprotein processing. Corresponding studies on endophytic fungi producing swainsonine have been isolated from a variety of locoweed, and these endophytic fungi and locoweed have a close relationship. Endophytic fungi can promote the growth of locoweed and increase swainsonine production. As a result, livestock that consume locoweed exhibit several symptoms, including dispirited behavior, staggering gait, chromatopsia, trembling, ataxia, and cellular vacuolar degeneration of most tissues by pathological observation. Locoism results in significant annual economic losses. Therefore, in this paper, we review the current research on locoweed, including that on locoweed species distribution in China, endophyte fungus in locoweed, the toxicology mechanism of locoweed, and the swainsonine effect on reproduction.

α-Mannosidase I Protein Expression in Peripheral Blood Mononuclear Cells Is Upregulated During Hepatitis B Virus Infection.

Hepatitis B virus (HBV) has been reported to be recognized by dendritic cell-specific ICAM-3-grabbing nonintegrin in the presence of the α-mannosidase I inhibitor kifunensine, whereas native HBV is not. The aim of our study was to determine whether changes in α-mannosidase I expression in peripheral blood mononuclear cells (PBMCs) occur in patients with HBV infection. Peripheral blood was collected from 90 HBV-infected patients (grouped into immune tolerance, chronic hepatitis B, or inactive carrier group based on their clinical states) and 30 healthy donors. Expression of the three α-mannosidase I subtypes, MAN1A1, MAN1A2, and MAN1C1, was measured using western blot analyses. Compared with the healthy controls, significant increases in the MAN1A1, MAN1A2, and MAN1C1 expression levels were observed in the three HBV-infected groups, among whom the immune tolerance group showed the largest increase. For the patients in the immune tolerance phase, the expression levels of both MAN1A1 and MAN1A2 were linearly and positively correlated with the hepatitis B e antigen (HBeAg) titer and HBV DNA level, although a positive correlation was only found between MAN1C1 expression and the HBeAg titer. These results indicate that increased α-mannosidase I expression in PBMCs may play an important role in HBV immune escape and that its expression level is closely related to viral replication activity.

Metal-ion dependent catalytic properties of Sulfolobus solfataricus class ii α-mannosidase.

The active site for the family GH38 class II α-mannosidase is constituted in part by a divalent metal ion, mostly Zn(2+), as revealed in the crystal structures of enzymes from both animal and bacterial sources. The metal ion coordinates to the bound substrate and side chains of conserved amino acid residues. Recently, evidence has accumulated that class II α-mannosidase is active in complex with a range of divalent metal ions. In the present work, with employment of the class II α-mannosidase, ManA, from the hyperthermophilic archaeon Sulfolobus solfataricus, we explored the influence of the divalent metal ion on the associated steady-state kinetic parameters, K(M) and k(cat), for various substrates. With p-nitrophenyl-α-d-mannoside as a substrate, the enzyme showed activity in the presence of Co(2+), Cd(2+), Mn(2+), and Zn(2+), whereas Ni(2+) and Cu(2+) were inhibitory and nonactivating. Co(2+) was the preferred metal ion, with a k(cat)/K(M) value of about 120 mM(-1) s(-1), 6 times higher than that with Cd(2+) and Zn(2+) and 10 times higher than that with Mn(2+). With α-1,2-, α-1,3-, α-1,4-, or α-1,6-mannobiose as a substrate, Co(2+) was the only metal ion promoting hydrolysis of all substrates; however, Mn(2+), Cd(2+), and Zn(2+) could substitute to a varying extent. A change in the divalent metal ion generally affected the K(M) for the hydrolysis of p-nitrophenyl-α-d-mannoside; however, changes in both k(cat) and K(M) for the hydrolysis of α-mannobioses were observed, along with changing preferences for the glycosidic linkage. Finally, it was found that the metal ion and substrate bind in that order via a steady-state, ordered, sequential mechanism.

Functionalized pyrrolidine inhibitors of human type II alpha-mannosidases as anti-cancer agents: optimizing the fit to the active site.

Refining the chemical structure of functionalized pyrrolidine-based inhibitors of Golgi alpha-mannosidase II (GMII) to optimize binding affinity provided a lead molecule that demonstrated nanomolar competitive inhibition of alpha-mannosidases II and an optimal fit in the active site of Drosophila GMII by X-ray crystallography. Esters of this lead compound also inhibited the growth of human glioblastoma and brain-derived endothelial cells more than the growth of non-tumoral human fibroblasts, suggesting their potential for anti-cancer therapy.

Family 47 alpha-mannosidases in N-glycan processing.

Alpha-mannosidases in eukaryotic cells are involved in both glycan biosynthetic reactions and glycan catabolism. Two broad families of enzymes have been identified that cleave terminal mannose linkages from Asn-linked oligosaccharides (Moremen, 2000), including the Class 1 mannosidases (CAZy GH family 47 (Henrissat and Bairoch, 1996)) of the early secretory pathway involved in the processing of N-glycans and quality control and the Class 2 mannosidases (CAZy family GH38 [Henrissat and Bairoch, 1996]) involved in glycoprotein biosynthesis or catabolism. Within the Class 1 family of alpha-mannosidases, three subfamilies of enzymes have been identified (Moremen, 2000). The endoplasmic reticulum (ER) alpha1,2-mannosidase I (ERManI) subfamily acts to cleave a single residue from Asn-linked glycans in the ER. The Golgi alpha-mannosidase I (GolgiManI) subfamily has at least three members in mammalian systems (Herscovics et al., 1994; Lal et al., 1994; Tremblay and Herscovics, 2000) involved in glycan maturation in the Golgi complex to form the Man(5)GlcNAc(2) processing intermediate. The third subfamily of GH47 proteins comprises the ER degradation, enhancing alpha-mannosidase-like proteins (EDEM proteins) (Helenius and Aebi, 2004; Hirao et al., 2006; Mast et al., 2005). These proteins have been proposed to accelerate the degradation of misfolded proteins in the lumen of the ER by a lectin function that leads to retrotranslocation to the cytosol and proteasomal degradation. Recent studies have also indicated that ERManI acts as a timer for initiation of glycoprotein degradation via the ubiquitin-proteasome pathway (Hosokawa et al., 2003; Wu et al., 2003). This article discusses methods for analysis of the GH47 alpha-mannosidases, including expression, purification, activity assays, generation of point mutants, and binding studies by surface plasmon resonance.

Crystallization and preliminary X-ray analysis of cytosolic alpha-mannosidase from Thermotoga maritima.

Class II alpha-mannosidase cleaves off alpha-1,2-, alpha-1,3- and alpha-1,6-mannose residues. In this paper, the crystallization and preliminary X-ray analysis of cytosolic class II alpha-mannosidase from Thermotoga maritima (TM1851), a family 38 glycoside hydrolase, is described. The crystal of recombinant TM1851 belongs to the C-centred monoclinic space group C2, with unit-cell parameters a = 244.7, b = 87.4, c = 166.6 A, beta = 124.7 degrees. X-ray diffraction data were collected to a resolution of 2.9 A.