A Mannosylated,PEGylated Albumin as a Drug Delivery System for the Treatment of Cancer Stroma Cells

Mannose receptors that are expressed on macrophages and fibroblasts in cancer stroma are promising therapeutic targets for cancer treatment. Albumin can be used as a drug carrier in chemotherapeutics due to its accumulation in the tumor tissue by the enhanced permeability and retention effects. A mannosylated albumin was recently developed as a new drug carrier targeting cells that express mannose receptors such as macrophages and fibroblasts in cancer stroma. The mannosylated albumin is specifically distributed to hepatic macrophages in vivo, leading to an extremely short residence time in the blood. Here, a dual-modified albumin, i.e., mannosylated and polyethylene glycosylated (PEGylated) is reported, to improve its blood circulating time and stromal cell targeting. The product efficiently delivers paclitaxel to stromal cells in a mouse melanoma model, thus resulting in the disruption of stromal cells and suppressed tumor growth, which is seven times stronger than that for PEGylated albumin. The findings suggest that the dual-modified albumin has the potential to provide maximal therapeutic efficacies of chemotherapeutics for the treatment of intractable cancer.     

Mannosylated solid lipid nanoparticles for lung-targeted delivery of Paclitaxel

Objective: The present study discusses paclitaxel (PTX)-loaded mannosylated-DSPE (Distearoyl-phosphatidyl-ethanolamine) solid lipid nanoparticles (M-SLNs) using mannose as a lectin receptor ligand conjugate for lung cancer targeting and to increase the anticancer activity of PTX against A549 lung’s epithelial cancer cells.

Materials and methods: The PTX-SLNs were prepared by solvent injection method and mannose was conjugated to the free amine group of stearylamine. The M-SLNs obtained were characterized for their particle size, polydispersity index, zeta potential and morphology by transmission electron microscope.

Results: The M-SLNs were spherical in shape with 254?±?2.3?nm average size, positive zeta potential (3.27?mV), 79.4?±?1.6 drug entrapment efficiency and showed the lower extent of drug release 40% over 48?h in vitro. Cytotoxicity study on A549 cell lines and biodistrubtion study of drug revealed that M-SLNs deliver a higher concentration of PTX as compared to PTX-SLNs in an alveolar cell site.

Discussion and conclusion: These results suggested that mannosylated M-SLNs are safe and potential vector for lung cancer targeting.     

Cooperative functions of the mannoprotein-encoding genes in the biogenesis and maintenance of the cell wall in Saccharomyces cerevisiae.

To elucidate the roles of genes involved in the cell wall biogenesis and function in Saccharomyces cerevisiae, we isolated and characterized mutants that were lethal in a strain in which the SED1 gene encoding a cell wall mannoprotein was disrupted. Thus, double mutants of SED1 and either MNN9 or MNN10 were unable to grow and YOL155c on a multicopy plasmid could suppress their synthetic lethality. A Yol155cp-GFP fusion protein was found to localize to the cell wall, suggesting that it might also be a cell wall mannoprotein. Subsequently, we analysed the effects of the shut-off of SED1 in a sed1 and mnn9 double mutant: cells after the shut-off showed anomalous cellular morphology and died in the mitotic M phase. From these and other results, we postulate that these genes function cooperatively with each other and in a cell cycle-dependent manner in the biogenesis and maintenance of cell wall in S. cerevisiae.     

Dissecting the Molecular Basis of the Role of the O‐Mannosylation Pathway in Disease: α‐Dystroglycan and Forms of Muscular Dystrophy

Dystroglycanopathies form a subgroup of muscular dystrophies that arise from defects in enzymes that are implicated in the recently elucidated O‐mannosylation pathway, thereby resulting in underglycosylation of α‐dystroglycan. The emerging identification of additional brain proteins modified by O‐mannosylation provides a broader context for interpreting the range of neurological consequences associated with dystroglycanopathies. This form of glycosylation is associated with protein mucin‐like domains that present numerous serine and threonine residues as possible sites for modification. Furthermore, the O‐Man glycans coexist in this region with O‐GalNAc glycans (conventionally associated with such protein sequences), thus resulting in a complex glycoconjugate landscape. Sorting out the relationships between the various molecular defects in glycosylation and the modes of disease presentation, as well as the regulatory interplay among the O‐Man glycans and the effects on other modes of glycosylation in the same domain, is challenging. Here we provide a perspective on chemical biology approaches employing synthetic and analytical methods to address these questions.     

Mutation of the protein-O-mannosyltransferase enhances secretion of the human urokinase-type plasminogen activator in Hansenula polymorpha

Human urokinase-type plasminogen activator (uPA) is poorly secreted and aggregates in the endoplasmic reticulum of yeast cells due to inefficient folding. A screen for Hansenula polymorpha mutants with improved uPA secretion revealed a gene encoding a homologue of the Saccharomyces cerevisiae protein-O-mannosyltransferase Pmt1p. Expression of the H. polymorpha PMT1 gene (HpPMT1) abolished temperature sensitivity of the S. cerevisiae pmt1 pmt2 double mutant. As in S. cerevisiae, inactivation of the HpPMT1 gene affected electrophoretic mobility of the O-glycosylated protein, extracellular chitinase. In contrast to S. cerevisiae, disruption of HpPMT1 alone caused temperature sensitivity. Inactivation of the HpPMT1 gene decreased intracellular aggregation of uPA, suggesting that enhanced secretion of uPA was due to improvement of its folding in the endoplasmic reticulum. Unlike most of the endoplasmic reticulum membrane proteins, HpPmt1p possesses the C-terminal KDEL retention signal.     

A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white

To study cell wall assembly, a simple screening method was devised for isolating cell wall mutants. Mutagenized cells were screened for hypersensitivity to Calcofluor White, which interferes with cell wall assembly. The rationale is that Calcofluor White amplifies the effect of cell wall mutations. As a result, the cells stop growing at lower concentrations of Calcofluor White than cells with normal cell wall. In this way, 63 Calcofluor White-hypersensitive (cwh), monogenic mutants were obtained, ordered into 53 complementation groups. The mannose/glucose ratios of the mutant cell walls varied from 0.15 to 3.95, while wild-type cell walls contained about equal amounts of mannose and glucose. This indicates that both low-mannose and low-glucose cell wall mutants had been obtained. Further characterization showed the presence of three low-mannose cell wall mutants with a mnn9-like phenotype, affected, however, in different genes. In addition, four new killer-resistant (kre) mutants were found, which are presumably affected in the synthesis of β1,6-glucan. Most low-glucose cell wall mutants were not killer resistant, indicating that they might be defective in the synthesis of β1,3-glucan. Eleven cwh mutants were found to be hypersensitive to papulacandin B, which is known to interfere with β1,3-glucan synthesis, and four cwh mutants were temperature-sensitive and lysed at the restrictive temperature. Finally, nine cwh mutants were hypersensitive to caffeine, suggesting that these were affected in signal transduction related to cell wall assembly.     

Isolation of new temperature-sensitive mutants of Saccharomyces cerevisiae deficient in mannose outer chain elongation.

We have isolated two temperature-sensitive Saccharomyces cerevisiae mutants which exhibit a deficiency in mannose outer chain elongation of asparagine-linked oligosaccharide. The size of yeast glycoprotein, secretory form of invertase, of one mutant (och1) was slightly larger than that of the sec18 mutant at the non-permissive temperature, while that of the other mutant (och2) was almost the same as that of the sec18 mutant. Unlike sec mutants, the och mutants were not deficient in secretion of invertase. The och1 mutant showed a 2+:2- cosegregation with regard to the temperature sensitivity and mannose outer chain deficiency, suggesting that a single gene designated as OCH1 is responsible for these two phenotypes. The och1 mutant stopped its growth at the early stage of bud formation and rapidly lost its viability at the non-permissive temperature. The och1 mutation was mapped near the ole1 on the left arm of chromosome VII. The och1 mutant cells accumulated the external invertase containing a large amount of core-like oligosaccharides (Man9-10GlcNAc2) and a small amount of high mannose oligosaccharides (greater than Man50GlcNAc2) at the non-permissive temperature. Production of the active form of human tissue-type plasminogen activator was increased in the och1 mutant compared with the parental strain, suggesting the potential advantage of this mutant for the production of mammalian-type glycoproteins which lack mannose outer chains in yeast.