Effects of signal sequences and folding accessory proteins on extracellular expression of carboxypeptidase Y in recombinant Saccharomyces cerevisiae.

Carboxypeptidase Y (CPY) is a yeast vacuolar protease with useful applications including C-terminal sequencing of peptides and terminal modification of target proteins. To overexpress CPY with the pro-sequence (proCPY) encoded by the Saccharomyces cerevisiae PRC1 gene in recombinant S. cerevisiae, the proCPY gene was combined with the gene coding for a signal sequence of S. cerevisiae mating factor α (MFα), invertase (SUC2), or Kluyveromyces marxianus inulinase (INU1). Among the three constructs, the MFα signal sequence gave the best specific activity of extracellular CPY. To enhance the CPY expression level, folding accessory proteins of Kar2p, Pdi1p and Ero1p located in the S. cerevisiae endoplasmic reticulum were expressed individually and combinatorially. A single expression of Kar2p led to a 28 % enhancement in extracellular CPY activity, relative to the control strain of S. cerevisiae CEN.PK2-1D/p426Gal1-MFαCPY. Coexpression of Kar2p, Pdi1p and Ero1p gave a synergistic effect on CPY expression, of which activity was 1.7 times higher than that of the control strain. This work showed that engineering of signal sequences and protein-folding proteins would be helpful to overexpress yeast proteins of interest.

Recent progress in development of transgenic silkworms overexpressing recombinant human proteins with therapeutic potential in silk glands.

Since 2000, transgenic silkworms have been developed to produce recombinant proteins with therapeutic potential for future clinical use, including antibody preparations. Lysosomal storage diseases (LSDs) are inherited metabolic disorders caused by mutations of lysosomal enzymes associated with excessive accumulation of natural substrates and neurovisceral symptoms. Over the past few years, enzyme replacement therapy (ERT) with human lysosomal enzymes produced by genetically engineered mammalian cell lines has been used clinically to treat several patients with an LSD involving multi-organ symptoms. ERT is based on the incorporation of recombinant glycoenzymes by their binding to glycan receptors on the surface of target cells and their subsequent delivery to lysosomes. However, ERT has several disadvantages, including difficulty mass producing human enzymes, dangers of pathogen contamination, and high costs. Recently, the current authors have succeeded in producing transgenic silkworms overexpressing human lysosomal enzymes in the silk glands and the authors have purified catalytically active enzymes from the middle silk glands. Silk gland-derived human enzymes carrying high-mannose and pauci-mannose N-glycans were endocytosed by monocytes via the mannose receptor pathway and were then delivered to lysosomes. Conjugates with cell-penetrating peptides were also taken up by cultured fibroblasts derived from patients with enzyme deficiencies to restore intracellular catalytic activity and reduce the excessive accumulation of substrates in patient fibroblasts. Transgenic silkworms overexpressing human lysosomal enzymes in the silk glands could serve as future bioresources that provide safe therapeutic enzymes for the treatment of LSDs. Combining recent developments in transglycosylation technology with microbial endoglycosidases will promote the development of therapeutic glycoproteins as bio-medicines.

Indiscriminate glycosylation of procarboxypeptidase Y expressed in Pichia pastoris.

To obtain large amounts of deglycosylated procarboxypeptidase Y (proCPY), in which all of the N-glycosylation sites were replaced by alanine residue by the point mutation method, an expression system was constructed using Pichia pastoris. The secreted enzyme was characterized by SDS-PAGE, native PAGE, MALDI-TOF mass spectrometry, and dynamic light scattering, and the results indicated heterogeneity. The recombinant proCPY contained 29 mol of glucose per mole of protein in average, according to the carbohydrate analysis by the phenol-sulfuric acid method. A large part of the recombinant enzyme absorbed on a Con A column: even the break-through fraction of the column contained 3 mol of glucose per mole of protein. These carbohydrates were removed by the mild alkaline treatment. Since the entire N-glycosylation site had been destructed in the present expression system, the carbohydrates contained in the recombinant proCPY are concluded to be O-linked ones, which bound indiscriminately to serine and/or threonine residues.

Expression and clinical significance of cathepsin B and stefin A in laryngeal cancer.

The aim of this study was to investigate the roles of the cathepsin B cysteine protease and its endogenous inhibitor stefin A in laryngeal cancer. Immunohistochemistry was employed to detect the expression of cathepsin B and stefin A in 84 patients with laryngeal cancer, respectively. The protein expression of stefin A was negatively associated with lymphatic metastasis, recurrence of laryngeal cancer and the survival rate, which was not observed with cathepsin B protein expression. Both down-regulation of cathepsin B and up-regulation of stefin A in vitro significantly inhibited the migration, invasion and proliferation of laryngeal cancer cells, respectively. Our results strongly suggest that stefin A may be a potential predictor of laryngeal cancer and may be used in the molecular diagnosis and gene therapy of laryngeal cancer. Cathepsin B may be used as a promising therapeutic target in the treatment of laryngeal cancer.