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

Itoh, Kohji; Kobayashi, Isao; Nishioka, So-ichiro; Sezutsu, Hideki; Machii, Hiroaki; Tamura, Toshiki

doi:10.5582/ddt.2016.01024

Cited by 19 publications

(10 citation statements)

References 32 publications

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“…Also large-scale production by molecular pharming in transgenic animals has been attempted, e.g., for production of α-glucosidase in milk of transgenic rabbits [184, 185], and for lysosomal acid lipase and α-N-acetylglucosaminidase in egg whites of transgenic hens [186, 187]. Transgenic silk-worms producing active cathepsin-A, able to bind to and be internalized into murine macrophages, have been developed [188]. Insect cells are another expression system that involve Baculovirus-mediated infection of the host cell line.…”

Section: Lysosomal Enzyme Replacement Therapymentioning

confidence: 99%

Lysosomal enzyme replacement therapies: Historical development, clinical outcomes, and future perspectives

Solomon

Muro

2017

Advanced Drug Delivery Reviews

126

113

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Lysosomes and lysosomal enzymes play a central role in numerous cellular processes, including cellular nutrition, recycling, signaling, defense, and cell death. Genetic deficiencies of lysosomal components, most commonly enzymes, are known as “lysosomal storage disorders” or “lysosomal diseases” (LDs) and lead to lysosomal dysfunction. LDs broadly affect peripheral organs and the central nervous system (CNS), debilitating patients and frequently causing fatality. Among other approaches, enzyme replacement therapy (ERT) has advanced to the clinic and represents a beneficial strategy for 8 out of the 50–60 known LDs. However, despite its value, current ERT suffers from several shortcomings, including various side effects, development of “resistance”, and suboptimal delivery throughout the body, particularly to the CNS, lowering the therapeutic outcome and precluding the use of this strategy for a majority of LDs. This review offers an overview of the biomedical causes of LDs, their socio-medical relevance, treatment modalities and caveats, experimental alternatives, and future treatment perspectives.

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Section: Lysosomal Enzyme Replacement Therapymentioning

confidence: 99%

Lysosomal enzyme replacement therapies: Historical development, clinical outcomes, and future perspectives

Solomon

Muro

2017

Advanced Drug Delivery Reviews

126

113

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“…The neuraminidase activity was measured with synthetic substrate 4methylumbelliferyl-α-N-acetyl neuraminic acid (4-MU-NANA) in 50 mM sodium acetate buffer (pH4.5), 2.5 mg/mL BSA, and the reaction was stopped by adding glycine-NaOH buffer (pH10.7). For purified NEU1 activity measurement, purified CTSA precursor derived from transgenic silkworm cocoons 22 was added to the reaction mix in molecular ratio NEU1:CTSA = 1:2. SDS-PAGE and Western Blotting.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

“…Hybridoma was produced by Merck as contract manufacturing. Purified CTSA produced in silkworm 22 was used as an immunogen. Transfected HEK293 NEU1 KO Cells were washed with PBS-0.1% Tween 20 and treated with Alexa Fluor 488 or 555 linked secondary antibody (Cell signaling technology), Alexa Fluor 488 or Cy5-linked streptavidin, and Hoechst 33258 (Merck) for nuclear staining.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

In Cellulo Crystallization of Human Neuraminidase 1 and Biological Roles of N-Glycans

Tsukimoto

Takeuchi

Horii

et al. 2021

ACS Appl. Bio Mater.

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Human neuraminidase 1 (NEU1) is a lysosomal glycosidase that cleaves the terminal sialic acids of sialylglycoconjugates. NEU1 is biosynthesized in the endoplasmic reticulum (ER) lumen as an N-glycosylated protein. NEU1 also associates with cathepsin A (CTSA) in ER, migrates to lysosomes, and exerts catalytic activity. Extraordinary in cellulo crystallization of NEU1 protein in ER despite carrying three N-glycans per molecule at N186, N343, and N352, respectively, were observed when the single human NEU1 gene was overexpressed in mammalian cells. In this study, we first purified the NEU1 from the isolated crystals produced by the HEK293 NEU1-KO cell transiently overexpressing the normal NEU1 and found that the N-glycans were high-mannose or complex types carrying terminal sialic acids. The result suggests that a part of NEU1 crystals were formed or transported to the Golgi apparatus. Second, we compared the effects of single amino acid substitution at the N-sequons, including N186Q, N343Q, and N352Q, each one N-glycan reduction from one NEU1 molecule. We demonstrated that N186Q mutant protein with low enzyme activity and formed a few amounts of smaller crystals. The N343Q mutant exhibited half of the normal intracellular activity, but the numbers and sizes of crystals were almost the same as those of normal NEU1. The N352Q mutant exhibited almost the same activity as the normal enzyme. The numbers of the N352Q crystals were smaller than those of normal NEU1. According to these findings, the N186Q NEU1 protein should have lower stability in ER due to abnormal folding. The second N-glycan at the N343-sequon has little effect on self-aggregation of NEU1. The third N-glycan at the N352-sequon contributes to the self-aggregation of NEU1. We also demonstrated that the three NEU1 mutants associate with the relatively excessive CTSA and migrate to lysosomes.

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“…Since the development of the first transgenic B. mori [15], various functional silks expressing useful fusion proteins have been reported [20,33,34]. The greatest advantage of using the transgenic silkworms is that the functional fibers and proteins can be prepared at low cost.…”

Section: Direct Recovery Of Re Using Silk Powdermentioning

confidence: 99%

Direct Recovery of the Rare Earth Elements Using a Silk Displaying a Metal-Recognizing Peptide

et al. 2020

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Rare earth elements (RE) are indispensable metallic resources in the production of advanced materials; hence, a cost-and energy-effective recovery process is required to meet the rapidly increasing RE demand. Here, we propose an artificial RE recovery approach that uses a functional silk displaying a RE-recognizing peptide. Using the piggyBac system, we constructed a transgenic silkworm in which one or two copies of the gene coding for the RE-recognizing peptide (Lamp1) was fused with that of the fibroin L (FibL) protein. The purified FibL-Lamp1 fusion protein from the transgenic silkworm was able to recognize dysprosium (Dy 3+ ), a RE, under physiological conditions. This method can also be used with silk from which sericin has been removed. Furthermore, the Dy-recovery ability of this silk was significantly improved by crushing the silk. Our simple approach is expected to facilitate the direct recovery of RE from an actual mixed solution of metal ions, such as seawater and industrial wastewater, under mild conditions without additional energy input.

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

Cited by 19 publications

References 32 publications

Lysosomal enzyme replacement therapies: Historical development, clinical outcomes, and future perspectives

Lysosomal enzyme replacement therapies: Historical development, clinical outcomes, and future perspectives

In Cellulo Crystallization of Human Neuraminidase 1 and Biological Roles of N-Glycans

Direct Recovery of the Rare Earth Elements Using a Silk Displaying a Metal-Recognizing Peptide

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