Human secretory signal peptide description by hidden Markov model and generation of a strong artificial signal peptide for secreted protein expression

Barash, Steve; Wang, Wei; Shi, Yijiang

doi:10.1016/s0006-291x(02)00566-1

Cited by 99 publications

(83 citation statements)

References 26 publications

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“…By immunocytochemistry we showed that angiogenin overexpressed in NSC34 cells localizes to a tunicamycin-sensitive perinuclear reticulate structure and Golgi function-dependent granular structures before secretion. This ER-Golgi trafficking is consistent with the angiogenin protein sequence, which contains a hydrophobic amino acid-rich signal peptide, strongly predicted to couple translation with ER translocation and secretion (Kurachi et al, 1985;Barash et al, 2002). Our data support previous studies showing similar perinuclear/ER localization of endogenous angiogenin and also suggest a regulated release of angiogenin protein by secretory granule exocytosis (Kurachi et al, 1985;Pavlov et al, 2003).…”

Section: Discussionsupporting

confidence: 90%

Motoneurons Secrete Angiogenin to Induce RNA Cleavage in Astroglia

Skorupa

King²,

Aparicio³

et al. 2012

J. Neurosci.

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Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder affecting motoneurons. Mutations in angiogenin, encoding a member of the pancreatic RNase A superfamily, segregate with ALS. We previously demonstrated that angiogenin administration shows promise as a neuroprotective therapeutic in studies using transgenic ALS mice and primary motoneuron cultures. Its mechanism of action and target cells in the spinal cord, however, are largely unknown. Using mixed motoneuron cultures, motoneuron-like NSC34 cells, and primary astroglia cultures as model systems, we here demonstrate that angiogenin is a neuronally secreted factor that is endocytosed by astroglia and mediates neuroprotection in paracrine. We show that wild-type angiogenin acts unidirectionally to induce RNA cleavage in astroglia, while the ALS-associated K40I mutant is also secreted and endocytosed, but fails to induce RNA cleavage. Angiogenin uptake into astroglia requires heparan sulfate proteoglycans, and engages clathrin-mediated endocytosis. We show that this uptake mechanism exists for mouse and human angiogenin, and delivers a functional RNase output. Moreover, we identify syndecan 4 as the angiogenin receptor mediating the selective uptake of angiogenin into astroglia. Our data provide new insights into the paracrine activities of angiogenin in the nervous system, and further highlight the critical role of non-neuronal cells in the pathogenesis of ALS.

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Section: Discussionsupporting

confidence: 90%

Motoneurons Secrete Angiogenin to Induce RNA Cleavage in Astroglia

Skorupa

King²,

Aparicio³

et al. 2012

J. Neurosci.

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“…A synthetic HP-NAP transgene sequence was purchased from GenScript (Piscataway, NJ). It was codon optimized for Homo sapiens; it contains the sequence for an artificial signal peptide for efficient secretion (27) and the sequence for an N-terminal tag (63His); and it is flanked by HpaI and XbaI restriction sites. This transgene was introduced and replaced E1A in the pShuttle-i/PPT-E1A (28).…”

Section: Recombinant Adenoviral Vectorsmentioning

confidence: 99%

Vector-Encoded Helicobacter pylori Neutrophil-Activating Protein Promotes Maturation of Dendritic Cells with Th1 Polarization and Improved Migration

Ramachandran

Jin

et al. 2014

The Journal of Immunology

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Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor involved in H. pylori infection. Both HP-NAP protein and oncolytic viruses encoding HP-NAP have been suggested as immunotherapeutic anticancer agents and adjuvants for vaccination but with little known about its mode of action to activate adaptive immunity. Dendritic cells (DCs) are key players in bridging innate and adaptive immune responses, and in this study we aim to evaluate the effect of HP-NAP on DC maturation, migration, and induction of adaptive immune response. Maturation markers CD83, CD80, CD86, HLA-DR, CD40, and CCR7 were upregulated on human DCs after treatment with supernatants from HP-NAP adenovirus–infected cells. HP-NAP–activated DCs had a Th1 cytokine secretion profile, with high IL-12 and relatively low IL-10 secretion, and migrated toward CCL19. Ag-specific T cells were efficiently expanded by Ag-presenting HP-NAP–activated DCs, which is an important property of functionally mature DCs. Furthermore, intradermal injections of HP-NAP–encoding adenovirus in C57BL/6 mice enhanced resident DC migration to draining lymph nodes, which was verified by imaging lymph nodes by two-photon microscopy and by phenotyping migrating cells by flow cytometry. In conclusion, therapeutic effects of HP-NAP are mediated by maturation of DCs and subsequent activation of Ag-specific T cells in addition to provoking innate immunity.

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“…Previous studies have shown that optimization of signal peptides effectively enhances the production of recombinant therapeutic proteins. [20][21][22] It has been shown that the native signal peptide is not always the most effect signal peptide for recombinant protein expression. 9 In this study, 5 signal peptides from different sources, including the native signal peptide, were fused to the N-terminal of rFVII and expressed in CHO cells.…”

Section: Discussionmentioning

confidence: 99%

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium

Lin

et al. 2016

Bioengineered

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(2016) Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium, Bioengineered, 7:3, 189-197, DOI: 10.1080/21655979.2016 ABSTRACTSignal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4CF4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.

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Human secretory signal peptide description by hidden Markov model and generation of a strong artificial signal peptide for secreted protein expression

Cited by 99 publications

References 26 publications

Motoneurons Secrete Angiogenin to Induce RNA Cleavage in Astroglia

Motoneurons Secrete Angiogenin to Induce RNA Cleavage in Astroglia

Vector-Encoded Helicobacter pylori Neutrophil-Activating Protein Promotes Maturation of Dendritic Cells with Th1 Polarization and Improved Migration

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium

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