Alteration in the IL‐2 signal peptide affects secretion of proteins <i>in vitro</i> and <i>in vivo</i>

Zhang, Lei; Leng, Qixin; Mixson, A. James

doi:10.1002/jgm.677

Cited by 84 publications

(71 citation statements)

References 41 publications

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“…Additional modifications of the BMP-2 signal sequence led to loss of potency in secretion when altering the length of the sequence rather than altering specific amino acids. Therefore, previously described observations of enhanced potency of a secretion signal by increasing the basicity and hydrophobicity of the amino acid composition (Zhang et al, 2005) are in contrast to our results obtained after modifications of the BMP-2 secretion signal. One of the few candidates which displayed a potency to mediate protein secretion comparable to, or higher than, the native BMP-2 signal sequence was a signal sequence designed in silico (Z), consisting of a typical short basic region followed by a hydrophobic area combined with an in silico designed cleavage site.…”

Section: Figcontrasting

confidence: 99%

“…The secretion of proteins can be a limiting step if the native protein secretion signal is weak. The different potencies of secretion signals to drive protein secretion can be related to their amino acid composition (Futatsumori-Sugai and Tsumoto, 2010;Klatt and Konthur, 2012;Knappskog et al, 2007;Kober et al, 2013;Wen et al, 2011;Zhang et al, 2005). Based on this, the present study was expanded by the addition of screening and modification of protein secretion signals in order to identify signal peptide candidates with beneficial activity for subsequent exchange of the native BMP-2 protein secretion sequence.…”

Section: Introductionmentioning

confidence: 99%

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Improved osteogenic vector for non-viral gene therapy

Hacobian

Posa-Markaryan²,

Sperger³

et al. 2016

eCM

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Therapeutic compensation of deficient bone regeneration is a challenging task and a topic of on-going search for novel treatment strategies. One promising approach for improvement involves non-viral gene delivery using the bone morphogenetic protein-2 (BMP-2) gene to provide transient, local and sustained expression of the growth factor. However, since efficiency of non-viral gene delivery is low, this study focused on the improvement of a BMP-2 gene expression system, aiming for compensation of poor transfection efficiency. First, the native BMP-2 gene sequence was modified by codon optimisation and altered by inserting a highly truncated artificial intron (96 bp). Transfection of multiple cell lines and rat adipose-derived mesenchymal stem cells with plasmids harbouring the improved BMP-2 sequence led to a several fold increased expression rate and subsequent osteogenic differentiation. Additionally, comparing expression kinetics of elongation factor 1 alpha (EF1α) promoter with a state of the art CMV promoter revealed significantly higher BMP-2 expression when under the influence of the EF1α promoter. Results obtained by quantification of bone markers as well as osteogenic assays showed reduced sensitivity to promoter silencing effects of the EF1α promoter in rat adiposederived mesenchymal stem cells. Finally, screening of several protein secretion signals using either luciferase or BMP-2 as reporter protein revealed no superior candidates for potential replacement of the native BMP-2 secretion signal. Taken together, by enhancing the exogenous BMP-2 expression system, low transfection efficiencies in therapeutic applications can be compensated, making safe non-viral systems even more suitable for tissue regeneration approaches.

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Section: Figcontrasting

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Improved osteogenic vector for non-viral gene therapy

Hacobian

Posa-Markaryan²,

Sperger³

et al. 2016

eCM

View full text Add to dashboard Cite

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“…Both leader sequences used in our studies did share those features. However, in the rat KC chemokine leader, the amino acid residues near the cleavage site have smaller side chains (Ser, Thr) than those of the H-2L d leader (Arg, Gln), which could account for its increased effectiveness (26,43). In conclusion, our findings demonstrate that leader sequences can be used to rationally design minigene DNA vaccines.…”

mentioning

confidence: 75%

The Signal Peptide Sequence Impacts the Immune Response Elicited by a DNA Epitope Vaccine

Vatakis

McMillan

2011

Clin. Vaccine Immunol.

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We examined the effect of two leader sequences, one from a transmembrane molecule (H2-L d ) and another from a secreted molecule (rat KC chemokine), on the immunogenicity of DNA epitope vaccines. The chemokine leader enhanced vaccine immunogenicity, thus underscoring the importance of the leader sequence in DNA epitope vaccine design.DNA vaccines have been shown to elicit strong humoral and cell-mediated immune responses in many animal models (2,12,13,23). Studies suggest that the inoculated DNA is internalized by the local antigen-presenting cells (APCs), which then express and present the plasmid-encoded antigen to cytotoxic lymphocytes (CTLs) or T helper (T H ) cells. Also, in vivo-transfected cells at the site of inoculation, such as keratinocytes and myocytes, can secrete the encoded antigen, which can be processed and presented by APCs (4,6,11,23,25).The immune responses elicited by DNA vaccines have had varied results in different animal models, underscoring the need for optimization (1,22,23). An advantage of DNA vaccines is the ease with which one can introduce changes to qualitatively and quantitatively affect their immunogenicity (13,23,35). A number of approaches have been employed to enhance the efficacy of DNA vaccines, which include optimizing antigen processing (24) and presentation (5, 9, 17, 27, 29-31, 33, 37), using epitope-carrier protein fusion molecules (41), employing different delivery methods (18,21,22,28,34), and introducing immunomodulatory sequences in the plasmid vaccines (3,8,10,13,19,22,23,40). In both full-length and minigene DNA vaccines, leader sequences have been used to directly target the encoded antigen/epitope to the endoplasmic reticulum (ER) for major histocompatibility complex class I (MHC I) presentation with mixed success (5,7,9,17,33). Studies have shown that signal sequences exhibit wide diversity, impacting their function and role in protein targeting, release, and maturation (15,20,32,38,39,42). Therefore, the alteration of the leader sequence can influence the immunogenicity of DNA epitope vaccines.In this study, we compared the immune responses elicited by two minigene DNA vaccines encoding different leaders, one derived from the murine H-2L d molecule and the other derived from the rat KC chemokine, a member of the platelet factor 4 neutrophil chemoattractant family (16). We rationalized that a leader from a secreted molecule (the rat KC) would target the class I epitope to the ER more efficiently than would the other leader. We have analyzed and compared the immune responses elicited by the two vectors and demonstrate that the KC chemokine leader improved the immunogenicity of the DNA epitope vaccines.To assess the role of the leader sequence in the DNA epitope vaccine immunogenicity, we constructed two vectors to be used in mouse immunizations (Fig. 1A). The first one, SAOLL, encodes a CTL epitope, HIV gp120 IIIB (318-327) (A), which is targeted to the ER by the murine H-2L d leader (S). The plasmid also contains a T H epitope, ovalbumin (OVA) (323-339) (O), ...

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“…Researchers showed that α-amylase, an enzyme native to the Bacillus stearothermophilus that degrades starch, can be secreted from mammalian cells by fusing a signal peptide 90 (METDTLLLWVLLLWVPGSTGD) derived from the murine Ig k-chain V-J2-C region [48,88]. Some of the commonly used secretion signals described in [8] are (i) ILCO1, which is an alteration of the interleukin-2 (IL-2) N-terminal signal peptide (ii) SS, which is the secretion signal originally derived from a piscine vitellogenin (Vtg) gene of Oreochromis aureus [88][89][90] (iii) Sec, which is a recently identified secretion signal that is a part of the C-terminal of the Engrailed-2 homeodomain protein present in Drosophila melanogaster. SEAP (Secreted Alkaline Phosphate) is naturally secreted from mammalian cells.…”

Section: (Vi) Type VI Secretion System (T6ss)mentioning

confidence: 99%

Genetically Programmable Pathogen Sense and Destroy

2013

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Twenty five percent of all the deaths worldwide are caused by infectious diseases. They are also the biggest cause of mortality among children under five years of age. Among them diarrheal diseases alone cause as many deaths as AIDS or TB and malaria combined. Also up to 80% of traveler's diarrhea is bacterial in nature. Vibrio cholerae (cholera), Salmonella spp (typhoid fever), Shigella spp (shigellosis) and a variety of enteropathogenic Escherichia coli strains are among the principle bacterial agents that cause this type of diarrhea.

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Alteration in the IL‐2 signal peptide affects secretion of proteins in vitro and in vivo

Abstract: Modification of the IL-2 signal peptide augments protein secretion both in vitro and in vivo. As a result, optimizing the signal peptide should be considered for increasing the therapeutic levels of secreted proteins.

Cited by 84 publications

References 41 publications

Improved osteogenic vector for non-viral gene therapy

Improved osteogenic vector for non-viral gene therapy

The Signal Peptide Sequence Impacts the Immune Response Elicited by a DNA Epitope Vaccine

Genetically Programmable Pathogen Sense and Destroy

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