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Stable Expression and Characterization of an Optimized Mannose Receptor.

Vigerust DJ, Vick S, Shepherd VL - J Clin Cell Immunol (2015)

Bottom Line: Current model systems available for the study of MR biology often demonstrate low levels of expression and do not retain many of the classical MR properties.To demonstrate the functional characteristics of the optimized receptor, we further show that the introduction of codon-optimized MR plasmid can confer MR-associated phagocytosis of S. aureus to non-phagocytic HeLa cells.This study describes a transfection capable, optimized MR receptor with functional characteristics similar to the wild type receptor and further demonstrates a new system for the continued study of MR biology and function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterans Affairs Medical Center, USA ; Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville TN 37212, USA.

ABSTRACT

The mannose receptor (MR) is a macrophage surface receptor that recognizes pathogen associated molecular patterns (PAMPs) from a diverse array of bacterial, fungal and viral pathogens. Functional studies of the MR are hampered by the scarcity of human cell lines that express the receptor. Current model systems available for the study of MR biology often demonstrate low levels of expression and do not retain many of the classical MR properties. Although several laboratories have reported transient and stable expression of MR from plasmids, preliminary data from our laboratory suggests that these plasmids produce a protein that lacks critical domains and is often not stable over time. In this current report we describe the generation and characterization of a novel human codon-optimized system for transient and stable MR expression. Rare codons and sequences that contribute to mRNA instability were modified to produce mRNA that is qualitatively and quantitatively improved. Confocal imaging of the transient and stably expressed optimized receptor demonstrates a distribution consistent with previous reports. To demonstrate the functional characteristics of the optimized receptor, we further show that the introduction of codon-optimized MR plasmid can confer MR-associated phagocytosis of S. aureus to non-phagocytic HeLa cells. We show that three molecules participate in the engagement and internalization of S. aureus. MR was found to colocalize with Toll-like receptor 2 (TLR2) and Rab5 following exposure to pHrodo-stained S. aureus, suggesting cooperation among the three molecules to engage and internalize the bacterial particle. This study describes a transfection capable, optimized MR receptor with functional characteristics similar to the wild type receptor and further demonstrates a new system for the continued study of MR biology and function.

No MeSH data available.


Related in: MedlinePlus

Codon optimization of human MR. (A). The histograms show the percentage of sequence codons, which fall into a certain quality class. The quality value of the most often used codon for a given amino acid in the desired expression system is set to 100, the remaining codons are scaled accordingly [52]. (B). The plots show the quality of the used codon at the indicated codon position. (C). The plots show the GC content in a 40 bp window centered at the indicated nucleotide position.
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Figure 1: Codon optimization of human MR. (A). The histograms show the percentage of sequence codons, which fall into a certain quality class. The quality value of the most often used codon for a given amino acid in the desired expression system is set to 100, the remaining codons are scaled accordingly [52]. (B). The plots show the quality of the used codon at the indicated codon position. (C). The plots show the GC content in a 40 bp window centered at the indicated nucleotide position.

Mentions: Several recent studies have shown that rational design of mammalian genes can lead to higher level expression of heterologous genes [53]. For example, rare codons can dramatically limit mammalian protein expression in E. coli-based systems. The most commonly described codons include arginine (AGG, AGA, CGA), leucine (CTA), isoleucine (ATA), proline (CCC, and glycine (GGA). If these codons occur at a frequency higher than 1%, it is likely that translation will be negatively affected. In addition, clusters of these codons can reduce both the quantity and quality of synthesized protein [54,55]. The wild type human MR gene contains all of these codons, with significantly high levels of glycine and arginine rare codons, as well as doublet and triplet clusters as shown in Table 1. Data in Figure 1A shows the codon adaptation index (CAI) for the wild type and optimized MR sequence. In this measure, a value of 1 indicates the use of more common codons; any decrease in this number indicates more usage of rare codons. The CAI of 0.73 for the wild type MR correlates with the high percentage of rare codon usage as shown in Table 1, with the optimized MR sequence reaching almost 1. Data from a recent study suggested that wild type genes with a CAI of less than 0.78 were much less likely to properly express. As the CAI approached 1 through optimization, the genes demonstrated high levels of expression suggesting that the CAI was a strong measure of successful expression in mammalian cells [51]. The codon quality at each nucleotide position is shown in Figure 1B. In addition, other studies have reported that low GC content as found in the MR (43%; Figure 1C) can lead to rapid mRNA turnover. Moreover, several negative cis-acting motifs were found which have been reported to hamper expression in mammals (Table 2; Figure 2A) [49,56–58].


Stable Expression and Characterization of an Optimized Mannose Receptor.

Vigerust DJ, Vick S, Shepherd VL - J Clin Cell Immunol (2015)

Codon optimization of human MR. (A). The histograms show the percentage of sequence codons, which fall into a certain quality class. The quality value of the most often used codon for a given amino acid in the desired expression system is set to 100, the remaining codons are scaled accordingly [52]. (B). The plots show the quality of the used codon at the indicated codon position. (C). The plots show the GC content in a 40 bp window centered at the indicated nucleotide position.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4645283&req=5

Figure 1: Codon optimization of human MR. (A). The histograms show the percentage of sequence codons, which fall into a certain quality class. The quality value of the most often used codon for a given amino acid in the desired expression system is set to 100, the remaining codons are scaled accordingly [52]. (B). The plots show the quality of the used codon at the indicated codon position. (C). The plots show the GC content in a 40 bp window centered at the indicated nucleotide position.
Mentions: Several recent studies have shown that rational design of mammalian genes can lead to higher level expression of heterologous genes [53]. For example, rare codons can dramatically limit mammalian protein expression in E. coli-based systems. The most commonly described codons include arginine (AGG, AGA, CGA), leucine (CTA), isoleucine (ATA), proline (CCC, and glycine (GGA). If these codons occur at a frequency higher than 1%, it is likely that translation will be negatively affected. In addition, clusters of these codons can reduce both the quantity and quality of synthesized protein [54,55]. The wild type human MR gene contains all of these codons, with significantly high levels of glycine and arginine rare codons, as well as doublet and triplet clusters as shown in Table 1. Data in Figure 1A shows the codon adaptation index (CAI) for the wild type and optimized MR sequence. In this measure, a value of 1 indicates the use of more common codons; any decrease in this number indicates more usage of rare codons. The CAI of 0.73 for the wild type MR correlates with the high percentage of rare codon usage as shown in Table 1, with the optimized MR sequence reaching almost 1. Data from a recent study suggested that wild type genes with a CAI of less than 0.78 were much less likely to properly express. As the CAI approached 1 through optimization, the genes demonstrated high levels of expression suggesting that the CAI was a strong measure of successful expression in mammalian cells [51]. The codon quality at each nucleotide position is shown in Figure 1B. In addition, other studies have reported that low GC content as found in the MR (43%; Figure 1C) can lead to rapid mRNA turnover. Moreover, several negative cis-acting motifs were found which have been reported to hamper expression in mammals (Table 2; Figure 2A) [49,56–58].

Bottom Line: Current model systems available for the study of MR biology often demonstrate low levels of expression and do not retain many of the classical MR properties.To demonstrate the functional characteristics of the optimized receptor, we further show that the introduction of codon-optimized MR plasmid can confer MR-associated phagocytosis of S. aureus to non-phagocytic HeLa cells.This study describes a transfection capable, optimized MR receptor with functional characteristics similar to the wild type receptor and further demonstrates a new system for the continued study of MR biology and function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterans Affairs Medical Center, USA ; Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville TN 37212, USA.

ABSTRACT

The mannose receptor (MR) is a macrophage surface receptor that recognizes pathogen associated molecular patterns (PAMPs) from a diverse array of bacterial, fungal and viral pathogens. Functional studies of the MR are hampered by the scarcity of human cell lines that express the receptor. Current model systems available for the study of MR biology often demonstrate low levels of expression and do not retain many of the classical MR properties. Although several laboratories have reported transient and stable expression of MR from plasmids, preliminary data from our laboratory suggests that these plasmids produce a protein that lacks critical domains and is often not stable over time. In this current report we describe the generation and characterization of a novel human codon-optimized system for transient and stable MR expression. Rare codons and sequences that contribute to mRNA instability were modified to produce mRNA that is qualitatively and quantitatively improved. Confocal imaging of the transient and stably expressed optimized receptor demonstrates a distribution consistent with previous reports. To demonstrate the functional characteristics of the optimized receptor, we further show that the introduction of codon-optimized MR plasmid can confer MR-associated phagocytosis of S. aureus to non-phagocytic HeLa cells. We show that three molecules participate in the engagement and internalization of S. aureus. MR was found to colocalize with Toll-like receptor 2 (TLR2) and Rab5 following exposure to pHrodo-stained S. aureus, suggesting cooperation among the three molecules to engage and internalize the bacterial particle. This study describes a transfection capable, optimized MR receptor with functional characteristics similar to the wild type receptor and further demonstrates a new system for the continued study of MR biology and function.

No MeSH data available.


Related in: MedlinePlus