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Histoplasma capsulatum encodes a dipeptidyl peptidase active against the mammalian immunoregulatory peptide, substance P.

Cooper KG, Zarnowski R, Woods JP - PLoS ONE (2009)

Bottom Line: Expression of HcDPPIVA under heterologous regulatory sequences in H. capsulatum resulted in increased secreted DppIV activity, indicating that the encoded protein can be expressed and secreted by its native organism.However, HcDPPIVA was not required for virulence in a murine model of histoplasmosis.This work reports a fungal enzyme that can function to cleave the immunomodulatory host peptide substance P.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin, United States of America.

ABSTRACT
The pathogenic fungus Histoplasma capsulatum secretes dipeptidyl peptidase (Dpp) IV enzyme activity and has two putative DPPIV homologs (HcDPPIVA and HcDPPIVB). We previously showed that HcDPPIVB is the gene responsible for the majority of secreted DppIV activity in H. capsulatum culture supernatant, while we could not detect any functional contribution from HcDPPIVA. In order to determine whether HcDPPIVA encodes a functional DppIV enzyme, we expressed HcDPPIVA in Pichia pastoris and purified the recombinant protein. The recombinant enzyme cleaved synthetic DppIV substrates and had similar biochemical properties to other described DppIV enzymes, with temperature and pH optima of 42 degrees C and 8, respectively. Recombinant HcDppIVA cleaved the host immunoregulatory peptide substance P, indicating the enzyme has the potential to affect the immune response during infection. Expression of HcDPPIVA under heterologous regulatory sequences in H. capsulatum resulted in increased secreted DppIV activity, indicating that the encoded protein can be expressed and secreted by its native organism. However, HcDPPIVA was not required for virulence in a murine model of histoplasmosis. This work reports a fungal enzyme that can function to cleave the immunomodulatory host peptide substance P.

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Expression and purification of recombinant HcDppIVA in Pichia pastoris.(A) Gly-pro-AMC zymogram of supernatants from P. pastoris transformed with empty vector and HcDppIVA-expressing Pichia (mcr#1). Molecular weights (kDa) are indicated on the right. (B) SDS-PAGE gel of supernatants from a non-expressing strain (vector) and mcr#1 as well as the purified fraction of HcDppIVA (rDppIV). The arrow indicates the band likely corresponding to the enzyme. The molecular weight markers (MWM) are in the first lane of the gel. The gel was stained with Coomassie Brilliant Blue.
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pone-0005281-g001: Expression and purification of recombinant HcDppIVA in Pichia pastoris.(A) Gly-pro-AMC zymogram of supernatants from P. pastoris transformed with empty vector and HcDppIVA-expressing Pichia (mcr#1). Molecular weights (kDa) are indicated on the right. (B) SDS-PAGE gel of supernatants from a non-expressing strain (vector) and mcr#1 as well as the purified fraction of HcDppIVA (rDppIV). The arrow indicates the band likely corresponding to the enzyme. The molecular weight markers (MWM) are in the first lane of the gel. The gel was stained with Coomassie Brilliant Blue.

Mentions: In order to test whether HcDPPIVA encoding a putative DppIV enzyme was functional, we expressed the HcDPPIVA open reading frame with its native signal sequence and a C-terminal histidine tag in the methylotrophic yeast Pichia pastoris. After transformation, we induced expression of HcDPPIVA by the addition of methanol to the medium. Culture supernatants were harvested and enzyme activity was detected using a zymogram assay. A fluorescent band appeared in HcDPPIVA-expressing Pichia supernatant but not a control strain transformed with empty vector, suggesting that HcDPPIVA encodes an active DppIV enzyme with a functional secretion signal (Figure 1A). The apparent molecular weight of the recombinant protein was larger than the predicted molecular weight of 88 kilodaltons, suggesting that the enzyme is assembled as a multimer and/or may be modified although we have not experimentally determined the basis for the size or whether multimerization or modification is necessary for function. Dimerization and/or glycosylation have been described for other DppIV enzymes, including Porphyromonas gingivalis DppIV [8] and human CD26 [13], [14]. To characterize the biochemical properties of HcDppIVA, we attempted to purify the C-terminal histidine-tagged enzyme using Ni2+ chromatography but were unsuccessful. Instead, we performed analytical-scale purification of recombinant enzyme from Pichia pastoris culture supernatant using anion exchange and size exclusion chromatography (Figure 1B). We measured DppIV activity of the purified preparation towards Gly-pro-AMC in different buffers and temperatures to determine optimal enzymatic conditions and stability of the enzyme. Similar to reported values for other DppIV enzymes, HcDppIVA is optimally active at 42°C and pH 8. Additionally, HcDppIVA is destabilized by low pH or high temperature (Figure 2). These data indicate that HcDPPIVA encodes a DppIV-type enzyme with biochemical properties similar to those previously reported for DppIV enzymes. As further demonstration of recombinant H. capsulatum enzyme expression in P. pastoris, we replaced the carboxyl-terminal his-tag with a c-myc epitope tag followed by transformation, expression, and supernatant isolation as described above. Western immunoblotting with anti-c-myc antibody revealed a reactive band from P. pastoris transformed with the expression plasmid but not empty vector (data not shown). Furthermore, immunoprecipitation of this recombinant P. pastoris supernatant with anti-c-myc antibody yielded DppIV activity (data not shown).


Histoplasma capsulatum encodes a dipeptidyl peptidase active against the mammalian immunoregulatory peptide, substance P.

Cooper KG, Zarnowski R, Woods JP - PLoS ONE (2009)

Expression and purification of recombinant HcDppIVA in Pichia pastoris.(A) Gly-pro-AMC zymogram of supernatants from P. pastoris transformed with empty vector and HcDppIVA-expressing Pichia (mcr#1). Molecular weights (kDa) are indicated on the right. (B) SDS-PAGE gel of supernatants from a non-expressing strain (vector) and mcr#1 as well as the purified fraction of HcDppIVA (rDppIV). The arrow indicates the band likely corresponding to the enzyme. The molecular weight markers (MWM) are in the first lane of the gel. The gel was stained with Coomassie Brilliant Blue.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2668075&req=5

pone-0005281-g001: Expression and purification of recombinant HcDppIVA in Pichia pastoris.(A) Gly-pro-AMC zymogram of supernatants from P. pastoris transformed with empty vector and HcDppIVA-expressing Pichia (mcr#1). Molecular weights (kDa) are indicated on the right. (B) SDS-PAGE gel of supernatants from a non-expressing strain (vector) and mcr#1 as well as the purified fraction of HcDppIVA (rDppIV). The arrow indicates the band likely corresponding to the enzyme. The molecular weight markers (MWM) are in the first lane of the gel. The gel was stained with Coomassie Brilliant Blue.
Mentions: In order to test whether HcDPPIVA encoding a putative DppIV enzyme was functional, we expressed the HcDPPIVA open reading frame with its native signal sequence and a C-terminal histidine tag in the methylotrophic yeast Pichia pastoris. After transformation, we induced expression of HcDPPIVA by the addition of methanol to the medium. Culture supernatants were harvested and enzyme activity was detected using a zymogram assay. A fluorescent band appeared in HcDPPIVA-expressing Pichia supernatant but not a control strain transformed with empty vector, suggesting that HcDPPIVA encodes an active DppIV enzyme with a functional secretion signal (Figure 1A). The apparent molecular weight of the recombinant protein was larger than the predicted molecular weight of 88 kilodaltons, suggesting that the enzyme is assembled as a multimer and/or may be modified although we have not experimentally determined the basis for the size or whether multimerization or modification is necessary for function. Dimerization and/or glycosylation have been described for other DppIV enzymes, including Porphyromonas gingivalis DppIV [8] and human CD26 [13], [14]. To characterize the biochemical properties of HcDppIVA, we attempted to purify the C-terminal histidine-tagged enzyme using Ni2+ chromatography but were unsuccessful. Instead, we performed analytical-scale purification of recombinant enzyme from Pichia pastoris culture supernatant using anion exchange and size exclusion chromatography (Figure 1B). We measured DppIV activity of the purified preparation towards Gly-pro-AMC in different buffers and temperatures to determine optimal enzymatic conditions and stability of the enzyme. Similar to reported values for other DppIV enzymes, HcDppIVA is optimally active at 42°C and pH 8. Additionally, HcDppIVA is destabilized by low pH or high temperature (Figure 2). These data indicate that HcDPPIVA encodes a DppIV-type enzyme with biochemical properties similar to those previously reported for DppIV enzymes. As further demonstration of recombinant H. capsulatum enzyme expression in P. pastoris, we replaced the carboxyl-terminal his-tag with a c-myc epitope tag followed by transformation, expression, and supernatant isolation as described above. Western immunoblotting with anti-c-myc antibody revealed a reactive band from P. pastoris transformed with the expression plasmid but not empty vector (data not shown). Furthermore, immunoprecipitation of this recombinant P. pastoris supernatant with anti-c-myc antibody yielded DppIV activity (data not shown).

Bottom Line: Expression of HcDPPIVA under heterologous regulatory sequences in H. capsulatum resulted in increased secreted DppIV activity, indicating that the encoded protein can be expressed and secreted by its native organism.However, HcDPPIVA was not required for virulence in a murine model of histoplasmosis.This work reports a fungal enzyme that can function to cleave the immunomodulatory host peptide substance P.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin, United States of America.

ABSTRACT
The pathogenic fungus Histoplasma capsulatum secretes dipeptidyl peptidase (Dpp) IV enzyme activity and has two putative DPPIV homologs (HcDPPIVA and HcDPPIVB). We previously showed that HcDPPIVB is the gene responsible for the majority of secreted DppIV activity in H. capsulatum culture supernatant, while we could not detect any functional contribution from HcDPPIVA. In order to determine whether HcDPPIVA encodes a functional DppIV enzyme, we expressed HcDPPIVA in Pichia pastoris and purified the recombinant protein. The recombinant enzyme cleaved synthetic DppIV substrates and had similar biochemical properties to other described DppIV enzymes, with temperature and pH optima of 42 degrees C and 8, respectively. Recombinant HcDppIVA cleaved the host immunoregulatory peptide substance P, indicating the enzyme has the potential to affect the immune response during infection. Expression of HcDPPIVA under heterologous regulatory sequences in H. capsulatum resulted in increased secreted DppIV activity, indicating that the encoded protein can be expressed and secreted by its native organism. However, HcDPPIVA was not required for virulence in a murine model of histoplasmosis. This work reports a fungal enzyme that can function to cleave the immunomodulatory host peptide substance P.

Show MeSH
Related in: MedlinePlus