Limits...
Novel transmembrane receptor involved in phagosome transport of lysozymes and β-hexosaminidase in the enteric protozoan Entamoeba histolytica.

Furukawa A, Nakada-Tsukui K, Nozaki T - PLoS Pathog. (2012)

Bottom Line: We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8.We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens.This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo, Japan.

ABSTRACT
Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and β-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes.

Show MeSH

Related in: MedlinePlus

The serine-rich region of CPBF8 is involved in the binding with β-hexosaminidase α-subunit and lysozymes.(A) Schematic diagram of the serine-rich region and transmembrane domain in CPBF8. Numbers indicate amino acid positions from the amino terminus. The filled or hatched box depicts the serine rich region or the transmembrane domain, respectively. (B) Comparison of the carboxyl-terminal region of CPBF proteins. Boxes indicate the putative transmembrane domain. The serine-rich region is underlined. (C) The amino acid sequences of the wild-type and mutated serine-rich regions (SRR). Note that the entire SRR was deleted in CPBF8ΔSRR-HA. The first or second stretch of three serine residues within SRR were substituted with alanines in CPBF8AAA1-HA and CPBF8AAA2-HA, respectively. (D) Localization of CPBF8ΔSRR-HA to phagosomes. Amoebae were incubated with Cell Tracker Blue-stained CHO cells (blue) for 60 min, fixed, and reacted with anti-HA antibody (green). Bar, 10 µm. (E–F) Isolation and identification of binding proteins of CPBF8-HA, CPBF8ΔSRR-HA, CPBF8AAA1-HA, and CPBF8AAA2-HA. Lysates of CPBF8-HA, CPBF8ΔSRR-HA, CPBF8AAA1-HA, and CPBF8AAA2-HA transformants were mixed with anti-HA-antibody-conjugated agarose, washed, and eluted with HA peptide. Immunoprecipitated samples were separated on SDS-PAGE and silver stained (The upper and lower arrow indicated that β-hexosaminidase α-subunit and lysozymes, respectively. (E), or blotted and reacted with anti-HA, β-hexosaminidase α-subunit and lysozyme2 antibody (F).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3285589&req=5

ppat-1002539-g006: The serine-rich region of CPBF8 is involved in the binding with β-hexosaminidase α-subunit and lysozymes.(A) Schematic diagram of the serine-rich region and transmembrane domain in CPBF8. Numbers indicate amino acid positions from the amino terminus. The filled or hatched box depicts the serine rich region or the transmembrane domain, respectively. (B) Comparison of the carboxyl-terminal region of CPBF proteins. Boxes indicate the putative transmembrane domain. The serine-rich region is underlined. (C) The amino acid sequences of the wild-type and mutated serine-rich regions (SRR). Note that the entire SRR was deleted in CPBF8ΔSRR-HA. The first or second stretch of three serine residues within SRR were substituted with alanines in CPBF8AAA1-HA and CPBF8AAA2-HA, respectively. (D) Localization of CPBF8ΔSRR-HA to phagosomes. Amoebae were incubated with Cell Tracker Blue-stained CHO cells (blue) for 60 min, fixed, and reacted with anti-HA antibody (green). Bar, 10 µm. (E–F) Isolation and identification of binding proteins of CPBF8-HA, CPBF8ΔSRR-HA, CPBF8AAA1-HA, and CPBF8AAA2-HA. Lysates of CPBF8-HA, CPBF8ΔSRR-HA, CPBF8AAA1-HA, and CPBF8AAA2-HA transformants were mixed with anti-HA-antibody-conjugated agarose, washed, and eluted with HA peptide. Immunoprecipitated samples were separated on SDS-PAGE and silver stained (The upper and lower arrow indicated that β-hexosaminidase α-subunit and lysozymes, respectively. (E), or blotted and reacted with anti-HA, β-hexosaminidase α-subunit and lysozyme2 antibody (F).

Mentions: CPBF family proteins show common structural organization: the signal peptide at the amino terminus, the transmembrane domain close to the carboxyl-terminal end, and the YxxL motif in the cytosolic tail located at the carboxyl terminus (Nakada-Tsukui K, et al., unpublished data). Besides, among 11 members, only 3 members, CPBF6, CPBF7, and CPBF8, have a stretch of serine-rich hydrophilic region prior to the transmembrane domain (Figure 6A and B). In order to investigate whether this region is involved in the binding of CPBF8 to the cargos and whether the region is involved in the phagosomal transport, we created a transformant that expressed HA-tagged CPBF8 lacking the 23-a.a.-long serine-rich region (CPBF8ΔSRR-HA). We immunoprecipitated CPBF8-HA and CPBF8ΔSRR-HA using anti-HA antibody from lysates of the corresponding strains. Both the silver-stained SDS-PAGE gel and immunoblot analysis with HA antibody showed that the size of CPBF8ΔSRR-HA (∼100 kDa) detected was ∼50 kDa smaller than that of CPBF8-HA (∼150 kDa), which was larger than predicted (Figure 6 E and F, see below “The nature of post-translational modifications of CPBF8”). The amount of the 75- and 25-kDa proteins, which correspond to β-hexosaminidase α-subunit and lysozymes, respectively, detected in the immunoprecipitated samples from the lysates of CPBF8ΔSRR-HA significantly decreased, compared to that from CPBF8-HA strain (Figure 6E and F). The identity of the precipitated proteins was confirmed by the immunoblots using anti-β-hexosaminidase α-subunit antibody and lysozyme 2 antibody (Figure 6F).


Novel transmembrane receptor involved in phagosome transport of lysozymes and β-hexosaminidase in the enteric protozoan Entamoeba histolytica.

Furukawa A, Nakada-Tsukui K, Nozaki T - PLoS Pathog. (2012)

The serine-rich region of CPBF8 is involved in the binding with β-hexosaminidase α-subunit and lysozymes.(A) Schematic diagram of the serine-rich region and transmembrane domain in CPBF8. Numbers indicate amino acid positions from the amino terminus. The filled or hatched box depicts the serine rich region or the transmembrane domain, respectively. (B) Comparison of the carboxyl-terminal region of CPBF proteins. Boxes indicate the putative transmembrane domain. The serine-rich region is underlined. (C) The amino acid sequences of the wild-type and mutated serine-rich regions (SRR). Note that the entire SRR was deleted in CPBF8ΔSRR-HA. The first or second stretch of three serine residues within SRR were substituted with alanines in CPBF8AAA1-HA and CPBF8AAA2-HA, respectively. (D) Localization of CPBF8ΔSRR-HA to phagosomes. Amoebae were incubated with Cell Tracker Blue-stained CHO cells (blue) for 60 min, fixed, and reacted with anti-HA antibody (green). Bar, 10 µm. (E–F) Isolation and identification of binding proteins of CPBF8-HA, CPBF8ΔSRR-HA, CPBF8AAA1-HA, and CPBF8AAA2-HA. Lysates of CPBF8-HA, CPBF8ΔSRR-HA, CPBF8AAA1-HA, and CPBF8AAA2-HA transformants were mixed with anti-HA-antibody-conjugated agarose, washed, and eluted with HA peptide. Immunoprecipitated samples were separated on SDS-PAGE and silver stained (The upper and lower arrow indicated that β-hexosaminidase α-subunit and lysozymes, respectively. (E), or blotted and reacted with anti-HA, β-hexosaminidase α-subunit and lysozyme2 antibody (F).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285589&req=5

ppat-1002539-g006: The serine-rich region of CPBF8 is involved in the binding with β-hexosaminidase α-subunit and lysozymes.(A) Schematic diagram of the serine-rich region and transmembrane domain in CPBF8. Numbers indicate amino acid positions from the amino terminus. The filled or hatched box depicts the serine rich region or the transmembrane domain, respectively. (B) Comparison of the carboxyl-terminal region of CPBF proteins. Boxes indicate the putative transmembrane domain. The serine-rich region is underlined. (C) The amino acid sequences of the wild-type and mutated serine-rich regions (SRR). Note that the entire SRR was deleted in CPBF8ΔSRR-HA. The first or second stretch of three serine residues within SRR were substituted with alanines in CPBF8AAA1-HA and CPBF8AAA2-HA, respectively. (D) Localization of CPBF8ΔSRR-HA to phagosomes. Amoebae were incubated with Cell Tracker Blue-stained CHO cells (blue) for 60 min, fixed, and reacted with anti-HA antibody (green). Bar, 10 µm. (E–F) Isolation and identification of binding proteins of CPBF8-HA, CPBF8ΔSRR-HA, CPBF8AAA1-HA, and CPBF8AAA2-HA. Lysates of CPBF8-HA, CPBF8ΔSRR-HA, CPBF8AAA1-HA, and CPBF8AAA2-HA transformants were mixed with anti-HA-antibody-conjugated agarose, washed, and eluted with HA peptide. Immunoprecipitated samples were separated on SDS-PAGE and silver stained (The upper and lower arrow indicated that β-hexosaminidase α-subunit and lysozymes, respectively. (E), or blotted and reacted with anti-HA, β-hexosaminidase α-subunit and lysozyme2 antibody (F).
Mentions: CPBF family proteins show common structural organization: the signal peptide at the amino terminus, the transmembrane domain close to the carboxyl-terminal end, and the YxxL motif in the cytosolic tail located at the carboxyl terminus (Nakada-Tsukui K, et al., unpublished data). Besides, among 11 members, only 3 members, CPBF6, CPBF7, and CPBF8, have a stretch of serine-rich hydrophilic region prior to the transmembrane domain (Figure 6A and B). In order to investigate whether this region is involved in the binding of CPBF8 to the cargos and whether the region is involved in the phagosomal transport, we created a transformant that expressed HA-tagged CPBF8 lacking the 23-a.a.-long serine-rich region (CPBF8ΔSRR-HA). We immunoprecipitated CPBF8-HA and CPBF8ΔSRR-HA using anti-HA antibody from lysates of the corresponding strains. Both the silver-stained SDS-PAGE gel and immunoblot analysis with HA antibody showed that the size of CPBF8ΔSRR-HA (∼100 kDa) detected was ∼50 kDa smaller than that of CPBF8-HA (∼150 kDa), which was larger than predicted (Figure 6 E and F, see below “The nature of post-translational modifications of CPBF8”). The amount of the 75- and 25-kDa proteins, which correspond to β-hexosaminidase α-subunit and lysozymes, respectively, detected in the immunoprecipitated samples from the lysates of CPBF8ΔSRR-HA significantly decreased, compared to that from CPBF8-HA strain (Figure 6E and F). The identity of the precipitated proteins was confirmed by the immunoblots using anti-β-hexosaminidase α-subunit antibody and lysozyme 2 antibody (Figure 6F).

Bottom Line: We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8.We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens.This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo, Japan.

ABSTRACT
Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and β-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes.

Show MeSH
Related in: MedlinePlus