Kex2 protease converts the endoplasmic reticulum alpha1,2-mannosidase of Candida albicans into a soluble cytosolic form

Hector Manuel Mora-Montes, Oliver Bader, Everardo López-Romero, Samuel Zinker, Patricia Ponce-Noyola, Bernhard Hube, Neil Andrew Robert Gow, Arturo Flores-Carreón

Research output: Contribution to journalArticle

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Abstract

Cytosolic alpha-mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free N-oligosaccharides. Two soluble alpha-mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched alpha-mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption. E-I corresponded to a polypeptide of 52 kDa that was associated with mannosidase activity and was recognized by an anti-alpha1,2-mannosidase antibody. The N-mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 alpha1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that alpha1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound alpha1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of alpha-mannosidase activity in a kex2Delta null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound alpha1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free N-oligosaccharides revealed that cytosolic alpha1,2-mannosidase E-I trims free Man8GlcNAc2 isomer B into Man7GlcNAc2 isomer B. This is believed to be the first report demonstrating the presence of soluble alpha1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell.
Original languageEnglish
Pages (from-to)3782-3794
Number of pages13
JournalMicrobiology
Volume154
Issue number12
DOIs
Publication statusPublished - 1 Dec 2008

Fingerprint

mannosyl-oligosaccharide 1,2-alpha-mannosidase
Mannosidases
Candida albicans
Endoplasmic Reticulum
alpha-Mannosidase
Peptide Hydrolases
Hydrolases
Oligosaccharides
Mannans
Monensin
Membranes
Density Gradient Centrifugation
Protoplasts
Proteolysis
Peptides
Antibodies
Enzymes

Keywords

  • Candida albicans
  • Cytosol
  • Endoplasmic Reticulum
  • Fungal Proteins
  • Monensin
  • Peptide Hydrolases
  • Solubility
  • alpha-Mannosidase

Cite this

Mora-Montes, H. M., Bader, O., López-Romero, E., Zinker, S., Ponce-Noyola, P., Hube, B., ... Flores-Carreón, A. (2008). Kex2 protease converts the endoplasmic reticulum alpha1,2-mannosidase of Candida albicans into a soluble cytosolic form. Microbiology , 154(12), 3782-3794. https://doi.org/10.1099/mic.0.2008/019315-0

Kex2 protease converts the endoplasmic reticulum alpha1,2-mannosidase of Candida albicans into a soluble cytosolic form. / Mora-Montes, Hector Manuel; Bader, Oliver; López-Romero, Everardo; Zinker, Samuel; Ponce-Noyola, Patricia; Hube, Bernhard; Gow, Neil Andrew Robert; Flores-Carreón, Arturo.

In: Microbiology , Vol. 154, No. 12, 01.12.2008, p. 3782-3794.

Research output: Contribution to journalArticle

Mora-Montes, HM, Bader, O, López-Romero, E, Zinker, S, Ponce-Noyola, P, Hube, B, Gow, NAR & Flores-Carreón, A 2008, 'Kex2 protease converts the endoplasmic reticulum alpha1,2-mannosidase of Candida albicans into a soluble cytosolic form', Microbiology , vol. 154, no. 12, pp. 3782-3794. https://doi.org/10.1099/mic.0.2008/019315-0
Mora-Montes, Hector Manuel ; Bader, Oliver ; López-Romero, Everardo ; Zinker, Samuel ; Ponce-Noyola, Patricia ; Hube, Bernhard ; Gow, Neil Andrew Robert ; Flores-Carreón, Arturo. / Kex2 protease converts the endoplasmic reticulum alpha1,2-mannosidase of Candida albicans into a soluble cytosolic form. In: Microbiology . 2008 ; Vol. 154, No. 12. pp. 3782-3794.
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abstract = "Cytosolic alpha-mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free N-oligosaccharides. Two soluble alpha-mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched alpha-mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption. E-I corresponded to a polypeptide of 52 kDa that was associated with mannosidase activity and was recognized by an anti-alpha1,2-mannosidase antibody. The N-mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 alpha1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that alpha1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound alpha1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of alpha-mannosidase activity in a kex2Delta null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound alpha1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free N-oligosaccharides revealed that cytosolic alpha1,2-mannosidase E-I trims free Man8GlcNAc2 isomer B into Man7GlcNAc2 isomer B. This is believed to be the first report demonstrating the presence of soluble alpha1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell.",
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T1 - Kex2 protease converts the endoplasmic reticulum alpha1,2-mannosidase of Candida albicans into a soluble cytosolic form

AU - Mora-Montes, Hector Manuel

AU - Bader, Oliver

AU - López-Romero, Everardo

AU - Zinker, Samuel

AU - Ponce-Noyola, Patricia

AU - Hube, Bernhard

AU - Gow, Neil Andrew Robert

AU - Flores-Carreón, Arturo

PY - 2008/12/1

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N2 - Cytosolic alpha-mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free N-oligosaccharides. Two soluble alpha-mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched alpha-mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption. E-I corresponded to a polypeptide of 52 kDa that was associated with mannosidase activity and was recognized by an anti-alpha1,2-mannosidase antibody. The N-mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 alpha1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that alpha1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound alpha1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of alpha-mannosidase activity in a kex2Delta null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound alpha1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free N-oligosaccharides revealed that cytosolic alpha1,2-mannosidase E-I trims free Man8GlcNAc2 isomer B into Man7GlcNAc2 isomer B. This is believed to be the first report demonstrating the presence of soluble alpha1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell.

AB - Cytosolic alpha-mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free N-oligosaccharides. Two soluble alpha-mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched alpha-mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption. E-I corresponded to a polypeptide of 52 kDa that was associated with mannosidase activity and was recognized by an anti-alpha1,2-mannosidase antibody. The N-mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 alpha1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that alpha1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound alpha1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of alpha-mannosidase activity in a kex2Delta null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound alpha1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free N-oligosaccharides revealed that cytosolic alpha1,2-mannosidase E-I trims free Man8GlcNAc2 isomer B into Man7GlcNAc2 isomer B. This is believed to be the first report demonstrating the presence of soluble alpha1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell.

KW - Candida albicans

KW - Cytosol

KW - Endoplasmic Reticulum

KW - Fungal Proteins

KW - Monensin

KW - Peptide Hydrolases

KW - Solubility

KW - alpha-Mannosidase

U2 - 10.1099/mic.0.2008/019315-0

DO - 10.1099/mic.0.2008/019315-0

M3 - Article

VL - 154

SP - 3782

EP - 3794

JO - Microbiology

JF - Microbiology

SN - 1350-0872

IS - 12

ER -