Heterologous expression and biochemical characterization of an α1,2-mannosidase encoded by the Candida albicans MNS1 gene

Mora-Montes, Héctor M.; López-Romero, Everardo; Zínker, Samuel; Ponce-Noyola, Patricia; Flores-Carreón, Arturo

doi:10.1590/s0074-02762008000700016

Cited by 8 publications

(6 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This relationship was further supported by the observation that a C. albicans mns1D null mutant lacks soluble and membrane-bound a-mannosidase activities [102]. To further investigate the ER a1,2-mannosidase, the MNS1 gene from C. albicans was cloned in E. coli, expressed and the enzyme purified [127]. The biochemical properties of the recombinant enzyme were closely comparable to those of Mns1 isolated from C. albicans membranes [127].…”

Section: A -Mannosidasesmentioning

confidence: 81%

Protein Glycosylation in Candida

et al. 2009

Self Cite

View full text Add to dashboard Cite

Candidiasis is a significant cause of invasive human mycosis with associated mortality rates that are equivalent to, or worse than, those cited for most cases of bacterial septicemia. As a result, considerable efforts are being made to understand how the fungus invades host cells and to identify new targets for fungal chemotherapy. This has led to an increasing interest in Candida glycobiology, with an emphasis on the identification of enzymes essential for glycoprotein and adhesion metabolism, and the role of N- and O-linked glycans in host recognition and virulence. Here, we refer to studies dealing with the identification and characterization of enzymes such as dolichol phosphate mannose synthase, dolichol phosphate glucose synthase and processing glycosidases and synthesis, structure and recognition of mannans and discuss recent findings in the context of Candida albicans pathogenesis.

show abstract

Section: A -Mannosidasesmentioning

confidence: 81%

Protein Glycosylation in Candida

et al. 2009

Self Cite

View full text Add to dashboard Cite

show abstract

“…As with α1,2-mannosidases from S. cerevisiae (Jelinek-Kelly & Herscovics 1988) and C. albicans (Mora-Montes et al 2006, 2008b), the enzyme activity purified here was susceptible to proteolysis by native aspartyl proteases. The thermostability of the enzyme allowed a non-traditional solubilisation procedure that was similar to the approach used for the purification of ER α1,2-mannosidase from C. albicans (Mora-Montes et al 2008c) and other proteins from prokaryotic cells (Novikov et al 1993, Heinz & Niederweis 2000.…”

Section: Discussionmentioning

confidence: 99%

Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

Mora-Montes

Robledo-Ortiz

Gonzalez-Sanchez

et al. 2010

Mem. Inst. Oswaldo Cruz

Self Cite

View full text Add to dashboard Cite

N-glycosylation of proteins is a common post-translational modification in eukaryotic cells. Following the translocation of nascent proteins into the endoplasmic reticulum (ER), the Glc 3 Man 9 GlcNAc 2 oligosaccharide is transferred to asparagine residues and sequentially processed by ER α-glucosidases, which remove the three glucose residues and by α-mannosidases that trim at least one mannose residue (Kornfeld & Kornfeld 1985, Herscovics 1999a.Alpha-mannosidases are grouped within families 38 and 47 of the glycosyl hydrolase classification (Henrissat 1991, Henrissat & Davies 1997. Members of family 38 are less specific than enzymes from family 47, removing α1,2, α1,3 and α1,6-linked mannose units (Daniel et al. 1994, Herscovics 1999a. Enzymes from family 47 are α1,2-mannosidases that participate in the trimming of N-glycans and in ER-associated degradation (Herscovics 1999a, b, 2001, Helenius & Aebi 2004. Two subgroups of enzymes comprise family 47: the ER α1,2-mannosidases that trim only one mannose residue from Man 9 GlcNAc 2 (M 9 ) generating Man 8 GlcNAc isomer B (M 8 B) (Ziegler & Trimble 1991, Tremblay & Herscovics 1999, Mora-Montes et al. 2004, Movsichoff et al. 2005; and Golgi α1,2-mannosidases from mammalian cells and filamentous fungi, which act after the ER enzyme to remove three α1,2-linked mannose units, generating Man 5 GlcNAc 2 , an intermediate required for the biosynthesis of complex and hybrid N-glycans (Yoshida et al. 1993, Lal et al. 1998, Ichishima et al. 1999, Eades & Hintz 2000, Tremblay & Herscovics 2000, Lobsanov et al. 2002, Akao et al. 2006. Lower eukaryotes, such as Saccharomyces cerevisiae, lack Golgi α1,2-mannosidases and synthesise only high-mannose N-glycans (Herscovics 1999b).Sporothrix schenckii, the etiological agent of sporotrichosis, is a dimorphic fungus that grows as filamentous and yeast cells during saprophytic and parasitic phases, respectively. This organism is closely related to Ophiostoma stenoceras, a non-pathogenic fungus that grows on sapwood from coniferous and hardwood trees (de Beer et al. 2003). Sporotrichosis is a subcutaneous mycosis caused by traumatic inoculation of colonised materials (soil, wood, decomposed vegetable matter) or inhalation of the conidia (Ramos-e-Silva et al. 2007). The disease has been reported as an emerging mycosis in HIV-infected humans (Durden & Elewski 1997). The cell wall of S. schenckii is being actively researched, as this structure has an important role in adhesion to host tissues and is the main source of antigens that can be exploited in the immuno-diagnosis of the disease (LopesBezerra et al. 2006). The cell wall of S. schenckii contains β1,3, β1,6 and β1,4-glucans (Lopes-Bezerra et al. In order to gain insight into the processing steps of Nglycan biosynthesis, a membrane-bound α-mannosidase from S. schenckii was targeted for purification and characterisation. Biochemical characterisation and intracellular localisation studies indicated that this enzyme is an ER α1,2-mannosidase and therefore a new member of glycosyl hydrol...

show abstract

“…1). The plasmid was propagated and maintained in E. coli One Shot Ò TOP10 cells, while BL21 Star TM (DE3) cells were used for gene expression as previously described (Mora-Montes et al 2008). …”

Section: Construction and Expression Of Pcwh41ctmentioning

confidence: 99%

Biochemical characterization of Candida albicans α-glucosidase I heterologously expressed in Escherichia coli

Frade-Pérez

Hernández‐Cervantes

Flores-Carreón

et al. 2010

Antonie van Leeuwenhoek

Self Cite

View full text Add to dashboard Cite

Protein glycosylation is one of the most common post-translational modifications present in the eukaryotic cell. The N-linked glycosylation is a biosynthetic pathway where an oligosaccharide is added to asparagine residues within the endoplasmic reticulum. Upon addition of the N-linked glycan to nascent proteins, alpha-glucosidase I removes the outermost alpha1,2-glucose unit from the N-linked core Glc(3)Man(9)GlcNAc(2). We have previously demonstrated that the endoplasmic reticulum α-glucosidase I is required for normal cell wall composition, and virulence of the human pathogen Candida albicans. In spite of the importance of this enzyme for normal cell biology, little is known about its structure and the amino acids participating in enzyme catalysis. Here, a DNA fragment corresponding to the 3'-end fragment of C. albicans CWH41, the encoding gene for α-glucosidase I, was expressed in a bacterial system and the recombinant peptide showed alpha-glucosidase activity, despite lacking 419 amino acids from the N-terminal end. The biochemical characterisation of the recombinant enzyme showed that presence of hydroxyl groups at carbons 3 and 6, and orientation of hydroxyl moiety at C-2 are important for glucose recognition. Additionally, results suggest that cysteine rather than histidine residues are involved in the catalysis by the recombinant enzyme.

show abstract

Heterologous expression and biochemical characterization of an α1,2-mannosidase encoded by the Candida albicans MNS1 gene

Cited by 8 publications

References 22 publications

Protein Glycosylation in Candida

Protein Glycosylation in Candida

Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

Biochemical characterization of Candida albicans α-glucosidase I heterologously expressed in Escherichia coli

Contact Info

Product

Resources

About