The GAS multigene family is constituted by 5 genes (GAS1 to GAS5). GAS1 is the best characterized gene to date. It encodes the major GPI-anchored plasma membrane protein in the yeast Saccharomyces cerevisiae. Gas1p is endowed of a β(1,3)-glucanosyltransferase activity that is essential for the proper assembly of the glucan network of the cell wall during vegetative growth. The absence of this activity causes a weakening of the cell wall that activates a salvage pathway. GAS2 and GAS4 are expressed during sporulation and are essential for the assembly of the spore wall. Gas proteins belong to a broader family of extracellular enzymes from fungal and yeast species that includes also Phr1 and Phr2 from Candida albicans and Gel proteins from Aspergillus fumigatus. At the moment 70 protein sequences similar to Gas1p were identified and constitute Family 72 of the Glycoside Hydrolase database. In this work we focused on the Gas family of proteins as representative of the GH72 family. Gas proteins share an N-terminal domain of about 330-350 amino acids, where two catalytic residues are located, whereas they are dissimilar in the C-terminal portion. Out of the five Gas proteins, only Gas1 and Gas2 proteins share a cysteine-enriched domain of about 100 amino acids in their C-terminal region. This module, named Cys-box is similar to a novel Carbohydrate Binding Module (CBM), namely CBM43, an independent module that tightly binds laminarin in some plant β(1,3)-glucanases. Family GH72 appears to be divided into two subfamilies: one comprehends proteins with the Cys-box (GH72+) and the other one includes proteins without the Cys-box (GH72-). First we tested the activity of all the Gas proteins. Recombinant forms were produced in soluble form and purified from P. pastoris medium. Gas2 protein exhibited in vitro a β(1,3)-glucan-transferase activity identical to that of Gas1p whereas Gas4 and Gas5 proteins an activity similar to Gas1p. In order to study the role of the Cys-box we carried out a truncation analysis from the C-terminal end of Gas1 and Gas2 proteins. The removal of the Cys-box did not affect the folding of the proteins, as assessed by different spectroscopic analysis, but totally abolished the activity and also slightly reduced the thermal stability of the proteins. More extensive truncations greatly affected folding of the recombinant proteins and the putative catalytic N-terminal domain could not be produced in a proper conformation indicating that it does not constitute a structurally and functionally independent module. The results obtained suggest a possible interdependent relation of the N-terminal and C-terminal region in the GH72+ enzymes indicating a different role of the Cys-box in the fungal glucan transferases with respect to the CBM43 of plant glucanases. The analysis of a phylogenetic tree of the N-terminal domains of Family GH72 revealed a distinct molecular evolution of the GH72- and GH72+ subfamilies providing support to the hypothesis that the type of C-terminal region imposed constraints to the evolution of the N-terminal portion.

A cysteine-rich domain related to the plant CBM43 is essential for the beta(1,3)-glucanosyltransferase activity of Gas family of proteins of Saccharomyces cerevisiae / E. Ragni, O. Palomares, E. Rolli, J. Calderon, L. Popolo. ((Intervento presentato al 1. convegno International fungal/plant cell wall meeting. Cell wall polysacchardes of fungi and plants tenutosi a Biarritz (France) nel 2007.

A cysteine-rich domain related to the plant CBM43 is essential for the beta(1,3)-glucanosyltransferase activity of Gas family of proteins of Saccharomyces cerevisiae

E. Ragni
Primo
;
E. Rolli;J. Calderon;L. Popolo
Ultimo
2007

Abstract

The GAS multigene family is constituted by 5 genes (GAS1 to GAS5). GAS1 is the best characterized gene to date. It encodes the major GPI-anchored plasma membrane protein in the yeast Saccharomyces cerevisiae. Gas1p is endowed of a β(1,3)-glucanosyltransferase activity that is essential for the proper assembly of the glucan network of the cell wall during vegetative growth. The absence of this activity causes a weakening of the cell wall that activates a salvage pathway. GAS2 and GAS4 are expressed during sporulation and are essential for the assembly of the spore wall. Gas proteins belong to a broader family of extracellular enzymes from fungal and yeast species that includes also Phr1 and Phr2 from Candida albicans and Gel proteins from Aspergillus fumigatus. At the moment 70 protein sequences similar to Gas1p were identified and constitute Family 72 of the Glycoside Hydrolase database. In this work we focused on the Gas family of proteins as representative of the GH72 family. Gas proteins share an N-terminal domain of about 330-350 amino acids, where two catalytic residues are located, whereas they are dissimilar in the C-terminal portion. Out of the five Gas proteins, only Gas1 and Gas2 proteins share a cysteine-enriched domain of about 100 amino acids in their C-terminal region. This module, named Cys-box is similar to a novel Carbohydrate Binding Module (CBM), namely CBM43, an independent module that tightly binds laminarin in some plant β(1,3)-glucanases. Family GH72 appears to be divided into two subfamilies: one comprehends proteins with the Cys-box (GH72+) and the other one includes proteins without the Cys-box (GH72-). First we tested the activity of all the Gas proteins. Recombinant forms were produced in soluble form and purified from P. pastoris medium. Gas2 protein exhibited in vitro a β(1,3)-glucan-transferase activity identical to that of Gas1p whereas Gas4 and Gas5 proteins an activity similar to Gas1p. In order to study the role of the Cys-box we carried out a truncation analysis from the C-terminal end of Gas1 and Gas2 proteins. The removal of the Cys-box did not affect the folding of the proteins, as assessed by different spectroscopic analysis, but totally abolished the activity and also slightly reduced the thermal stability of the proteins. More extensive truncations greatly affected folding of the recombinant proteins and the putative catalytic N-terminal domain could not be produced in a proper conformation indicating that it does not constitute a structurally and functionally independent module. The results obtained suggest a possible interdependent relation of the N-terminal and C-terminal region in the GH72+ enzymes indicating a different role of the Cys-box in the fungal glucan transferases with respect to the CBM43 of plant glucanases. The analysis of a phylogenetic tree of the N-terminal domains of Family GH72 revealed a distinct molecular evolution of the GH72- and GH72+ subfamilies providing support to the hypothesis that the type of C-terminal region imposed constraints to the evolution of the N-terminal portion.
12-mar-2007
Lievito ; Parete cellulare ; glucanosiltransferasi ; Proteine Gas
Settore BIO/11 - Biologia Molecolare
Settore BIO/10 - Biochimica
http://www.fungwall.org/anglet/abstracts.pdf
A cysteine-rich domain related to the plant CBM43 is essential for the beta(1,3)-glucanosyltransferase activity of Gas family of proteins of Saccharomyces cerevisiae / E. Ragni, O. Palomares, E. Rolli, J. Calderon, L. Popolo. ((Intervento presentato al 1. convegno International fungal/plant cell wall meeting. Cell wall polysacchardes of fungi and plants tenutosi a Biarritz (France) nel 2007.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/153720
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