title: Cell wall regeneration in Bangia atropurpurea (Rhodophyta) protoplasts observed using a mannan-specific carbohydrate-binding module.
abstract: The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (beta-1,4-mannan, beta-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized beta-mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding family 27 CBM (CBM27) of beta-1,4-mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP-CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP-CBM27 maintained its binding ability to soluble beta-mannans, while normal GFP could not bind to beta-mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP-CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete beta-mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, beta-mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP-CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP-CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of beta-mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.
authors.affiliation: Laboratory for the Utilization of Aquatic Bioresources, Department of Life Science, Graduate School of Bioresources, Mie University, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Umemoto
authors.forename: Toshiyoshi
authors.initials: T
authors.lastname: Araki
mesh_terms: D002462:Cell Membrane / Q000502:physiology*; D037161:Galectins / Q000378:metabolism*; D008351:Mannans / Q000378:metabolism*; D011523:Protoplasts / Q000502:physiology*; D012038:Regeneration / Q000502:physiology*; D000461:Rhodophyta / Q000502:physiology*
chemical_list: D037161:Galectins; D008351:Mannans