Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module

• Published in: Marine biotechnology (New York, N.Y.) N.Y.), 2010-02, Vol.12 (1), p.24-31
• Main Authors: Umemoto, Yoshiaki, Araki, Toshiyoshi
• Format: Article
• Language: English
• Subjects: Algae; Biomedical and Life Sciences; Carbohydrates; Cell Membrane - physiology; Cellular biology; E coli; Engineering; Enzymes; Fluorescence; Fluorescence microscopy; Freshwater & Marine Ecology; Fronds; Galectins - metabolism; Laboratories; Life Sciences; Mannans - metabolism; Microbiology; Original Article; Proteins; Protoplasts - physiology; Regeneration - physiology; Rhodophyta - physiology; Saccharides; Studies; Zoology
• ISSN: 1436-2228; 1436-2236
• DOI: 10.1007/s10126-009-9196-z

The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (β-1,4-mannan, β-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized β-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 β-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 β-mannans, while normal GFP could not bind to β-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 β-mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-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 β-mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.