- The cells of the red microalga Porphyridium sp. (UTEX 637) are encapsulated in a cell wall of a negatively charged mucilaginous polysaccharide complex composed of 10 different sugars, sulfate, and proteins. In this work, we studied the proteins associated with the cell-wall polysaccharide. A number of noncovalently associated proteins were resolved by SDS-PAGE, but no covalently bound proteins were detected. The most prominent protein detected was a 66-kDa glycoprotein consisting of a polypeptide of approximately 58 kDa and a glycan moiety of approximately 8 kDa containing N-linked terminal mannose. In size-exclusion chromatography, the 66-kDa protein was coeluted with the polysaccharide and could be separated from the polysaccharide only after denaturation of the protein, indicating that the 66-kDa protein was tightly bound to the polysaccharide. Western blot analysis revealed that the 66-kDa protein was specific to Porphyridium sp. and P. cruentum, because it was not detected in the other species of red microalgae examined. Indirect immunofluorescence assay confirmed the location of the protein in the algal cell wall. The sequence of cDNA clone encoding the 66-kDa glycoprotein, detected in our in-house expressed sequence tag database of Porphyridium sp., revealed that this is a novel protein with no similarity to any protein in the public domain databases and our in-house expressed sequence tag database of the red microalga Rhodella reticulata. The 66-kDa protein bound polysaccharides from red algae but not from those of other origins tested. Possible roles of the 66-kDa protein in the biosynthesis of the polysaccharide are discussed.