Gpa, a Calcium‐binding Protein in the Coccolithophorid Emiliania Huxleyi (Prymnesiophyceae)

Intracellular polysaccharide fractions were isolated from calcifying B‐type cells of Emiliania huxleyi and separated by electrophoretic fractionation. In all fractions, the polysaccharide was immunologically related to the polysaccharide of (extracellular) B‐type coccoliths (CP‐B) and not to polysaccharides of A‐type coccoliths (CP‐A). Most polysaccharide fractions also contained protein material. The fraction with the largest proportion of protein was used to raise antibodies. The resulting antiserum, α‐BP, contained antibodies against both CP‐B‐ and protein‐epitopes. The antibodies specific for polysaccharide‐epitopes reacted with intracellular polysaccharide fractions of B‐type cells only. In contrast, the antibodies specific for protein‐epitopes reacted with the intracellular fractions of B‐type as well as A‐type cells. With immunolocalization, the presence of protein antigen in a layer surrounding both types of cells was demonstrated. A cDNA library of E. huxleyi was screened with α‐BP, and a gene called gpa was isolated. The open reading frame of gpa was found to encode a protein (GPA) of 36,608 D, containing, inter alia, 24% acidic residues (18% glutamic acid and 6% aspartic acid), 12% proline, and 23% alanine. GPA has two repeats, one containing a sequence resembling the Ca2+‐binding loop of EF‐hands. Overproduction of GPA in a prokaryotic system yielded a dimeric product capable of binding Ca2+. The possible role of GPA in the formation of coccoliths in E. huxleyi is discussed.