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 frac tionation. In all fractions, the polysaccharide was immunologically re lated to the polysaccharide of (extracellular) B-type coccoliths (CIP- B) and not to polysaccharides of A-type coccoliths (CP-A). Most polysa ccharide fractions also contained protein material. The fraction with the largest proportion of protein was used to raise antibodies. The re sulting antiserum, alpha-BP, contained antibodies against both CP-B- a nd protein-epitopes. The antibodies specific for polysaccharide-epitop es reacted with intracellular polysaccharide fractions of B-type cells only. In contrast, the antibodies specific for protein-epitopes react ed with the intracellular fractions of B-type as well as A-type cells. With immunolocalization, the presence of protein antigen in a layer s urrounding both types of cells was demonstrated. A cDNA library off. h uxleyi was screened with alpha-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 ac id and 6% aspartic acid), 12% pro line, and 23% alanine. GPA has two r epeats, one containing a sequence resembling the Ca2+-binding loop of EF-hands. Overproduction of GPA in a prokaryotic system yielded a dime ric product capable of binding Ca2+. The possible role of GPA in the f ormation of coccoliths in E. huxleyi is discussed.