Isolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family

The abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-anal., spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large no. of cleavage peptides revealed a high level of similarity of the blue protein with arylphorins from a no. of other moth species. In particular, there is a high abundance of the arom. amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatog., anal. ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for arylphorins. The protein was further characterized as a weakly acidic (pI .apprx. 5.5) glycoprotein contg. mannose, glucose and N-acetylglucosamine in an approx. ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in arylphorins from other moths. The intense blue color of the Cerura protein is due to non-covalent assocn. with a bilin of novel structure at an estd. protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diam. and 9 nm in height. A small fraction of particles of the same diam. but half the height was likely a trimeric arylphorin dissocn. intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.