Arylphorin from Manduca sexta: carbohydrate structure and immunological studies.

The major hemolymph protein in the last larval stage of Manduca sexta is a hexameric glycoprotein, arylphorin (Mr = 450,000). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified arylphorin reveals the presence of two subunits, A1 and A2. Both subunits are glycosylated and have apparent Mr = 77,000 and 72,000, respectively. Pronase digestion of arylphorin yielded a single major glycopeptide. 250 MHz NMR spectroscopy of arylphorin glycopeptide revealed a Man9GlcNAc2 oligosaccharide structure similar to that observed in mammalian glycoproteins. Endoglycosidase-H treatment of arylphorin was employed to remove covalently linked carbohydrate residues. The carbohydrate removal lowered the apparent Mr of subunits A1 and A2 to 72,000 and 69,000, respectively, indicating that the difference in arylphorin subunit size is not due to levels of glycosylation. Immunoblotting experiments with anti-arylphorin antiserum and Bombyx mori storage proteins indicated cross reactivity with the corresponding arylphorin of this insect. Preparation of subunit A2 monospecific antibodies, followed by immunoblotting of arylphorin showed a close immunological relationship between subunits A1 and A2.