Arylphorin from Manduca sexta: carbohydrate structure and immunological studies

Area of Science:

• Biochemistry
• Insect Physiology
• Glycobiology

Background:

• Arylphorin is the primary hemolymph protein in Manduca sexta larvae.
• It is a hexameric glycoprotein with a molecular weight of 450,000.
• Purified arylphorin shows two subunits, A1 and A2, with apparent molecular weights of 77,000 and 72,000, respectively.

Purpose of the Study:

• To investigate the structural basis for the size difference between arylphorin subunits A1 and A2.
• To characterize the glycosylation of arylphorin.
• To examine the immunological relationship between arylphorin subunits and homologous proteins in other insects.

Main Methods:

• Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to analyze subunits.
• Pronase digestion to isolate glycopeptides.
• Nuclear Magnetic Resonance (NMR) spectroscopy to determine oligosaccharide structure.
• Endoglycosidase-H treatment to assess glycosylation.
• Immunoblotting with specific antisera.

Main Results:

• A single major glycopeptide was obtained after pronase digestion.
• NMR revealed a Man9GlcNAc2 oligosaccharide structure, similar to mammalian glycoproteins.
• Endoglycosidase-H treatment reduced subunit molecular weights, indicating glycosylation, but did not account for the initial size difference.
• Immunoblotting showed cross-reactivity with Bombyx mori storage proteins and a close immunological relationship between arylphorin subunits A1 and A2.

Conclusions:

• The size difference between arylphorin subunits A1 and A2 is not solely due to variations in glycosylation.
• Arylphorin shares structural and immunological similarities with storage proteins of other insects.
• Further investigation is needed to elucidate the precise structural differences between the subunits.