CHARACTERIZATION OF 2 HIGH-MOLECULAR-WEIGHT CATECHOL-CONTAINING GLYCOPROTEINS FROM PHARATE PUPAL CUTICLE OF THE TOBACCO HORNWORM, MANDUCA-SEXTA

Two high molecular weight cuticular proteins (MSCP120 and MSCP246) wer e extracted in acidic guanidine hydrochloride solution from tanning ab dominal cuticle of Manduca sexta pharate pupae and purified by size ex clusion high performance liquid chromatography. The apparent molecular weights were ca. 120 and 246 kDa as determined by sodium dodecyl sulf ate polyacrylamide gel electrophoresis. Both proteins contained high l evels of glutamate/glutamine, glycine, serine, alanine and aspartate/a sparagine. MSCP120 was enriched in histidine relative to MSCP246, but the reverse was true for valine and proline. Small quantities of beta- alanine and 3,4-dihydroxyphenylalanine (DOPA), as well as other catech ols and carbohydrates, also were detected in the hydrolysates. The pro teins became radiolabeled when [1-C-14]-beta-alanine was injected into pharate pupae, presumably by the formation of adducts with N-beta-ala nyldopamine metabolites during sclerotization. Mild acid hydrolysis re leased N-beta-alanylnorepinephrine and 3,4-dihydroxyphenylketoethanol from both proteins. Strong acid hydrolysis yielded predominantly 3,4-d ihydroxyphenylketoethylamine (arterenone), but also DOPA and dopamine. The N-terminal amino acid sequences of the two cuticular proteins wer e dissimilar, and that of MSCP246 was more hydrophobic than MSCP120. B oth of these proteins mere glycosylated with glucose, N-acetylglucosam ine and traces of N-acetylgalactosamine, and MSCP246 also contained ga lactose. These structural glycoproteins, which occur in cuticle underg oing sclerotization, apparently react post-translationally with quinon oid tanning agents to yield catecholamine-protein adducts. Small amoun ts of peptidyl DOPA probably are formed by hydroxylation of tyrosyl re sidues. Results from this study are consistent with the hypothesis tha t these catechol-containing glycoproteins participate in cross-linking reactions in M. sexta pupal cuticle during sclerotization.