Determination of the covalent structure of an N‐ and C‐terminally blocked glycoprotein from endocuticle of Locusta migratoria

Abstract

The complete structure of a protein isolated from endocuticle of sexually mature locusts, Locusta migratoria, has been determined by a combination of automatic Edman degradation and plasma desorption mass spectrometry. The protein is extensively post-translationally modified. The N-terminal is 5-oxoproline (pyroglutamic acid) and the C-terminal proline residue is amidated. Furthermore, the protein is glycosylated by a single N-acetylgalactosamine residue at one, two or three threonines. The N-terminal sequence was obtained by analysing the N-acetylated N,O-permethylated derivative using plasma desorption mass spectrometry. The position and type of carbohydrate were determined by combining an HPLC-based carbohydrate analysis with the peak pattern of the phenylthiohydantoin derivative in automatic sequencing and with mass information on peptides.The protein has pronounced similarity to cuticular proteins from larvae of diptera and lepidoptera, but only slight resemblance to the previously sequenced locust exocuticular proteins. This indicates a similarity between soft larval cuticles and locust endocuticle, a similarity which may extend to their mechanical properties.Locust cuticle consists of a relatively soft endocuticle, which contains mainly acidic proteins, and a harder exocuticle, containing more basic proteins [l]. The proteins in the exocuticle of the migratory locust Locusta migratoria can only be extracted immediately after ecdysis, before cuticular sclerotization is initiated. Several of the exocuticular proteins have been purified and a few have been sequenced using a combination of "' Cf plasma desorption mass spectrometry and Edman degradation [2, 31. The extractable, endocuticular proteins from mature locust abdominal cuticle have been characterised using gel electrophoresis and amino acid analysis [l]. The proteins resemble the exocuticular proteins in the lack of sulphur-containing amino acids and a relatively high content of proline. The main differences are low isoelectric points (PI 4 -6) and high contents of hydrophilic amino acids in endocuticular proteins.The presence of glycosylated proteins in soft larval lepidopteran cuticle has been described [4], but the specific glycosylation sites and the detailed structures of the glycosyl residues have not been reported. We have now determined the complete structure of a glycosylated endocuticular protein, Correspondence to P.

Cloning and Sequencing of a cDNA Encoding a Larval‐Pupal‐Specific Cuticular Protein in Tenebrio Molitor (Insecta, Coleoptera)

“…The presence of TMLPCP-22 in epidermal regions secreting hard and flexible articulating cuticle indicates that it does not belong to hard or soft cuticular proteins classes, contrary to many other cuticular proteins which are clearly identified as hard or flexible cuticle components (Rebers and Riddiford, 1988;Willis, 1989;Talbo et al, 1991).…”

Isolation and Identification of Multiple Neuropeptides of the Allatostatin Superfamily in the Shore Crab Carcinus Maenas

“…The method has an accuracy of 0.1 5%. The possible amidation of the C-terminus, in those peptides where sufficient material remained, was assessed by determining the total number of free carboxyl groups by methylation of an aliquot and remeasurement of the molecular mass (Talbo et al, 1991). This method involves adding SO p1 of freshly prepared 2 M HCI in methanol to the dried peptide sample and allowing the mixture to react in a capped tube for 2 h at room temperature.…”

Callatostatins: neuropeptides from the blowfly Calliphora vomitoria with sequence homology to cockroach allatostatins.

“…For callatostatin 1, the number of free carboxyl groups was determined by subjecting an aliquot to methylation and remeasuring the mass. The method requires a 2-hr reaction of the peptide with 2 M HCl in methanol, obtained by addition of acetyl chloride to dry methanol (10).…”