Primary structure and unusual carbohydrate moiety of functional unit 2-c of keyhole limpet hemocyanin (KLH)

The complete amino acid sequence of the Megathura crenulata hemocyanin func tional unit KLH2-c was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of the protein, and of peptid es obtained by cleavage with EndoLysC proteinase, chymotrypsin and cyanogen bromide. This is the first complete primary structure of a functional unit c from a gastropod hemocyanin. KLH2-c consists of 420 amino acid residues. Circular dichroism spectra indicated approx. 31% beta-sheet and 29% alpha- helix contents. A multiple sequence alignment with other molluscan hemocyan in functional units revealed average identities between 41 and 49%, but 55% in case of Octopus hemocyanin functional unit c which is the structural eq uivalent to KLH2-c. KLH2-c has a molecular mass of approx. 48 kDa as calcul ated from its sequence and a measured mass of approx. 56 kDa; the mass diff erence is attributed to the sugar side chains usually decorating molluscan hemocyanin. However, inspection of the sequence of KLH2-c revealed no poten tial N-linked carbohydrate attachment sites, and this was supported by its inability to bind concanavalin A. Also KLH1-c was unreactive, whereas most, if not all, other functional units of KLH1 and KLH2 reacted positively to this lectin. On the other hand, peanut agglutinin specifically binds KLH2-c , indicating the presence of O-glycosidically linked carbohydrates in this functional unit. This contrasts to all other KLH functional units (includin g KLH1-c), which lack O-linked glycosides. The present results are discusse d in view of the recent X-ray structure of the functional unit g from Octop us hemocyanin, and a published record of the Thomsen Friedenreich tumor ant igenic epitope in KLH. (C) 1999 Elsevier Science B.V. All rights reserved.