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 functional unit KLH2-c was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of the protein, and of peptides 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 hemocyanin functional units revealed average identities between 41 and 49%, but 55% in case of Octopus hemocyanin functional unit c which is the structural equivalent to KLH2-c. KLH2-c has a molecular mass of approx. 48 kDa as calculated from its sequence and a measured mass of approx. 56 kDa; the mass difference is attributed to the sugar side chains usually decorating molluscan hemocyanin. However, inspection of the sequence of KLH2-c revealed no potential 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 (including KLH1-c), which lack O-linked glycosides. The present results are discussed in view of the recent X-ray structure of the functional unit g from Octopus hemocyanin, and a published record of the Thomsen Friedenreich tumor antigenic epitope in KLH.
Stoeva
S
S
Abteilung für Physikalische Biochemie, Physiologisch-chemisches Institut der Universität Tübingen, Hoppe-Seyler-Str. 4, D-72076, Tübingen, Germany. stanka.stoeva@unij-tuebingen.de
Schütz
J
J
Gebauer
W
W
Hundsdörfer
T
T
Manz
C
C
Markl
J
J
Voelter
W
W
D000595:Amino Acid Sequence; D000818:Animals; D002241:Carbohydrates / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006433:Hemocyanins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008974:Mollusca; D012333:RNA, Messenger / Q000737:chemistry; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002241:Carbohydrates; D012333:RNA, Messenger; D006433:Hemocyanins; D002918:Chymotrypsin; C032808:keyhole-limpet hemocyanin