title: The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum.
abstract: Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α(D)-HlH, α(N)-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α(D)-HlH and β-HlH was obtained, including the 5' and 3' UTR, 180bp of the 5' end and around 900bp at the 3' end are missing for the third subunit. The subunits α(D)-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α(N)-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α(D)-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.
authors.affiliation: Laboratory of Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Physiology, Ghent University, Belgium.
authors.forename: Lina
authors.initials: L
authors.lastname: De Smet
authors.forename: Ivan
authors.initials: I
authors.lastname: Dimitrov
authors.forename: Griet
authors.initials: G
authors.lastname: Debyser
authors.forename: Pavlina
authors.initials: P
authors.lastname: Dolashka-Angelova
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Dolashki
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D006372:Helix, Snails / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006433:Hemocyanins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017385:Sequence Homology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D018076:DNA, Complementary; D021122:Protein Subunits; D006433:Hemocyanins