Amino acid sequence and glycosylation of functional unit RtH2-e from Rapana thomasiana (gastropod) hemocyanin.
The complete amino acid sequence of Rapana thomasiana hemocyanin functional unit RtH2-e was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of peptides obtained by cleavage with EndoLysC proteinase, chymotrypsin, and trypsin. The single-polypeptide chain of RtH2-e consists of 413 amino acid residues and contains two consensus sequences NXS/T (positions 11-19 and 127-129), potential sites for N-glycosylation. Monosaccharide analysis of RtH2-e revealed a carbohydrate content of about 1.1% and the presence of xylose, fucose, mannose, and N-acetylglucosamine, demonstrating that only N-linked carbohydrate chains of high-mannose type seem to be present. On basis of the monosaccharide composition and MALDI-MS analysis of native and PNGase-F-treated chymotryptic glycopeptide fragment of RtH2-e the oligosaccharide Man(5)GlcNAc(2), attached to Asn(127), is suggested. Multiple sequence alignments with other molluscan hemocyanin e functional units revealed an identity of 63% to the cephalopod Octopus dofleini and of 69% to the gastropod Haliotis tuberculata. The present results are discussed in view of the recently determined X-ray structure of the functional unit g of the O. dofleini hemocyanin.
Stoeva
Stanka
S
Department of Physical Biochemistry, Institute of Physiological Chemistry, University of Tuebingen, Hoppe-Seyler-Strasse 4, D-72076 Tuebingen, Germany. stanka.stoeva@uni-tuebingen.de
Idakieva
Krasimira
K
Betzel
Christian
C
Genov
Nicolay
N
Voelter
Wolfgang
W
D000595:Amino Acid Sequence; D000818:Animals; D002241:Carbohydrates / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D006433:Hemocyanins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D008974:Mollusca / Q000737:chemistry*; D009005:Monosaccharides / Q000737:chemistry; D021122:Protein Subunits; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D002241:Carbohydrates; D006020:Glycopeptides; D009005:Monosaccharides; D021122:Protein Subunits; D006433:Hemocyanins