Mass spectrometric characterisation of post-translational modification and genetic variation in human tetranectin.
Tetranectin, a plasminogen-binding trimeric C-type lectin-like protein primarily involved in tissue remodeling and development, was scanned for covalent modifications and sequence heterogeneity, using a combination of mass spectrometric and classical protein chemical analytical methods. Electrospray ionisation mass spectrometry showed the presence of eight components of different mass and abundance in plasma tetranectin, all of higher mass than that calculated from the cDNA sequence. To identify and locate residues accounting for the heterogeneity, samples of tetranectin were subjected to proteolytic cleavage. Peptide fragments, in mixtures or in purified form, were analysed by matrix-assisted-laser-desorption-ionisation mass spectrometry and, where required, by Edman sequencing and compared to the cDNA sequence. Our results show that the mass heterogeneity in plasma tetranectin is due to sequence heterogeneity at position 85 and the presence of a partially sialylated oligosaccharide prosthetic group attached to Thr-4. Residue 85 is encoded in the cDNA as a Ser residue, but plasma tetranectin is a 1:1 mixture of Ser85 and Gly-85 sequence variants. Mass spectrometric analysis of enzymatic and mild acid hydrolysates of an N-terminal glycopeptide showed that the composition and partial covalent structure of the O-linked oligosaccharide prosthetic group is < or =N-acetylhexosamine < or =[hexose, (sialic acid)0-3].
Jaquinod
M
M
Department of Molecular Biology, University of Southern Denmark, Odense University.
Holtet
T L
TL
Etzerodt
M
M
Clemmensen
I
I
Thøgersen
H C
HC
Roepstorff
P
P
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002241:Carbohydrates / Q000032:analysis / Q000737:chemistry; D014644:Genetic Variation; D006801:Humans; D037181:Lectins, C-Type; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D001798:Blood Proteins; D002241:Carbohydrates; D037181:Lectins, C-Type; D010446:Peptide Fragments; C048648:tetranectin; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin