O-linked L-fucose is present in Desmodus rotundus salivary plasminogen activator.
DSPAalpha1 (Desmodus rotundus salivary plasminogen activator), a plasminogen activator from the saliva of the vampire bat Desmodus rotundus, is an effective thrombolytic agent. An unusual type of posttranslational modification, in which L-fucose is O-glycosidically linked to threonine 61 in the epidermal growth factor domain was found for natural DSPAalpha1 and its recombinant form isolated from Chinese hamster ovary cells. In the present study a combination of carbohydrate and amino acid composition analysis, amino acid sequencing, and mass spectrometry revealed that the L-fucose is bound to residues 56-68 of DSPAalpha1. The amino acid sequence of this glycosylation site agreed with the suggested consensus sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys described for other proteins. Anew strategy for the identification of the modified amino acid was established. Direct evidence for the occurrence of fucosyl-threonine was obtained by mass spectrometry after digestion of the glycopeptide with a mixture of peptidases. On the basis of these results, DSPAalpha1 is a suitable model for studying the influence of O-fucosylation on clearance rates, particularly in comparative studies with the identically fucosylated and structurally related tissue plasminogen activator.
Gohlke
M
M
Institut für Molekularbiologie und Biochemie der Freien, Unviersitat Berlin, Berlin-Dahlem, Germany.
Baude
G
G
Nuck
R
R
Grunow
D
D
Kannicht
C
C
Bringmann
P
P
Donner
P
P
Reutter
W
W
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002417:Cattle; D002685:Chiroptera; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D005643:Fucose / Q000032:analysis*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010960:Plasminogen Activators / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012463:Saliva / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013912:Threonine; D010959:Tissue Plasminogen Activator / Q000737:chemistry; D014162:Transfection
D011994:Recombinant Proteins; D005643:Fucose; D013912:Threonine; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator