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Abstract
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.
Highlights
In the present study a combination of carbohydrate and amino acid composition analysis, amino acid sequencing, and mass spectrometry revealed that the Lfucose is bound to residues 56 – 68 of DSPA␣1
The resulting peptides were separated by reversed-phase HPLC (RP-HPLC) (Fig. 1), and their carbohydrate content was analyzed by monosaccharide composition analysis
Comparative Study of Natural and Recombinant DSPA␣1—To determine if O-fucosylation was restricted to DSPA␣1 when overexpressed in Chinese hamster ovary (CHO) cells, we studied natural DSPA␣1 derived from the Mexican vampire bat D. rotundus
Summary
Materials—rDSPA␣1 from CHO cells was obtained from Schering AG (Berlin, Germany). Mexican DSPA␣1 (80 g) was purified from 30. Large scale tryptic digestion of rDSPA␣1 (3 mg) was performed overnight at 37 °C after exchange of the buffer into 1% NH4HCO3, pH 8.0, by the addition of two portions of trypsin (1/100, w/w) at 0 and 8 h. Digestion was performed for 48 h at 37 °C in 1% NH4HCO3, pH 7.0, containing 10 mM CaCl2, the Pronase E (approximately 100 g/nmol of peptide) being added in two portions, one at 0 h and the other at 24 h. Defucosylation—Digestion with ␣-L-fucosidase from chicken liver was performed with 0.2 units/ϳ3 nmol of peptide in a buffer containing 100 mM sodium citrate-phosphate, pH 6.0, for 18 h at 37 °C according to the protocol of the manufacturer. Digestion with ␣-L-fucosidase from C. lampas was carried out with 0.05 units/ϳ3 nmol of peptide in a buffer containing 200 mM sodium citrate-phosphate, pH 4.1, for 18 h at 37 °C according to the manufacturer’s protocol
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