O-Linked L-Fucose Is Present in Desmodus rotundus Salivary Plasminogen Activator (*)

DSPAα1 (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 DSPAα1 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 DSPAα1. 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. A new 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, DSPAα1 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.

[1]  C. Hawkey.  Plasminogen Activator in Saliva of the Vampire Bat Desmodus rotundus , 1966, Nature.

[2]  G. Edelman,et al.  The covalent structure of a human gamma G-immunoglobulin. II. Isolation and characterization of the cyanogen bromide fragments. , 1968, Biochemistry.

[3]  B. Keil,et al.  Proteolytic activity of pseudotrypsin , 1971, FEBS letters.

[4]  T. Cartwright,et al.  The plasminogen activator of vampire bat saliva. , 1974, Blood.

[5]  P. Bornstein,et al.  [14] Cleavage at AsnGly bonds with hydroxylamine , 1977 .

[6]  K. Kurachi,et al.  Isolation and characterization of a cDNA coding for human factor IX. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[7]  P. Fernlund,et al.  Beta-hydroxyaspartic acid in vitamin K-dependent proteins. , 1983, The Journal of biological chemistry.

[8]  K. Fujikawa,et al.  The occurrence of beta-hydroxyaspartic acid in the vitamin K-dependent blood coagulation zymogens. , 1983, Biochemical and biophysical research communications.

[9]  P. Seeburg,et al.  Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli , 1983, Nature.

[10]  P. Fernlund,et al.  beta-Hydroxyaspartic acid in vitamin K-dependent protein C. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[11]  T. Suyama,et al.  Primary structure of single-chain pro-urokinase. , 1985, The Journal of biological chemistry.

[12]  K Fujikawa,et al.  Amino acid sequence of the heavy chain of human alpha-factor XIIa (activated Hageman factor). , 1985, The Journal of biological chemistry.

[13]  T. Wun,et al.  Spontaneous fibrinolysis in whole human plasma. Identification of tissue activator-related protein as the major plasminogen activator causing spontaneous activity in vitro. , 1985, The Journal of biological chemistry.

[14]  K. Kurachi,et al.  Characterization of a cDNA coding for human factor VII. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[15]  B. Dahlbäck,et al.  beta-Hydroxyasparagine in domains homologous to the epidermal growth factor precursor in vitamin K-dependent protein S. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[16]  A. Stern,et al.  Occurrence of beta-hydroxylated asparagine residues in non-vitamin K-dependent proteins containing epidermal growth factor-like domains. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[17]  K. Nakagawa,et al.  Bovine factor VII. Its purification and complete amino acid sequence. , 1988, The Journal of biological chemistry.

[18]  S. Kawabata,et al.  A new trisaccharide sugar chain linked to a serine residue in bovine blood coagulation factors VII and IX. , 1988, Journal of biochemistry.

[19]  R. Townsend,et al.  Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection. , 1988, Analytical biochemistry.

[20]  W. Schneider,et al.  beta-Hydroxyaspartic acid or beta-hydroxyasparagine in bovine low density lipoprotein receptor and in bovine thrombomodulin. , 1988, The Journal of biological chemistry.

[21]  B. Furie,et al.  The molecular basis of blood coagulation , 1988, Cell.

[22]  W. Kisiel,et al.  Identification of a disaccharide (Xyl-Glc) and a trisaccharide (Xyl2-Glc) O-glycosidically linked to a serine residue in the first epidermal growth factor-like domain of human factors VII and IX and protein Z and bovine protein Z. , 1989, The Journal of biological chemistry.

[23]  R. Fears.  Binding of plasminogen activators to fibrin: characterization and pharmacological consequences. , 1989, The Biochemical journal.

[24]  D. Smith,et al.  Strategies for locating disulfide bonds in proteins. , 1990, Methods in enzymology.

[25]  S. Kawabata,et al.  The structure of (xylose)2glucose-O-serine 53 found in the first epidermal growth factor-like domain of bovine blood clotting factor IX. , 1990, The Journal of biological chemistry.

[26]  A. Buko,et al.  Carbohydrate composition and presence of a fucose-protein linkage in recombinant human pro-urokinase. , 1990, Biochemical and biophysical research communications.

[27]  Deborah L. Higgins,et al.  Tissue plasminogen activator: the biochemistry and pharmacology of variants produced by mutagenesis. , 1990, Annual review of pharmacology and toxicology.

[28]  B. Haendler,et al.  The plasminogen activator family from the salivary gland of the vampire bat Desmodus rotundus: cloning and expression. , 1991, Gene.

[29]  A. Petros,et al.  Characterization of a posttranslational fucosylation in the growth factor domain of urinary plasminogen activator. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[30]  R J Harris,et al.  Tissue plasminogen activator has an O-linked fucose attached to threonine-61 in the epidermal growth factor domain. , 1991, Biochemistry.

[31]  W. Kisiel,et al.  Human plasma and recombinant factor VII. Characterization of O-glycosylations at serine residues 52 and 60 and effects of site-directed mutagenesis of serine 52 to alanine. , 1991, The Journal of biological chemistry.

[32]  D. Rijken,et al.  Binding of tissue-type plasminogen activator by the mannose receptor. , 1991, The Journal of biological chemistry.

[33]  B. Haendler,et al.  Plasmhogen Activators from the Saliva of Desmodus rotundus (Common Vampire Bat): Unique Fibrin Specificity , 1992, Annals of the New York Academy of Sciences.

[34]  R J Harris,et al.  O-linked fucose is present in the first epidermal growth factor domain of factor XII but not protein C. , 1992, The Journal of biological chemistry.

[35]  W. Reutter,et al.  Incorporation of the hexose analogue 2-deoxy-D-galactose into membrane glycoproteins in HepG2 cells. , 1992, Archives of biochemistry and biophysics.

[36]  T. Takao,et al.  Human factor IX has a tetrasaccharide O-glycosidically linked to serine 61 through the fucose residue. , 1992, The Journal of biological chemistry.

[37]  S. Iwanaga,et al.  Evidence for the existence of O-linked sugar chains consisting of glucose and xylose in bovine thrombospondin. , 1992, Journal of biochemistry.

[38]  David F. Smith,et al.  The selectin family of carbohydrate‐binding proteins: Structure and importance of carbohydrate ligands for cell adhesion , 1992, BioEssays : news and reviews in molecular, cellular and developmental biology.

[39]  B. Haendler,et al.  High-level secretion of the four salivary plasminogen activators from the vampire bat Desmodus rotundus by stably transfected baby hamster kidney cells. , 1992, Gene.

[40]  R. Cummings,et al.  O-linked fucose in glycoproteins from Chinese hamster ovary cells. , 1993, Glycobiology.

[41]  Reed J. Harris,et al.  O-linked fucose and other post-translational modifications unique to EGF modules. , 1993, Glycobiology.

[42]  A. Schwartz,et al.  39-kD protein inhibits tissue-type plasminogen activator clearance in vivo. , 1993, The Journal of clinical investigation.

[43]  Kenneth J. Smith,et al.  Identification and structural analysis of the tetrasaccharide NeuAc.alpha.(2.fwdarw.6)Gal.beta.(1.fwdarw.4)GlcNAc.beta.(1.fwdarw.3)Fuc.alpha.1.fwdarw.O-linked to serine 61 of human factor IX , 1993 .

[44]  W. Schleuning,et al.  Coronary Thrombolysis With Desmodus Salivary Plasminogen Activator in Dogs: Fast and Persistent Recanalization by Intravenous Bolus Administration , 1994, Circulation.

[45]  alpha-Fucose-mediated binding and degradation of tissue-type plasminogen activator by HepG2 cells. , 1994, The Journal of clinical investigation.

[46]  W. Schleuning,et al.  Production of vampire bat plasminogen activator DSPA α1 in CHO and insect cells , 1995 .