ABSTRACT
The recombinant plasminogen activator (rDSPA alpha 1) from the vampire bat Desmodus rotundus is a promising new thrombolytic agent that exhibits a superior pharmacological profile if compared to tissue-type plasminogen activator (t-PA) or streptokinase. In the present study the structures of the carbohydrate moieties at the two N-glycosylation sites (Asn-117, Asn-362) of rDSPA alpha 1 expressed in Chinese hamster ovary cells were determined. N-Linked glycans were enzymatically released from isolated tryptic glycopeptides by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F digestion and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by analysis of carbohydrate composition and linkage, by mass spectrometry, and by sequence analysis in which the fluorescently labeled glycans were cleaved with an array of specific exoglycosidases. More than 30 different oligosaccharides were identified. The results revealed that Asn-117 carried a mixture of one high-mannose structure (17% of site-specific glycosylation), three hybrid glycans (26%) and predominantly biantennary complex N-glycans (54%). Glycosylation site Asn-362 was found to comprise complex glycans with biantennary (50%), 2,4- and 2,6-branched triantennary (21%, 11%), and tetraantennary structures (10%), which were fucosylated at the innermost residue of N-acetylglucosamine. Mainly neutral and monosialylated glycans, and smaller quantities of disialylated glycans, were detected at both glycosylation sites. Sialic acid was alpha 2-3 linked to galactose exclusively. As shown in this study the N-glycans attached to Asn-117 of rDSPA alpha 1 are more processed during biosynthesis than the high-mannose structures linked to Asn-117 of t-PA, to which the polypeptide backbone of rDSPA alpha 1 is structurally closely related.
Subject(s)
Plasminogen Activators/metabolism , Saliva/metabolism , Amino Acid Sequence , Animals , CHO Cells , Chiroptera , Cricetinae , Cricetulus , Glycopeptides/analysis , Glycosylation , Molecular Sequence Data , Oligosaccharides/analysis , Peptide Mapping , Plasminogen Activators/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
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.
Subject(s)
Fucose/analysis , Plasminogen Activators/chemistry , Saliva/enzymology , Amino Acid Sequence , Animals , CHO Cells , Cattle , Chiroptera , Chromatography, High Pressure Liquid , Cricetinae , Gas Chromatography-Mass Spectrometry , Humans , Mass Spectrometry , Molecular Sequence Data , Peptide Mapping , Plasminogen Activators/isolation & purification , Plasminogen Activators/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Threonine , Tissue Plasminogen Activator/chemistry , TransfectionABSTRACT
We have used in vitro techniques to study the metabolism of dexamethasone. Tissue slices, homogenates and microsomal fractions of various mammalian organs from rats and humans have been used. We focused particularly on the question of whether or not dexamethasone (Dexa) is oxidized at the C11-OH group by 11 beta-hydroxysteroid-dehydrogenase. High activities of this enzyme system for Dexa were localized in renal cortex and rectum. Material from both human and murine liver was ineffective. The main metabolite formed from Dexa in renal and intestinal systems was identified by different mass-spectrometric techniques including on line HPLC mass spectrometry as 11-dehydro-dexamethasone. This finding was corroborated by the observation that both corticosterone and glycyrrhetinic acid block the metabolic transformation of Dexa.
