Expression of biological activity of draculin, the anticoagulant factor from vampire bat saliva, is strictly dependent on the appropriate glycosylation of the native molecule Citation for published version (APA): Fernandez, A. Z., Tablante, A., Bartoli, F., Beguin, S., Hemker, H. C., & Apitz-Castro, R. (1998). Expression of biological activity of draculin, the anticoagulant factor from vampire bat saliva, is strictly dependent on the appropriate glycosylation of the native molecule. Biochimica et Biophysica Acta-general Subjects, 1425(2), 291-299. https://doi.org/10.1016/S0304-4165(98)00082-8 Document status and date: Published: 23/10/1998 DOI: 10.1016/S0304-4165(98)00082-8 Document Version: Publisher's PDF, also known as Version of record Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. 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Apartado 21827, Caracas 1020A, Venezuela b Department of Biochemistry, University of Maastricht, Maastricht, The Netherlands Received 13 May 1998; revised 1 July 1998; accepted 16 July 1998 Abstract Draculin, a glycoprotein isolated from vampire bat (Desmodus rotundus) saliva, is a natural anticoagulant which inhibits activated coagulation factors IX (IXa) and X (Xa). The observation that under captivity conditions, the anticoagulant activity present in vampire bat saliva is dependent upon the salivation protocol, led us to investigate the possible relationship between the expression of biological activity of native draculin and the post-translational glycosylation of the protein backbone. Daily salivation of vampire bats yields a saliva that progressively decreases in anticoagulant activity, without any significant change in overall protein content, or in the amount of protein specifically recognized by a polyclonal anti-draculin antibody. Anticoagulant activity of the saliva is restored after a 4-day period of rest. Besides the marked difference in anticoagulant activity, purified native draculin, obtained from high- and low-activity saliva, shows significant differences in: (a) composition of the carbohydrate moiety, and (b) Glycosylation pattern. Furthermore, controlled chemical deglycosylation of native draculin, under conditions that do not affect the polypeptide backbone, progressively leads to complete loss of the biological activity. Our present results implicate that correct glycosylation of draculin is a seminal event for the expression of the biological activity of this glycoprotein. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Draculin; Glycosylation; Natural glycoform; Anticoagulant 1. Introduction Abbreviations: FIXa, activated factor IX; FXa, activated fac- tor X; TFA, tri£uoroacetic acid; TFMS, tri£uoromethanesul- Draculin, which is a selective inhibitor of the bio- fonic acid; FACE, £uorophore-assisted carbohydrate electropho- resis; SDS^PAGE, sodium dodecyl sulfate^polyacrylamide gel logical activity of activated coagulation factors IX electrophoresis; ELISA, enzyme-linked immunosorbent assay; (FIXa) and X (FXa), was ¢rst reported by Apitz- TMS, trimethylsilyl; GC^MS, gas chromatography^mass spec- Castro et al. as a glycoprotein of 88.5 kDa obtained trometry analysis; 2-AB, 2-aminobenzamide; GalNAc, N-acetyl- from saliva of vampire bat Desmodus rotundus [1]. galactosamine; GlcNAc, N-acetylglucosamine; Man, mannose; This natural anticoagulant displays a microheteroge- Gal, galactose; AD, active draculin; ID, inactive draculin; PNA, peanut agglutinin; WGA, wheat germ agglutinin neous band pattern in SDS^PAGE, commonly found * Corresponding author. Fax: +58-2-504-1093; in most glycoproteins [2]. Since the initial isolation of E-mail: [email protected] draculin, it was observed that the anti-FXa biologi- 0304-4165 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S0304-4165(98)00082-8 BBAGEN 24676 12-10-98 292 A.Z. Fernandez et al. / Biochimica et Biophysica Acta 1425 (1998) 291^299 cal activity of native draculin is not similar for all the highest quality commercially available. For batches, even when saliva is obtained from the same graphics and mathematical approaches, Origin 5.0 animals. Among possible explanations to this fact Software was used (Microcal Software, MA, USA). one can invoke: (a) diminished production of the native protein, or (b) Impairment of relevant post- 2.2. Animals translational modi¢cations of the protein, both due to environmental changes that vampire bats undergo Vampire bats (Desmodus rotundus) were regularly when kept in captivity. captured from wild colonies in the region of Choron|¨ It is now well established that the appropriate gly- (State of Aragua, Venezuela). The bats were kept in cosylation of proteins may be relevant to the expres- captivity, in individual, metabolic-type cages (Acme sion of their biological activity and that the set of Metal Products, Chicago, IL, USA), under con- glycoforms of a given polypeptide expressed by an trolled light and temperature (25³C). The animals organism is dependent on its physiological state [3,4]. were maintained on bovine blood anticoagulated In this paper, we report results that indicate that the with 3.8% sodium citrate at a ratio of 1:9. Food anticoagulant activity of draculin is highly dependent was given every 24 h and water was given ad libitum on the proper glycosylation of the polypeptide back- [5]. bone. 2.3. Saliva collection 2. Materials and methods For saliva collection, animals were anaesthetized with a mixture of 2.5% 2-bromo-2-chloro, 1,1,1-tri- 2.1. Materials and reagents £uorethane (Halothan, Hoechst) and 30% nitrous oxide in oxygen [5,6]. Once anesthetized, 20 Wl of Hydroxyapatite (Biogel-HTP), acrylamyde and sil- 1% pilocarpine (Isopto Carpin, Alcon, Ft. Worth, ver staining reagents were purchased from Bio-Rad TX, USA) was placed in the mouth in order to stim- (Richmond, CA, USA). Coagulation factors were ulate salivation. Saliva was collected in 2-ml micro- kindly provided by Dr. Rob Wagenvoord (Depart- centrifuge tubes and stored at 320³C until use. Each ment of Biochemistry, University of Limburg, Neth- sample was assayed for protein concentration and erlands). The activity of FXa was measured using the FXa inhibitory activity, and similar batches were chromogenic substrates S2222 (Km=200 WM, Chro- kept separate and eventually pooled for draculin pu- mogenix, Sweden) and S2337 (Km=200 WM, Kabi ri¢cation. Diagnostica, Sweden). For continuous measurement of thrombin generation we used the substrate SQ68. 2.4. Draculin puri¢cation The following reagents were purchased from Oxford GlycoSystems (USA): GlycoFree deglycosylation kit, Draculin was puri¢ed with a modi¢cation of the GlycoRelease kit, Signal 2-AB kit and HPLC Glyco- original method [1]. Brie£y, the saliva was thawed Sep. For the electrophoresis of the £uorophore-la- and dialyzed for 14 h against distilled water at 4³C. beled carbohydrates we used the FACE N-linked oli- The dialysate, 1/3 diluted, was adjusted to 1 mM gosaccharide pro¢ling system provided by Glyco NaCl and loaded onto a hydroxyapatite column (CA, USA). Neuraminidase X type (EC 3.2.1.18) equilibrated with NaCl at a £ow rate of 0.3 ml/ was a gift from Dr. Raimundo Villegas (IDEA, Bar- min. The column was washed with 5 column volumes uta, Venezuela). Freund's complete adjuvant (FCA) of 1 mM NaCl (pH 6.8), followed by 4 column vol- was from Calbiochem (CA, USA) and Freund's in- umes of 200 mM potassium phosphate bu¡er (pH complete adjuvant (FIA) was from Gibco (OH, 6.8). After column washing, the elution was initiated USA). EGGstract puri¢cation system and Anti- using a phosphate bu¡er gradient from 200 mM (pH chicken IgY AP conjugate were supplied by Promega 6.8) to 1 M (pH 7.4), and 1-ml fractions were col- (WI, USA). WGA and PNA lectins were provided by lected at a £ow rate of 0.3 ml/min. Anti-FXa activity Vector (CA, USA). All other reagents used were of of each fraction was assayed using a microplate BBAGEN 24676 12-10-98 A.Z. Fernandez et al. / Biochimica et Biophysica Acta 1425 (1998) 291^299 293 adaptation of the FXa assay [1].