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First published online January 3, 2006
Journal of Experimental Biology 209, 343-352 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02003

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Vasotab, a vasoactive peptide from horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary glands

Peter Taká^1,*,, Miles A. Nunn^2,*, János Mészáros³, Olga Pecháová⁴, Norbert Vrbjar⁵, Petra Vlasáková¹, Milan Kozánek¹, Mária Kazimírová¹, George Hart³, Patricia A. Nuttall² and Milan Labuda¹

¹ Institute of Zoology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 06 Bratislava, Slovak Republic
² Centre of Ecology and Hydrology, NERC, Oxford, UK
³ Department of Medicine, University Clinical Departments, The Duncan Building, Daulby Street, Liverpool, L69 3GA, UK
⁴ Institute of Normal and Pathological Physiology
⁵ Institute for Heart Research, Department of Biochemistry, Slovak Academy of Sciences, Bratislava, Slovak Republic

View larger version (57K): [in a new window]   Fig. 1. Head showing salivary glands of horse fly Hybomitra bimaculata. Bar, 1 mm.

View larger version (18K): [in a new window]   Fig. 2. Reverse-phase HPLC of crude salivary gland extract (SGE) of horse fly H. bimaculata. The peaks containing vasodilator activity are indicated by their retention times. The purification procedures are described in the text. (A) Crude SGE purified on a Vydac C4 column; (B) active fraction from A with retention time 13.77 min., second purification on the Beckman Ultrasphere C-18 column; (C) active fraction from B with retention time 14.40 min, third purification on the Vydac C18 column.

View larger version (16K): [in a new window]   Fig. 3. Vasodilating activity of protein HPLC fractions (purification A, Fig. 1) in the retention time range 5–28 min of H. bimaculata SGE on rat arterial rings without endothelium.

View larger version (13K): [in a new window]   Fig. 4. Relaxation of contracted rat femoral artery induced by purified vasotab EV048, corresponding to the peak at 14.51 min (purification C, Fig. 2). The rat femoral artery was contracted with phenylephrine (5x10-6 mol l-1).

View larger version (11K): [in a new window]   Fig. 5. Coronary flow record after application of H. bimaculata SGE (equivalent of 10 salivary glands).

View larger version (17K): [in a new window]   Fig. 6. In vitro effect of H. bimaculata (H.b.) SGE on cardiac Na,K-ATPase activity. SGE is expressed as total protein concentration. (A) Actual data of representative measurements. Estimation at each concentration was performed in triplicate. (B) Transformation of the data to a Hanes plot.

View larger version (37K): [in a new window]   Fig. 7. Primary structure of peptide vasotab. The signal sequence is underlined, cysteine residues are shown in bold type, and the stop codon is indicated by an asterisk.

View larger version (54K): [in a new window]   Fig. 8. Pfam alignment of vasotab with representative Kazal type proteins. The residues in lower case are outstandingly different from the overall consensus. Residues identical in more than half of the sequences depicted are highlighted in grey. Cysteines are highlighted in black. Identity of sequences shown: Anemonia sulcata inhibitor of elastase (IELA_ANESU/4–48); Rhodnius prolixus thrombin inhibitor domain 1 and 2 (THBI_RHOPR/6–48 and 57–101); AGRI, agrin; IAC, acrosin inhibitor; FSA, follistatin; PE60, PEC60; IPST, pancreatic secretory trypsin inhibitor; IPSG, double headed protease inhibitor; IOV, ovomucoid inhibitor; QR1, quail retinal 1; SC1, secreted calcium binding 1 matrix glycoprotein; SPRC, secreted protein acidic and rich in cysteine, also called basement membrane protein 40.

View larger version (8K): [in a new window]   Fig. 9. Schematic representation of the primary structure of a Kazal-type protein. Disulphide bridging pattern of the conserved cysteine (C) residues is indicated by solid lines. Asterisks show the position of the Pfam consensus pattern: C–x(7–10)–C–x(6)–Y–x(3)–C–x(2–6)–C. All six cysteines are conserved in mature vasotab, although the spacing between cysteine residues within the consensus pattern is unusual: C–x(7)–C–x(13)–F–x(3)–C–x(6)–C. The active site residue of protease inhibitors is marked by a hash (#).

View larger version (96K): [in a new window]   Fig. 10. Coomassie Blue stained NuPAGETM 4–12% Bis-Tris gel showing purified peptide vasotab. Lane 1, protein markers (molecular masses in kDa are indicated at left); lanes 2–5, serial fractions that elute at about 0.15 mol l-1 NaCl from a SP sepharose column.

View larger version (11K): [in a new window]   Fig. 11. Representative whole-cell voltage-clamp recordings of ICa at –5 mV depolarizing voltage steps in control myocytes and myocytes treated with 0.4 µg ml-1 native protein (A) and 0.4 µg ml-1 recombinant protein (B).