Cloning of a salivary gland metalloprotease and characterization of gelatinase and fibrin(ogen)lytic activities in the saliva of the Lyme disease tick vector Ixodes scapularis

Abstract

The full-length sequence of tick salivary gland cDNA coding for a protein similar to metalloproteases (MP) of the reprolysin family is reported. The Ixodes scapularis MP is a 488 amino acid (aa) protein containing pre- and pro-enzyme domains, the zinc-binding motif HExxHxxGxxH common to metalloproteases, and a cysteine-rich region. In addition, the predicted amino-terminal sequences of I. scapularis MPs were found by Edman degradation of PVDF-transferred SDS/PAGE-separated tick saliva proteins, indicating that these putative enzymes are secreted. Furthermore, saliva has a metal-dependent proteolytic activity towards gelatin, fibrin(ogen), and fibronectin, but not collagen or laminin. Accordingly, I. scapularis saliva has a rather specific metalloprotease similar to the hemorrhagic proteases of snake venoms. This is the first description of such activity in tick saliva and its role in tick feeding and Borrelia transmission is discussed.

Fig. 1

Full-length nucleotide sequence and deduced amino acid sequence of Ixodes scapularis metalloprotease (I. scapularis MP1)(gi AY264367). The nucleotides and aminoacids are numbered from the translation starting site ATG. The signal peptide sequence or pre-enzyme (1–19 aa) is in bold underlined. The pro-enzyme (20–169 aa) is in bold italicized. The mature protein (170–488 aa) starting with the sequence YKIPL obtained by Edman degradation of salivary proteins [14] is in bold.

Fig. 2

(A) Alignment of I. scapularis MP1 sequence (Ix_scapula_MP1; gi AY264367) having similarity to other Ixodes MPs: I. scapularis MP2 (Ix_scapula_MP2; gi 22164294), I. scapularis MP3 (Ix_scapula_MP3; gi 22164296), and I. ricinus MP (Ix_ricinus_MP; gi 5911708). Peptides YKIP… and AADT…(shown in reversed background) were found by Edman degradation of protein bands from SDS-PAGE gels separating tick saliva and indicate the N-terminus of the mature protein [14]. The box indicates, above the aligned sequences, the metzincin zinc-binding motif HEXXHXXGXXH [22] and, below the aligned sequences, the snake venom metalloprotease active site consensus sequence [17,18]. The reversed background residues in these motifs indicate identities in the tick sequences. The triad MSY following the zinc-binding box is the putative methionine turn found after the zinc-binding domain [22]. All cysteines are shown in reversed background. (B) Alignment of I. scapularis putative reprolysins with atrolysins to show conservation of the carboxy terminal M (arrow) if two insertions flanked by glycine and proline residues (in reverse background) are considered. The line above the sequences indicates the zinc-binding domain of the active center. The symbols under the sequences indicate identity (*), highly conserved (:), and conserved (.) residues. The NCBI accession numbers of non-tick sequences are shown after the gi|.

Fig. 3

Gelatinase activity of I. scapularis saliva. (A) Graph represents increase in fluorescence of fluoresceinated gelatin (0.1 mg/ml in Hepes buffer 25 mM NaCl 150 mM containing either 5 mM CaCl₂ or 5 mM EDTA) following addition of diluted tick saliva to give a total amount of 0.125 µl in a 50-µl reaction mixture. Similar results were obtained with four other pools of tick saliva. (B) Exponential loss of activity of tick gelatinase activity in the presence of 5 mM EDTA. Maximal fluorescence (MaxFluor) was estimated as the average of the last five fluorescence level determinations in the presence of EDTA. Each time point was plotted in the natural logarithmic scale as the ratio of the difference between MaxFluor minus the fluorescence at each time point (Fluor) divided by MaxFluor. Similar results were obtained with four other pools of saliva, each done at three different saliva dilutions.

Fig. 4

(A) Hydrolysis of the Aα peptide chain of FNG by I. scapularis saliva. FNG was incubated with tick saliva for the indicated times (in minutes) in the presence of CaCl₂ (5 mM) or EDTA (5 mM) before being heated at 70°C with sample buffer to terminate the reaction. Lane MW contains the mol wt markers, with their numbers indicated on the right. The Aα, Bβ, and γ chains of FNG are indicated. Arrows indicate degradation products observed when saliva was incubated with FNG in the presence of Ca⁺⁺. Two other experiments with two different saliva pools gave identical results. For more details, see Materials and methods. (B) Hydrolysis of FN by I. scapularis saliva. Fibronectin was incubated with tick saliva for the indicated times (in minutes) in the presence of CaCl₂ (5 mM) or EDTA (5 mM) before being heated at 70°C with sample buffer to terminate the reaction. Lane MW contains the mol wt markers, with their numbers indicated on the left. Two other experiments with two different saliva pools gave identical results. For more details, see Materials and methods. (C) Fibrinolytic activity of I. scapularis saliva. The gel that has not been digested is Coomassie blue stained and shown in black in the Figure. The central digested gel is not stained and represents the fibrinolytic activity of saliva. For more details, see Materials and methods. Bar, 1 cm length.