Amblyomma americanum tick calreticulin binds C1q but does not inhibit activation of the classical complement cascade

Abstract

In this study we characterized Amblyomma americanum (Aam) tick calreticulin (CRT) homolog in tick feeding physiology. In nature, different tick species can be found feeding on the same animal host. This suggests that different tick species found feeding on the same host can modulate the same host anti-tick defense pathways to successfully feed. From this perspective it's plausible that different tick species can utilize universally conserved proteins such as CRT to regulate and facilitate feeding. CRT is a multi-functional protein found in most taxa that is injected into the vertebrate host during tick feeding. Apart from it's current use as a biomarker for human tick bites, role(s) of this protein in tick feeding physiology have not been elucidated. Here we show that annotated functional CRT amino acid motifs are well conserved in tick CRT. However our data show that despite high amino acid identity levels to functionally characterized CRT homologs in other organisms, AamCRT is apparently functionally different. Pichia pastoris expressed recombinant (r) AamCRT bound C1q, the first component of the classical complement system, but it did not inhibit activation of this pathway. This contrast with reports of other parasite CRT that inhibited activation of the classical complement pathway through sequestration of C1q. Furthermore rAamCRT did not bind factor Xa in contrast to reports of parasite CRT binding factor Xa, an important protease in the blood clotting system. Consistent with this observation, rAamCRT did not affect plasma clotting or platelet aggregation. We discuss our findings in the context of tick feeding physiology.

Figure 1. Multiple sequence alignment of Amblyomma americanum (Aam) CRT and amino acid sequences of functionally characterized CRT in human and other parasites

Downloaded CRT amino acid (AA) sequences of A. americanum (GenBank:AY395246), human (NP_004334) and other parasite CRT, T. cruzi (EKG06053), T. carrasi (ACV30040), T. solium (AF340232), H. contortus (AAR99585), N. americanus (AJ006790) and H. polygyrus (CAL30086) were subjected to multiple sequence alignment using ClustalW in MacVector. The aligned sequences were visually inspected for, globular N- domain, the proline rich P-domain and the C- domain noted in figure 1. The P- domain contains the nuclear localization signal [NLS] (solid box and noted) and three calreticulin P domain repeated motifs [CPRM1-3] (dotted box and noted). The C-domain contains the endoplasmic reticulum retention signal [ERRS, boxed and noted]. The six C1q binding site AA motifs [C1qBS1-6] are noted and boxed with solid lines. CRT conserved histidine (H) AA residues are marked with an asterisks (*) sign above, the proline (P) AA residues are noted with filled circles (●) above, and the characteristic acidic AA residues in AamCRT c-domain are marked with filled squares (■).

Figure 2. Multiple sequence alignment of functionally annotated amino acid motifs of Amblyomma americanum and other parasite CRT proteins

Thirty four tick CRT amino acid sequences from NCBI consisting of Metastriata: A. americanum (GenBank:AY395246), A. scutatum_AAR29938.1, A. rotundatum_AAR29937.1, A. geayi_AAR29935.1, A. cooperi_AAR29934.1, A. brasiliense_AAR29933.1, A. variegatum_DAA34570.1, A. triste_JAC35302.1, A. parvum_JAC26090.1, A. cajennense_JAC22751.1, A. maculatum_AEO34580.1), Rhipicephalus (R. microplus_AAR29940, R. sanguineus_AAR29961.1), Hyalomma (H. anatolicum anatolicum_AFQ98396.1, H. dromedarii_AEW43369.1, H. anatolicum excavatum_AAR29945.1), Dermacentor (D. variabilis_AAR29944.1, D. occidentalis_AAR29943.1, D. andersoni_AAR29942.1, D. albipictus_AAR29941.1), Haemaphysalis (H. qinghaiensis_AAY42204.1, H. longicornis_AAQ18695.1, H. leporispalustris_AAR29947.1), and Prostriata ticks: Ixodes (I. ricinus_AAR29958.1, I. persulcatus_AAR29957.1, I. pararicinus_AAR29956.1, I. pacificus_AAR29955.1, I. pavlovskyi_AAR29954.1, I. ovatus_AAR29953.1, I. nipponensis_AAR29952.1, I. muris_AAR29951.1, I. minor_AAR29950.1, I. jellisoni_AAR29949.1, I. affinis_AAR29948.1, I. scapularis_XP_002402080.1), six other parasites: T. cruzi (EKG06053), T. carrasi (ACV30040), T. solium (AF340232), H. contortus (AAR99585), N. americanus (AJ006790) and H. polygyrus (CAL30086) and human: H. sapiens (NP_004334) CRTs, were subjected to multiple sequence alignment using T-coffee in MacVector software. The aligned sequences were visually inspected for presence of the six C1q binding site amino acid motifs (C1qBS1-6), the nuclear localization signal (NLS), three calreticulin P domain repeated motifs (CPRM1-3), and the endoplasmic reticulum retention signal [ERRS].

Figure 3. Phylogeny analysis of Amblyomma americanum, other parasites, and human CRT amino acid sequences

A guide phylogeny tree of AamCRT protein sequence with other tick, parasite, and human CRT sequences was constructed using the Maximum Likelihood method with bootstrap replicates set to 1000. Number at each node represents bootstrap values that signify the level of confidence in the branch. Three main clusters: X (tick CRTs), Y (Homo sapien [GenBank:NP004334.1]), and Z other parasite and an outgroup of human calnexin (GenBank:AAA2103.1) noted with asterisk (*). The overall amino acid identity percentage levels are noted for cluster A–F.

Figure 4. Expression and affinity purification of recombinant Amblyomma americanum CRT in Pichia pastoris

The coding domain for mature AamCRT amplified as described in materials and methods was sub-cloned into pPICZαA yeast expression plasmid using routine directional approach. Following confirmation of sequence, recombinant pPICZαA-AamCRT was expressed and affinity purified as described in materials and methods. Expression and affinity purification of rAamCRT was respectively confirmed using routine western blotting analysis using antibodies C-terminus hexa-histidine tag and detected with chemiluminescent substrates (A) and SDS-PAGE with Coommassie blue staining (B). Lanes 1–5 in “4A” = daily expression levels of rAamCRT. In “4B”, AP1 and AP2 = affinity purified fractions 1 and 2.

Figure 5. Western blotting analysis of rAamCRT using antibodies to 48h Amblyomma americanum tick saliva proteins

Affinity purified rAamCRT loaded into triplicate lanes was electrophoresed on a 10% Tris-glycine acrylamide gel and electroblotted onto PVDF membranes using routine methods. The three lanes were cut into individual strips, which were exposed to three different concentrations (noted in Fig. 5) of pre-immune and antibodies to 48h A. americanum tick saliva proteins as indicated.

Figure 6. Recombinant AamCRT binding of the C1q protein

Different amounts of rAamCRT (indicated) were diluted in 200μL ELISA coating buffer (carbonate buffer, pH 9.5) and bound onto ELISA plates overnight at 4°C. Control wells received buffer only or ligand (C1q or factor Xa). Following coating, wells were blocked as described in materials and methods and subsequently incubated with 100μL (0.5μg) of C1q protein with the exception of the ligand (C1q) only control wells, overnight at 4°C. Following appropriate washing, wells were incubated with antibodies to human C1q for 2h at room temperature (RT). Following incubation and appropriate washing, plates were incubated for 2h at RT with 100μL of anti-rabbit peroxidase conjugated IgG. Following appropriate washing bound peroxidase activity, proxy for bound C1q protein was detected by adding OPD substrate as described and level of bound C1q quantified at A₄₅₀ using the VersaMax mircoplate reader. Shown in the figure is bound C1q OD with background removed.