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. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Ticks Tick Borne Dis. 2014 Sep 18;6(1):16–30. doi: 10.1016/j.ttbdis.2014.08.002

Bioinformatic analyses of male and female Amblyomma americanum tick expressed serine protease inhibitors (serpins)

Lindsay Porter 1,2,3, Zeljko Radulovic 1,2,3, Tae Kim 1,2,3, Gloria R C Braz 1,2,3, Itabajara Da Silva Vaz Jr 1,2,3, Albert Mulenga 1,2,3,*
PMCID: PMC4252504  NIHMSID: NIHMS631433  PMID: 25238688

Abstract

Serine protease inhibitors (serpins) are a diverse family of proteins that is conserved across taxa. The diversity of Amblyomma americanum serpins (AAS) is far more complex than previously thought as revealed by discovery of 57 and 33 AAS transcripts that are respectively expressed in male and female A. americanum ticks, with 30 found in both. While distinct reproductively, both male and female metastriate ticks, such as A. americanum, require a blood meal. Thus, 30 AAS sequences found in both male and female ticks could play important role(s) in regulating tick feeding and thus represent attractive candidates for anti-tick vaccine development. Of significant interest, 19 AAS sequences expressed in male and female ticks are also part of the 48 AAS sequences expressed in fed female tick salivary glands or midguts; two organs through which the tick interacts with host blood and immune response factors. Considered the most important domain for serpin function, the reactive center loop (RCL) is further characterized by a single ‘P1’ site amino acid residue, which is central to determining the protease regulated by the serpin. In this study, a diversity of 17 different P1 site amino acid residues were predicted, suggesting that A. americanum serpins potentially regulate a large number of proteolytic pathways. Our data also indicate that some serpins in this study could regulate target protease common to all tick species, in that more than 40% of AAS show 58–97% inter-species amino acid conservation. Of significance, 24% of AAS showed 62–100% inter-species conservation within the functional RCL domain, with 10 RCLs showing ≥90–100% conservation. In vertebrates, serpins with basic residues at the P1 site regulate key host defense pathways, which the tick must evade to feed successfully. Interestingly, we found that AAS sequences with basic or polar uncharged residues at the putative P1 site are more likely to be conserved across tick species. Another notable observation from our data is that AAS sequences found only in female ticks and those found in both males and females, but not those found only in male ticks, were highly conserved in other tick species. While descriptive, this study provides the basis for more in-depth studies exploring the roles of serpins in tick feeding physiology.

Keywords: Amblyomma americanum, serine protease inhibitors (serpins), tick feeding physiology, orthologous serpins

INTRODUCTION

Ticks are among the most successful ectoparasites of humans and animals. In livestock production, an estimated 80% of the world’s cattle are affected by tick-borne diseases (TBD), of which the most important include theileriosis, babesiosis, anaplasmosis, and heartwater (Marcelino et al., 2012). In addition to pathogen transmission, tick feeding has been documented to cause severe direct effects including paralysis, exsanguination, reduced livestock productivity, and damage to skin impacting the economic value of hides (Klompen et al., 1996; Jongejan and Uilenberg, 2004). In public health, ticks have been implicated in the transmission of 17 diseases affecting humans, and the list continues to grow (Day, 2011; Dantas-Torres et al., 2012; Savage et al., 2013).

Amblyomma americanum has emerged among the most important tick species in public health in the United States. A. americanum transmits multiple TBD agents, including Ehrlichia chaffeensis, E. ewingii, Rickettsia amblyommii, Francisella tularensis, the as yet undescribed causative agent of southern tick associated rash illness (STARI), Cytauxzoon felis, Theileria cervi, and the emerging human Heartland virus (Waldrup et al., 1992; James et al., 2001; Childs and Paddock, 2003; Telford and Goethert, 2004; Telford III et al., 2008; de la Fuente et al., 2008; Goddard, 2009; Schulze et al., 2011; Savage et al., 2013). For many years, this tick was mainly distributed in the southeastern United States, but has now been reported as established in 32 states throughout the Southeast, South Central, and Midwest regions as well as along the eastern seaboard as far north as Maine (Springer et al., 2014). Its emerging geographic expansion and role as a vector of many important human disease agents makes A. americanum an important consideration in strategies to improve public health.

With a lack of effective vaccines against TBD agents, the prevention of these infections in humans and animals depends on the control of ticks, which is currently acaricide based. However, acaricide use comes with many disadvantages, including the threat of food and environmental contamination and resistance development to these chemicals by ticks. A promising alternative strategy to the chemical control of ticks is to vaccinate hosts against the ticks themselves. The prerequisite to the development of an effective vaccine however, is the identification of effective molecular targets against tick feeding success and/or pathogen transmission. Among the emerging candidates for vaccine target antigens are members of the serine protease inhibitor (serpin) family. In animals, pathways critical to life, such as blood coagulation, complement activation, and inflammation are tightly regulated by serpins (Moore et al., 1993; Gettins, 2002; Tekin et al., 2005; Huntington, 2006; Huntington, 2011; Gatto et al., 2013). Furthermore, dysfunctional serpin activity in humans has been cited to cause numerous diseases including cirrhosis, emphysema, blood coagulation disorders, and dementia (Stein and Carrell, 1995; Davis et al., 1999; Gooptu and Lomas, 2009; Mocchegiani et al., 2011; Benson and Wilkes, 2012; Bosche et al., 2012; Gatto et al., 2013). The tick feeding style of lacerating host tissue and imbibing host blood which bleeds into the feeding site, is expected to provoke tissue repair and immune response mechanisms such as platelet aggregation, inflammation, blood clotting, and complement pathways (Ribeiro, 1989; Wikel et al., 1994), all of which are serpin-regulated. Thus, to complete feeding, ticks have to overcome serpin-regulated host defense pathways. From this perspective, it is conceivable that ticks may utilize serpins to block these host defenses against tick feeding (Muleng et al., 2001; Mulenga et al., 2002).

Serpin-encoding cDNAs have now been cloned from several tick species (Nene et al., 2002; Sugino et al., 2003; Mulenga et al., 2003a; Mulenga et al., 2003b; Imamura et al., 2005; Ribeiro et al., 2006; Imamura et al., 2006; Prevot et al., 2007; Chalaire et al., 2011; Yu et al., 2013). On the basis of unique putative functional domain reactive center loops (RCLs), at least 45 serpins are expressed in I. scapularis (Mulenga et al., 2009). Similarly, Mulenga et al., (Mulenga et al., 2007) and Karim et al., (Karim et al., 2011) have reported at least 17 and 32 different serpin transcripts expressed in A. americanum and A. maculatum, respectively. Data are now emerging which support the idea that some tick-encoded serpins are functional inhibitors associated with counter defense against anti-tick responses in the host, such as inflammation, complement activation, platelet aggregation, and blood clotting (Imamura et al., 2005; Prevot et al., 2009; Chmelar et al., 2011; Chmelar et al., 2012; Mulenga et al., 2013). In other studies, a significant reduction in feeding efficiency has been observed in ticks which fed on animals immunized with recombinant tick serpins (Imamura et al., 2005; Imamura et al., 2006; Prevot et al., 2007; Imamura et al., 2008; Kaewhom et al., 2009; Jittapalapong et al., 2010) suggesting a prime importance of serpins in tick feeding physiology.

An observation in our lab is that while overall amino acid conservation levels for serpins are around 35–45%, there are some serpins which show much higher conservation across all tick species investigated. We believe that highly conserved serpins could play crucial role(s) in tick physiology. The goal of this study was two-fold: first, to identify serpin transcripts expressed in unfed and fed A. americanum ticks, and second, to conduct a global intra- and inter-tick species bioinformatic analysis of A. americanum serpins (AAS) and other tick serpins. This study has described 57 and 33 AAS sequences that were respectively found only in male and female ticks, and a further 30 that were found in both. Nearly half of the serpin sequences expressed in A. americanum are predicted to regulate pathways important to all tick species, as they show 58–97% amino acid conservation in both metastriate and prostriate ticks. Although this study is descriptive, data presented here provide a foundation for further in depth studies on the roles of serpins in tick physiology.

MATERIALS AND METHODS

Identification and sequence analysis of A. americanum serpin (AAS) transcripts

AAS sequences used in this study were obtained by data mining of de novo assembled A. americanum transcriptomes (unpublished data), serendipitously while attempting to clone other targets, and from GenBank (Mulenga et al., 2007). A. americanum transcriptomes were assembled from Illumina sequence reads (BioProject accession number PRJNA226980) of 24 and 96h fed female phage display cDNA expression libraries, unfed and fed male and unfed and 24h fed female whole ticks, as well as 48, 96 and 120h fed tick dissected salivary gland (SG) and midgut (MG) tissues, using two approaches. In the first approach libraries were individually assembled with source library information for each contig retained, and in the second approach reads from all sources were combined and assembled (unpublished).

Mining and identification of putative AAS sequences was accomplished in two steps. In the first step, assembled contigs were subjected to batch blastx screening against tick sequences in GenBank. In the second step, contig sequences with matches to serpin sequences were manually inspected to confirm the presence of two consensus amino acid motifs: the reactive center loop (RCL) “"p17 [E]-p16 [E/K/R]-p15 [G]-p14 [T/S]-p13 [X]-p12-9 [AGS]-p8-1 [X]-p1' -4'” in the C-terminus, and the ‘NAVYFKG’ motif in the N-terminus (Carrell et al., 1987; Miura et al., 1995; Gettins, 2002). Sequences with a unique RCL sequence were identified as new and assigned an AAS number. Sequences without an RCL region were declared partial in the C-terminus region. For these sequences, two comparisons were made using Bl2seq-blastp (NCBI). First, these sequences were compared to each other to cluster contigs representing the same serpin. Next, we compared these clusters to AAS containing an RCL to eliminate redundancy between these groups. In addition to consensus amino acid motifs and secondary structure, a typical serpin ranges from 350–450 amino acids long (Gettins, 2002). Thus, sequences that had a starting methionine and were at least 350 amino acid residues long were considered putatively full-length. Full-length sequences were subjected to SignalP Version 4 web server to detect signal peptides (Petersen et al., 2011).

Relative AAS transcript abundance

To get insight into relative abundance, Illumina reads were mapped back to assembled contigs using the map reads to reference option in CLC genomics workbench vers. 6.4.2. Relative abundance values were adjusted to account for contig size and total library reads using the following equation: ey = (nyNxL1x/nxNyLy) × ex, where ex and ey represent normalized relative abundance levels of AAS transcript in libraries X and Y, Nx and Ny represent the total number of reads in libraries X and Y, nx and ny represent the number of reads related to the specific AAS transcript in libraries X and Y, and Lx and Ly represent the length of the contig related to the specific AAS transcript in libraries X and Y.

Phylogeny and comparative sequence analysis among AAS sequences

To determine relationships between AAS sequences, a guide phylogeny tree was constructed using the neighbor-joining method in MacVector vers 12 DNA analysis software (MacVector Inc., Cary, North Carolina). Sequences were first aligned using T-coffee, then a phylogeny tree out-rooted from human antithrombin (CAA48690), was constructed using the neighbor-joining method set to the default bootstrap setting of 1000 replications and differences adjusted using the absolute # differences setting. Subsequently, AAS sequences that clustered together on the phylogeny tree were subjected to pairwise sequence alignment analyses using MacVector. For partial sequences, amino acid identity levels were determined based on available sequences.

Comparative analyses of AAS to other tick serpin sequences

To investigate relationships among all available tick serpins, AAS and other tick serpins from publically available databases were subjected to phylogeny analysis and multiple sequence alignment analyses using alignment tools at NCBI and MacVector vers 12. This analysis was done at the whole amino acid sequence and RCL levels. At the whole amino acid sequence level, amino acid sequences were subjected to batch pairwise comparisons using Bl2seq-blastp (NCBI) to identify AAS orthologs in other tick species. The serpin RCL is an important functional domain, which determines what protease is regulated by a candidate serpin. Thus, to investigate the relationship of AAS sequences with other tick serpins at the functional level, the neighbor-joining method in MacVector (MacVector Inc.), was used to construct guide phylogeny trees using putative RCLs. To manage the huge dataset, RCLs were first divided into four groups based on charge and polarity characteristics of the amino acid residue at the putative P1 site: polar basic, polar acidic, polar uncharged, and hydrophobic. A separate tree was constructed for each group. Next intra-clade pairwise alignments of RCLs were performed using MacVector to determine identity levels.

RESULTS

Amblyomma americanum male and female ticks express large numbers of serpin transcripts

Data mining of transcriptomes from fed and unfed male and female whole ticks, as well as dissected 48, 96 and 120h female SG and MG transcriptomes, identified 28 and 57 AAS sequences respectively found only in females and males, respectively, and an additional 30 found in both (Table 1, Supplemental Tables 1 and 2). Mulenga et al., (2007) described 17 AAS (here after identified as AAS1–17) sequences that were expressed in 120h fed ticks. Of these 17 AAS sequences, this study found 7 sequences in both males and females, and five AAS sequences in females, while the remaining five were not found at all. Taken together, these studies show the total number of AAS sequences found only in female ticks to be 33 (Table 1, Supplemental Table 1). Please note that AAS 77 and 78 were found only in the combined A. americanum transcriptome were source library information for assembled contigs was not retained, and thus source information is unknown (Supplemental Table 1). Overall, 87 and 63 AAS sequences were found in male and female ticks, respectively (Supplemental Table 1 and 2).

Table 1.

Updated list of Amblyomma americanumserpin (AAS) transcripts

Table 1A: AAS sequences found in male ticks
AAS# ID Accession# Comment AAS# ID Accession# Comment AAS# ID Accession# Comment
44 GAYW01000312 Partial 73 GAYW01000241 Partial 95 GAYW01000268 Partial
46 GAYW01000194° Partial 74 GAYW01000254 Partial 96 GAYW01000269 Partial
48 GAYW01000205° Full, NSP 75 GAYW01000255 Partial 97 GAYW01000270 Partial
49 GAYW01000207 Partial 79 GAYW01000231 Partial 98 GAYW01000271 Partial
50 GAYW01000208° Partial 80 GAYW01000243 Partial 99 GAYW01000272 Partial
52 GAYW01000285 Partial 81 GAYW01000232 Partial 100 GAYW01000273 Partial
53 GAYW01000287 Full, NSP 82 GAYW01000247 Partial 101 GAYW01000275 Partial
55 GAYW01000292° Partial 83 GAYW01000249 Partial 102 GAYW01000277 Partial
56 GAYW01000294 Full, NSP 84 GAYW01000251 Partial 103 GAYW01000279 Partial
57 GAYW01000295 Full, NSP 85 GAYW01000235 Partial 104 GAYW01000280 Partial
58 GAYW01000298 Full, NSP 86 GAYW01000237 Partial 105 GAYW01000281 Partial
59 GAYW01000302 Full, NSP 87 GAYW01000256 Partial 106 GAYW01000282 Partial
60 GAYW01000304 Full, NSP 88 GAYW01000258 Partial 107 GAYW01000283 Partial
61 GAYW01000310 Partial 89 GAYW01000259 Partial 111 GAYW01000225 Partial
62 GAYW01000311 Partial 90 GAYW01000262 Partial 113 GAYW01000240 Partial
64 GAYW01000246 Partial 91 GAYW01000264 Partial 114 GAYW01000230 Partial
68 GAYW01000308° Partial 92 GAYW01000265 Partial 120 GAYW01000253° Partial
70 GAYW01000197 Partial 93 GAYW01000266 Partial 121 GAYW01000257° Partial
71 GAYW01000313 Partial 94 GAYW01000267 Partial 122 GAYW01000239 Partial
Table 1B: AAS sequences found in female ticks
AAS# ID Accession# Comment AAS# ID Accession# Comment AAS# ID Accession# Comment
2 ABS87354 Full, SP 20 GAYW01000324° Full, SP 43 GAYW01000156 Partial
5 ABS87357 Full, SP 22 GAYW01000149° Partial 45 GAYW01000131 Partial
9 ABS87361 Full, SP 26 GAYW01000039° Partial 65 GAYW01000368 Partial
11 ABS87363 Full, NSP 29 GAYW01000365° Partial 69 GAYW01000330 Partial
12 ABS87364 Full, SP 32 GAYW01000130 Partial 76 ^ Full, NSP
13 ABS87365 Full, SP 36 GAYW01000364° Full, NSP 109 GAYW01000172° Partial
14 ABS87366 Full, SP 37 GAYW01000322° Partial 112 GAYW01000397 Partial
15 ABS87367 Full, SP 38 GAYW01000044 Partial 115 GAYW01000125 Partial
16 ABS87368 Full, SP 40 GAYW01000047 Partial 116 GAYW01000070 Partial
17 ABS87369 Full, SP 41 GAYW01000021 Partial 117 GAYW01000067 Partial
19 GAYW01000076° Full, SP 42 GAYW01000022 Partial 118 GAYW01000073 Partial
Table 1C: AAS sequences found in both male and female ticks
AAS# ID Accession# Comment AAS# ID Accession# Comment AAS# ID Accession# Comment
1 ABS87353 Full, SP 24 GAYW01000037° Partial 47 GAYW01000316° Full, NSP
3 ABS87355 Full, SP 25 GAYW01000015° Full, NSP 51 GAYW01000367° Partial
4 ABS87356 Full, SP 27 GAYW01000017° Full, SP 54 GAYW01000363° Partial
6 ABS87358 Full, SP 28 GAYW01000019° Full, NSP 63 GAYW01000286° Partial
7 ABS87359 Full, NSP 30 GAYW01000321° Full, SP 66 GAYW01000309° Partial
8 ABS87360 Full, SP 31 GAYW01000315° Full, SP 67 GAYW01000317° Full, NSP
10 ABS87362 Full, SP 33 GAYW01000184 Partial 72 GAYW01000344° Partial
18 GAYW01000325° Full, NSP 34 GAYW01000293° Full, NSP 108 GAYW01000260° Partial
21 GAYW01000077° Full, SP 35 GAYW01000288° Partial 110 GAYW01000276° Partial
23 GAYW01000078° Full, SP 39 GAYW01000018° Full, SP 119 GAYW01000221° Partial
Table 1D: AAS sequences with no source information
AAS# ID Accession# Comment AAS# ID Accession# Comment
77 ^ Partial 78 Clust1-1-153 Partial

Full = full length open reading frame, SP = signal peptide is present, NSP = no signal peptide;

°

indicates more than one accession number associated with AAS (see Supplemental Table 1);

^

contig sequence was too short for GenBank submission (<200bp), hyperlinked in Supplemental Table 1.

Assignment of AAS identification numbers was done arbitrarily in three steps. In the first step, assembled A. americanum transcriptomes were subjected to batch blastx scanning against tick serpin sequence entries at NCBI. This analysis identified 388 contigs that encoded putative serpins. A typical serpin is characterized by a unique RCL region (Gettins, 2002). Thus in the second step, we conducted a manual inspection of all 388 contigs and found 61 previously unknown AAS RCL sequences that did not show identity to previously described AAS1–17 (Mulenga et al., 2007). AAS sequences encoding the 61 new RCLs were assigned the identifications of AAS18–78, according to the order in which they were discovered. 233 of the 388 contigs did not have RCL regions. These sequences were subjected to intra-contig comparisons using the bl2seq-blastn function at NCBI. This analysis identified 44 contig sequences that encoded previously unreported AAS sequences and were designated as AAS79–122 (Table 1). This brought the total number of unique transcripts identified in this study to 105. A typical serpin molecule is 350–450 amino acids long (Gettins, 2002). On this basis, 40 of the 122 AAS sequences were determined to have complete open reading frames (ORF), as well as the consensus serpin amino-terminus motif (NAVYFKG), and the start methionine. Of the 40 AAS ORFs, 23 are predicted to have signal peptides (Table 1).

Majority of female AAS transcripts expressed in fed salivary glands and midgut tissues

Figures 1A–C summarizes the different AAS sequences found in MG and SG of 48, 96 and 120h fed female ticks. Data previously reported for AAS1–17 are indicated with an asterisk in Figure1A, and data for MG and SG at 120h is taken from Mulenga et. al., (2007). Of the 63 AAS sequences found in female tick transcriptomes to date, 48 have been found in 48, 96, and 120h MG and/or SG. Of these 48, 10 and 12 AAS sequences were respectively found only in MG or in SG, while the remaining 26 were found in both (Figure 1A). This translates to a total of 36 and 38 different AAS sequences found in MG and in SG, respectively. Of the 36 AAS found in MG, seven (AAS1, 4, 7, 8, 21, 31, and 109) were found at all-time points, four (AAS27, 28, 72, and 110) were found at both 48 and 96h time points, and four (AAS19, 20, 22 and 43), one (AAS108), and 16 (AAS9–18, 23, 29, 32, 36, 45, and 115) were found at the 48, 96, or 120h time points, respectively (Figure 1B). Likewise, of the 38 AAS found in SG (Figure 1C), five (AAS7, 19, 21, 23, and 110) were found at all-time points, seven (AAS3, 9, 25, 27, 28, 31, and 39) were found at the 96 and 120h time points, and five (AAS20, 36, 116, 117, and 118), five (AAS24, 26, 37, 38 and 40), and 12 (AAS1, 2, 5, 8, 10–17, 41, and 42) were found in 48, 96, or 120h SG, respectively.

Figure 1. Adult female A. americanum salivary gland (SG) and midgut (MG) expressed serpin (AAS) transcripts.

Figure 1

(A) Total AAS sequences found in MG and SG and both at all tested time points, (B) Apparent temporal and spatial distribution of AAS transcripts found in MG (B), and SG (C). Please note that 17 previously characterized SG and MG expressed AAS transcripts [42] are not included here.

Relative AAS transcript abundance

We successfully mapped reads back to de novo assembled AAS contigs. This analysis identified 20 of 122 AAS transcripts with variable sequence reads in different libraries (Figure 2A and 2B, Supplemental Table3). For 8 (AAS25, 28, 39, 51, 66, 108, 110, and 119) of the 20 AAS sequences, differences between sequence reads were minimal (Supplemental Table 3) and we concluded that these apparently occurred in equivalent abundance in all libraries. Figure 2A summarizes differential abundance for ten (AAS19, 21, 23, 27, 29, 30, 31, 50, 54, 67, 72, 121) in unfed and fed, male and female whole tick libraries. Of ten AAS detected at unfed and fed time points, five are more abundant in unfed ticks (AAS19, 30, 31, 54, and 67), four are more abundant in fed ticks (AAS21, 29, 50, and 121), and one (AAS72) showed a mixed pattern with abundance in unfed females, but not in unfed males, and a transcript increase in fed male ticks (Figure 2A). It is also notable that AAS19, 21, 29, and 30 are abundant in female ticks, while AAS50, 67, and 121 are predominant in male ticks. Of the 31 AAS sequences summarized in Figure 1, we determined differential abundance in SG and MG for five transcripts, AAS19, 21, 23, 27, and 31 (Figure 2B), with differences for the remaining sequences being minimal to negligible (Supplemental Table 3). While AAS23 transcript abundance increases with feeding in both SG and MG, the remaining show an apparent dichotomous pattern. Transcripts for AAS19, 21, 27, and 31 are highest at the 96h time point, and lower at the 120h time point in SG. In MG, AAS19 and 21 are abundant at 48h, and absent or low at 96 and 120h time points, while AAS27 and 31 increase with feeding (Figure 2B).

Figure 2. Relative abundance of A. americanum serpin (AAS) transcripts: Sequence reads were mapped back to de novo assembled contigs in different libraries.

Figure 2

Figure 2

Relative abundance values in: (2A) unfed (empty bars) and fed (black filled bars) male and female ticks, and (2B) dissected 48–120h salivary glands (SG) and midguts (MG) were calculated using the formula described in materials and methods..

A diversity of seventeen amino acid residues is predicted at P1 sites of AAS putative RCLs

Table 2 lists 78 predicted reactive center loops (RCLs) from 27 (Table 2A) and 22 (Table 2B) AAS sequences found in female and male ticks, the 27 found in both (Table 2C), and 2 of an undetermined source (Table 2D). Please note that Table 2A includes previously characterized AAS1–17 (Mulenga et al., 2007). We would like to note that the RCL regions for AAS68 and 71 (Table 2B), and AAS77 (Table 2D) are partial (marked with an asterisk in Table 2), however based on the available sequence we were still able to conclude these RCLs to be unique. Additionally, while the predicted RCL for AAS8 and 9, 4 and 12, and 13 and 15 are identical, these sequences differ in the N-terminus region by 13–25 amino acids (Mulenga et al., 2007). Numbering of amino acid residues in the RCL is based on the standard nomenclature developed by Schechter and Berger (Schechter and Berger, 1967), in which amino acid residues at the N-terminal end of the scissile bond (P1-P1) are not primed and those on the C-terminal end are primed: “"p17 [E]-p16 [E/K/R]-p15 [G]-p14 [T/S]-p13 [X]-p12-9 [AGS]-p8-1 [X]-p1' -4'” (Miura et al., 1995; Gettins, 2002). Molecular analysis predictions of the P1 site assume that there are 17 amino acid residues between the beginning of the RCL hinge region (P17), and the scissile bond (P1-P1’), (Hopkins and Stone, 1995). Based on these conventions, a diversity of 17 different amino acid residues is predicted at the P1 sites of AAS1–78 (except for AAS71 which has a partial RCL excluding the P1 site, Table 2B). The P1 residues for AAS1–78 have the following charge and polarity properties: 27 (~ 35%) are polar uncharged [S (10/26), C (5/26), T (6/26), Q (4/26), Y (2/26)], 25 (~32%) are hydrophobic [L (8/25), I (4/25), P (5/25), G (3/25), M (1/25), A (3/25), V (1/25)], 21 (27%) are polar basic [R (11/21), K (9/21), H (1/21)], and four residues (~ 5%) are polar acidic [D (3/4), E (1/4)] (Figure 3). It is noteworthy that 11 of the 22 AAS sequences found in MG and SG have basic residues at the predicted P1 site.

Table 2.

Reactive Center Loops (RCLs) of A. americanumserpins

Table 2A: Predicted RCLs found in female ticks
AAS ID RCL amino acid sequence AAS ID RCL amino acid sequence
2 EEGTVAAGVTSVRVKPKSFAR 29 EEGTEAAAATAVTVVDGCMPR
5 EEGTVAAAVTGLSVTPLVVPP 32 EDGTEPEVAPTNAVFQPAVRT
9 EEGSPATAVTGVIMYTQSAFV 36 EEGTEAAAATGMRIQLKTRVK
11 EEGTVPTAVPGILLVGLVARH 37 EEGTEAAAATAVVMMCRSAAM
12 EEGTIAAAVTGLSFVPISALH 38 EEGTIATAVTGLSFAPISALH
13 EEGTVAAAVTGLSFPHLVVPP 40 EKGRAAAGVAARAFYTRAGDH
14 EEGTVATAVTGISLVALSALH 41 EEGSEAAGATGVVFVELIAVR
15 EEGTVAAAVTGLSFPHLVVPP 42 EKGTEAAAATAVMMMACCMSA
16 EEGTAAEAVTGLSITPLAVPP 43 EDGTEPEAAATNAVFQPAVRT
17 EEGTVAAAVTGLSSIALSSVG 45 EEGTEAAAATGVVMMCDSLPM
19 EEGSEAAAVTGFVIQLRTAAF 65 EEGSETDSATLMRISGKAXCE
20 EVGTRAVAATEAQFVSKSLVH 69 EEGTIAAAVTGLSFVATASFN
22 EEGSEAAGATGVIFYTKSAIV 76 EEGTIAAAVTGSLFRAHLGSP
26 EEGTEAAAATATVAMFGSAPS
Table 2B: Predicted RCLs Found in male ticks
AAS ID RCL amino acid sequence AAS ID RCL amino acid sequence
44 EEGTEAAAATAIITTECCIMP 59 EEGSWPVTYTEHVLSTGDPVT
46 EEGSEAAGATGVIFVETIAVR 60 ENGSSAAAVTGTTLHKSVHVP
48 EEGTEAAAGSASILTRRDAVE 61 EGGTDASSATAMTSLACSATM
49 EDGTDSEAARANEVPEPAFSI 62 EEGTEPEAATANALIESAGSI
50 EEGSETDSATLMRISGKAAEE 64 ARGGRAVSNEVQSTTSATTAA
52 ENGTVAAAASAAIGVGSAGPS 68 EDGTVAASTAALAFHADRPF*
53 EGGTEPGPATAGEASAPAGPE 70 EEGTIATAVTGLSFVATASFN
55 ENGTVAAAATAAEGGSSSGIM 71 EVGTKA*
56 EEGLGEACRPPNPLPATMIAF 73 GAGGRPPSSNDSREAGTSPAK
57 ENGTGAPAAEGSIYSPAFRRR 74 ERSTSRMPKYTGAQGAPGTSS
58 EEGTEAAAATGMTLMMCGAMV 75 RRGPKTVAAQAVAKEAARTAK
Table 2C: Predicted RCLs found in both male and female ticks
AAS ID RCL amino acid sequence AAS ID RCL amino acid sequence
1 EEGTVAAGVTSVRVKQKHSAR 30 EEGTVATAVTGLSNTRILDDS
3 EEGTGAAGVPSVGGKPKSFAR 31 EEGSEAAAVTGVVINTRTIGG
4 EEGTIAAAVTGLSFVPISALH 33 ENGTVAAAASAALLVGSAGPN
6 EEGTVATAVTGISLALSALHT 34 ENGTKAAAATTALGSNSFYVP
7 EEGTEAAAATGIAMMLMCARF 35 EKGTVAAASAAAAGGSSLYNP
8 EEGSQAAAVTGVIIYTQSAFV 39 EKGTVASASTVAIIVSRIGTP
10 EEGSQAAAVTGVIIYTQSAFV 47 EEGTEAAAATAMPAANSCEMF
18 EEGSEAAGATAVIFFTRGGSS 51 EEGTEAAAATAIIATECCIMP
21 EKGTEAVALSSGIIRHSKTPG 54 EDGVEGLFLTPLIMMCYAGVS
23 EEGTVAAAVTSIRMRMKSSRR 63 EDGNEIARTSALVTEVVSKVA
24 EEGTVATAVTGLSFVATASFN 66 EKGRAAAGVAARTFYTRAGDH
25 EEGTEAAAATAVVMMCYSLPM 67 EEGSEAAAATAVLIETRSDVP
27 EEGTEAAAASGVVGVNRIGID 72 EEPVRLDLHVSTLRLAERTDL
28 EEGTEAAAATGMVAMARCAII
Table 2D: Predicted RCLs, source undetermined
AAS ID RCL amino acid sequence AAS ID RCL amino acid sequence
77 EEGSQATAVTGVIIYTQS* 78 EEGTEAAAATSVVMMCDSLPM

Amino acid residues at putative P1 site are bolded;

*

indicates a partial RCL sequence.

Figure 3. Diversity and counts for amino acid residues at the putative P1 position in reactive center loops of the first 78 A. americanum serpin sequences.

Figure 3

Amino acid residues at putative P1 sites were determined based on molecular analysis predictions of the P1 site that assume that there are 17 amino acid residues between the beginning of the RCL hinge region (P17), and the scissile bond (P1-P1’) [54].

Some AAS sequences are highly identical

To gauge the relationships between AAS sequences, amino acid sequences were subjected to phylogeny analysis, except for AAS87 and 109, which were too short for an informative alignment (Figure 4). We would like to note that due to the large number of sequences, we split the tree into two parts, Figures 4P1 and 4P2. As shown in Figure 4, 68 of the 122 AAS sequences segregated into 19 clusters labeled A-S, and the remaining sequences did not cluster. Of the 19 clusters, eight have more than two sequences: E (AAS8–10, 18, 22, 41, 46, and 67), F (AAS4–6, 11–17, 24, 30, and 112), G (AAS1–3, 23), H (AAS75, 87, and 95), M (AAS32, 43, and 62), O (AAS33, 35, 52 and 79), P (AAS28, 36, 47, and 108), and R (AAS7, 25, 26, 37, 45, and 78), and the remaining 11 clusters have single pairs. When subjected to pairwise sequence alignment analysis, sequences in clusters A, B, D, E, F, G, K, M, O, P, R, and S showed variable amino acid identity levels of 15, 96, 93, 66–98, 61–98, 40–93, 98, 84 (excluding AAS62), 66–98, 56–78, 61–98, and 96%, respectively (Figure 4P1 and 4P2). In the remaining clusters (marked with asterisks), amino acid identity levels were below 15%. We would like to caution here that some sequences being compared are partial, and therefore the picture of these relationships might be incomplete. From pairwise alignment analyses, two general patterns emerged. In the first pattern, differences between two sequences were scattered throughout the alignment (not shown). In the second pattern, found between AAS25 and AAS45, and AAS33 and AAS52, differences were restricted to the C-terminus region within the RCL (not shown).

Figure 4. Phylogeny relationship of A. americanum serpin (AAS) sequences.

Figure 4

Translated AAS amino acid sequences and human antithrombin were aligned using T-coffee in in MacVector version 12. A bootstrap supported phylogeny tree was then constructed with human antithrombin (CAA48690) as the out-group using neighbor-joining method. Clades containing more than 2 AAS sequences are labeled “A” to “T”. Amino acid identity levels within each cluster are indicated. Clusters where amino acid identities were below 15% are marked with an asterisk (*) sign.

Close to half of AAS sequences have orthologs in other tick species

To determine if any AAS sequences in this study had orthologs in other tick species, inter-species comparisons were performed. A search for other tick serpin sequences in publically available databases retrieved 165 serpin sequences across nine tick species (Table 3). Pairwise comparisons of these serpins to AAS1–122 identified 50 AAS sequences that were conserved in other tick species. To manage the high number of sequences, the data are presented in separate tables: A. americanum versus A. maculatum (Karim et al., 2011) and A. variegatum (Ribeiro et al., 2011) (Table 4A), and versus R. pulchellus (direct submission), R. appendiculatus (Mulenga et al. 2003b), R. microplus (Tirloni et al., 2014), The Gene Index Project, http://compbio.dfci.harvard.edu/tgi/], R. haemaphysaloides (direct submission), H. longicornis (Imamura et al., 2005), I. scapularis (GenBank direct submissions, The Gene Index Project), and I. ricinus (Leboulle et al., 2002b) (Table 4B). As shown in Table 4A, AAS4–6, 8–19, 22–27, 29, 30, 41, 46, and 47 show ≥75–96% amino acid identity to A. maculatum serpin sequences AEO35533, AEO35520, AEO34312, AEO34447, AEO34313, AEO34314, AEO32541, AEO34349, AEO34279, AEO34218, AEO34217, AEO33019, and AEO32217, while the remaining sequences showed <75% amino acid conservation. Likewise, in Table 4B, AAS19–22, 41, 42, and 65 show amino acid identities of ≥75–91% to 7 R. pulchellus serpins: JAA54307, JAA54309, JAA543410, JAA54167, JAA54314, JAA54313, respectively, while the rest showed amino acid identities of <75%. Additionally, AAS7 shows 75 and 77% identity to R. microplus EST89704 and R. pulchellus JAA54312, and AAS19 and 21 show 82–96% identity to R. microplus TC17409, TC22658, and EST767976, while AAS41, 42, and 54 show 65, 79 and 90% identity to partially characterized R. appendiculatus AAK61378, and AAK61376, and R. microplus TC16456, respectively. Four AAS (24, 38, 69, and 70) showed 70% or greater identity to the same partially characterized R. appendiculatus AAK61377. Of the 50 AAS sequences conserved in other tick species, only 11 sequences (AAS19–21, 25, 37, 42, 44, 45, 54, 66 and 78) appear to be conserved in prostriate ticks (Table 4B). Except for AAS19, which showed 81 and 82% amino acid identity to I. ricinus ABI94058 and I. scapularis XP_00245308, respectively, all other AAS sequences showed 58–70% conservation with prostriate tick sequences (Table 4B).

Table 3.

Other tick serpins downloaded from publically available databases

Source tick species Accession# Source tick species Accession# Source tick species Accession#
Amblyomma maculatum AEO35533 R. microplus AHC98664 I. scapularis TC36982
AEO35320 AHC98665 TC38196
AEO34447 AHC98666 TC40138
AEO34349 AHC98667 TC40197
AE034314 AHC98668 TC40226
AE034313 AHC98669 TC40411
AE034312 AAP75707 TC40562
AE034279 TC16456_2 TC40843
AE034218 EST767976_2 TC40980
AE032154 EST896705_3 TC41916
AE032160 ADK62395 TC48141
AE032217 ADK62396 TC50198

AE032541 Ixodesricinus ABI94058 XP_002402925
AE032759 JAA66227 EW874987
AE032774 JAA66279 DN974443
AE033019 JAA66964 EW860426



AE034217 JAA67593 XP_002416236

A. variegatum DAA34267 JAA67756 XP_002415307
DAA34257 JAA69032 XP_002407493
DAA34183 JAA71154 XP_002399564
DAA34478 JAA71155 XP_002435393

Haemaphysalislongicornis BAD11156 JAA71156
JAA72548
XP_002434763
EW869079

Rhipicephalushaemaphysaloides AFX65224 gi|310689892 XP_002434444
AFX65225 gi|310689891 XP_002433376

R. appendiculatus AAK61377 JAA71163 XP_002415208
AAK61378 JAA66228 XP_002413438
AAK61376 JAA73759 XP_002416263
AAK61375 JAA72989 XP_002411933

R. pulchellus JAA63611 JAA72940 XP_002411931
JAA63258 JAA72595 XP_002402370

JAA54387 EW949864 XP_002411045
JAA54315 I. scapularis EW837780 XP_002401187
JAA54314 EW881762 XP_002400954
JAA54313 EW901007 XP_002403236
JAA54312 EW814209 XP_002401986



JAA54311 EW860426 XP_002415891
JAA54310 EW882018 XP_002415890
JAA54309 ISCW017295 XP_002415888
JAA54308 ISCW011017 XP_002415887
JAA54307 ISCW024435 XP_002415886
JAA54306 ISCW006062 XP_002415308
JAA54167 ISCW023208 XP_002416681
JAA53966 ISCW024387 XP_002416635

R. microplus AHC98652 ISCW023617 XP_002408111
AHC98653 ISCW024109 XP_002416150
AHC98654 ISCW023621 XP_002415892
AHC98655 ISCW010422 XP_002411932
AHC98656 EW851734 XP_002402368
AHC98657 EW959899 XP_002399745
AHC98658 EW955724 XP_002405749
AHC98659 AAM93649 ABJB011030283
AHC98660 AAV80788 ABJB010229324
AHC98661 ACI446630 ABJB011108013
AHC98662 TC36450

AHC98663 TC54356

Table 4.

A. americanum serpins (AAS) that have orthologs in other Amblyomma spp ticks
AAS
ID
Other tick best match %
ID
AAS
ID
Other tick best match %
ID
1 Amac-AEO32759 71 25 Amac-AEO35533 81

2 Amac-AEO32759 71 Amac-AEO35520 76

3 Amac-AEO32759 70 Amac-AEO32774 70

4 Amac-AEO34312 79 Amac-AEO32541 63
Amac-AEO34279 89 Amac-AEO32217 64

5 Amac-AEO34312 75 Amac-AEO32160 67
Amac-AEO34279 77 Amac-AEO32154 70


6 Amac-AEO34312 75 26 Amac-AEO35533 64
Amac-AEO34279 82 Amac-AEO35520 64

7 Amac-AEO35533 87 Amac-AEO32774 62
Amac-AEO35520 86 Amac-AEO32541 77
Amac-AEO32774 72 Amac-AEO32217 79
Amac-AEO32541 65 Amac-AEO32160 62
Amac-AEO32217 66 Amac-AEO32154 61

Amac-AEO32160 70 27 Amac-AEO34349 81

Amac-AEO32154 71 28 Amac-AEO34314 62


8 Amac-AEO34447 82 29 Amac-AEO35533 62
Amac-AEO34313 64 Amac-AEO35520 59
Amac-AEO33019 71 Amac-AEO32774 67

9 Amac-AEO34447 79 Amac-AEO32160 61
Amac-AEO34313 62 Amac-AEO32154 65

Amac-AEO33019 69 30 Amac-AEO34312 76

10 Amac-AEO34447 82 Amac-AEO34279 76

Amac-AEO34313 64 36 Amac-AEO34314 59

Amac-AEO33019 72 Amac-AEO35533 67

11 Amac-AEO34447 63 37 Amac-AEO35520 65
Amac-AEO34313 69 Amac-AEO32774 71

12 Amac-AEO34447 78 Amac-AEO32541 70


Amac-AEO34313 89 Amac-AEO32217 73

13 Amac-AEO34447 74 Amac-AEO32160 68
Amac-AEO34313 78 Amac-AEO32154 71


14 Amac-AEO34447 74 41 Avar-DAA34478 73
Amac-AEO34313 80
15 Amac-AEO34447 73 46 Avar-DAA34478 73
Amac-AEO34313 77 Amac-AEO34447 69

Amac-AEO34313 76
Amac-AEO33019 75 Amac-AEO33019 75

16 Amac-AEO34447 72
Amac-AEO34313 75 47 Amac-AEO34314 79


17 Amac-AEO34447 75 67 Amac-AEO34313 70
Amac-AEO34313 79 Amac-AEO33019 74


18 Amac-AEO34447 68
Amac-AEO34313 76

19 Amac-AEO32217 91
Amac-AEO34218 91

22 Avar-DAA34478 71
Amac-AEO34447 69
Amac-AEO34313 74
Amac-AEO33019 86

23 Amac-AEO32759 66

24 Amac-AEO34312 77
Amac-AEO34279 86
B: AAS amino acid identity to serpins in metastriata and prostriata ticks
AAS ID Other tick best match % ID AAS ID Other tick best match % ID AAS ID Other tick best match %ID
1 Rhaem-AFX65224 61 25 45 Rpulc-JAA54306 66
Rapp-AAK61377 69 Rapp-AAK61377 66 Rpulc-JAA54314 71
Rmic-AAP75707 68 Rapp-AAK61375 62 Rpulc-JAA54313 71
Rmic-TC16466 73 Rmic-TC24850 66 Rpulc-JAA54312 68

Rmic-EST89704 69 Ir-XP_02399745 61
4 Rhaem-AFX65224 59 Rpulc-JAA54306 70 Ir-XP_002408111 60
Rapp-AAK61377 66 Rpulc-JAA54314 63
Rmic-AAP75707 65 Rpulc-JAA54313 66 46 Rapp-AAK61378 64
Rmic-TC16466 67 Rpulc-JAA54312 71 Rpulc-JAA54312 73
Hlong-BAD11156 73 Rpulc-JAA54311 64

Ir-CAB55818.2 61 47 Rpulc-JAA63611 59
6 Rapp-AAK61377 68 Is-XP_002434444 59
Rmic-AAP75707 66 Ir-00245308 70 50 Rpulc-JAA54309 76
Rmic-TC16466 69 Ir-EW874987 70
Hlong-BAD11156 69 54 Rmic-TC16466
Is-XP_002401187
90
58



65 Rpulc-JAA54309 80

Rhaem-AFX65224 74 Rmic-TC24850 61
Rmic-TC24850 69 Rmic-EST89704 61 66 Rpulc-JAA54310
Is-XP_002415891
70
59
Rmic-EST89704 75 Rpulc-JAA54306 61
7 Rpulc-JAA54306 72 26 Rpulc-JAA54314 66 67 Rpulc-JAA54310 68
Rpulc-JAA54314 69 Rpulc-JAA54313 63
Rpulc-JAA54313 69 Rpulc-JAA54312 62 Rapp-AAK61377 70
Rpulc-JAA54312 77 Rpulc-JAA54311 66 69 Rmic-AAP75707 70
Hlong-BAD11156 74 Hlong-BAD11156 68


Rmic-TC24850 60 Rapp-AAK61377 71

8 Rpulc-JAA54310 68 29 Rmic-EST89704 62 70 Rmic-AAP75707 71

9 Rpulc-JAA54310 66 Rpulc-JAA54310 74 Hlong-BAD11156 73



10 Rpulc-JAA54310 66 30 Rapp-AAK61377 66 78 Rpulc-JAA54306 65



11 Rmic-TC16466
Hlong-BAD11156
62
62
Rmic-AAP75707
Rmic-TC16466
66
68
Rpulc-JAA54313
Rpulc-JAA54312
61
70

Hlong-BAD11156 70 Rpulc-JAA54314 63
12 Rmic-TC16466
Hlong-BAD11156
72
74

Rmic-AHC98652 62

36 Rpulc-JAA63611 69 Rmic-AHC98653 68
13 Rmic-TC16466 67 Rpuc-JAA62387 68 Rmic-AHC98662 61


14 Rmic-TC16466 70 Rapp-AAK61376 61
15 Rmic-TC16466 66 Rmic-EST89704 65 Rhaem-AFX65225 68

Rpulc-JAA54306 63 Is-XP_00240811 61
16 Rmic-TC16466 66

Rpulc-JAA54306 60 Is-XP_002399745 61
17 Rmic-TC16466 69 Rpulc-JAA54314 67 Ir-JAA66228 61

18 Rpulc-JAA54310 72 37 Rpulc-JAA54313 64 Ir-JAA72595 61

Rpulc-JAA54312 63 Ir-JAA66227 61
Rmic-TC22658 95 Rpulc-JAA54311 67
Rmic-EST67697 82 Ir-CAB55818.2 60
19 Rpulc-JAA54307 87 Is-ACI46630 61

Ir-ABI94058 81 114 Rpulc-JAA54310 75
Is-XP_00245308 82

Rapp-AAK61377 71 Rpulc-JAA63611 67

38 Rmic-AAP75707 70 121 Rpulc-JAA62387 67
Rpulc-JAA54310 79
20 Is-XP_002401986 58 39 Ir-XP_002401986 68


Rmic-TC17409 96 40 Rpulc-JAA54310 67

Rpulc-JAA63258 91 Rapp-AAK61378 65
21 Ir-JAB72483 67 41 Rpulc-JAA54310 76

Is-XP_002401986 70

Rapp-AAK61376 79
22 Rapp-AAK61378 61 Rpulc-JAA54314 79

23 Rhaem-AFX65224 71 42 Rpulc-JAA54313 77
Rpulc-JAA54310 75 Is-XP_002403236 61

Is-XP_002407493 61
Rapp-AAK61377 71
24 Rmic-AAP75707 71 44 Rpulc-JAA54314 59
Rmic-TC16466 73 Ir-XP_02399745 58

Amac = A. maculatum, Avar = A. variegatum, % amino acid identities are bed

Rapp = R. appendiculatus, Rmic = R. microplus, Rhaem = R. haemaphysaloides, Rpulc = R. pulchellus, Ir = I. ricinus, Is = I. scapularis, and Hlong = H. longicornis. % amino acid identities are bolded

Table 5 summarizes the 29 AAS RCL sequences which show at least 62% sequence conservation in other tick species. Preliminary manual inspection of RCLs showed high identities between sequences where the predicted P1 site is of the same charge and polarity, therefore all 212 tick serpin sequences for which an RCL could be determined were first divided into one of four groups: polar uncharged, polar basic, polar acidic, and hydrophobic. RCL sequences (Figure 5A–D) were then subjected to phylogeny analysis (not shown). Lastly, RCLs from all tick species were subjected to pairwise sequence alignments. Identity levels for the 29 highly conserved AAS RCLs, (AAS4, 7, 12, 14, 18–23, 25–29, 31, 37, 38, 42, 44, 45, 47, 50, 52, 54, 58, 65, 69, and 70) ranged from approximately 62–100% (Table 5). Of these 29 RCLs, seven (AAS7, 19–21, 23, 27, and 42) are conserved in both metastriate and prostriate ticks, with the remaining being conserved only in metastriate ticks. Seven AAS RCLs (AAS18, 20–22, 27, 42, and 50) showed 95–100% conservation with at least one RCL from another species. For AAS25, RCL identity to its partially characterized ortholog in R. appendiculatus (AAK61375), was higher, at 81% identity, than for whole sequence identity, at 62%. Three AAS RCLs (AAS7, 19, and 20) showed >80% identity to at least one RCL from a prostriate species. The identities between remaining RCL sequences ranged between ~62–76% (Table 5). It is notable that the RCL for AAS19 is 100% identical to serpin RCLs across several tick species in multiple genera, and including both metastriate and prostriate ticks. Of the 29 conserved AAS RCLs, 38% (11/29) (AAS18–20, 22, 23, 27, 28, 31, 37, 50, 65) have basic amino acid residues, 35% (10/29) (AAS21, 25, 42, 44, 47, 52, 54, 58, 69, and 70) have polar uncharged residues, and 24% (7/29) (AAS4, 7, 12, 14, 26, 29, 38, and 42) have hydrophobic residues, while only one sequence, AAS45, has a polar acidic residue at the putative P1 site. It is interesting to note that for the 10 most highly inter-species conserved RCL sequences (those which are ≥90%), the majority (7/10) have basic P1 residues, while the remaining three have polar uncharged P1 residues.

Table 5.

Cross-tick species conserved A. americanum serpin reactive center loops

Serpin ID Conserved RCL amino acid
sequence
*%
ID
Serpin ID Conserved RCL amino acid
sequence
*%
ID
AAS4* EEGTIAAAVTGLSFVPISALH - AAS26 EEGTEAAAATATVAMFGSAPS -
Amac-AE034279 EEGTIATAVTGLSFVALSALS 81 Amac-AE032541 EEGTEAAAATAVIMVFGSASS 76
Rapp-AAK61377 EEGTIATAVTGLGFVPLSAHY 76 Amac-AE032217 EEGTEAAAATAVIMLFGSASS 76

Rmic-AAP75707 EEGTIATAVTGLGFVPLSVHY 71 AAS27 EEGTEAAAASGVVGVNRIGID -
Rmic-AHC98654 EEGTIATAVTGLGFVPLSAHY 71 Is-EW860426 EEGTEAAAASGVVAVNRLIGV 66
Rmic-AHC98661 EEGTVAAAVTGLFVRPTAPLP 71 Is-XP_002411045 EEGTEAAAASGVVMTRRAIEG 75
Rmic-AHC98655 EEGTIAAAVTGLFVMPSSSLY 81 Amac-AE033019 EEGTQAAAASGVVGVNRIGIE 90
Hlong-BAD11156 EEGTVATAVTGISFIPLSAHY 67 Rpulc-JAA54308 EEGTEAAAVSGVVSVNRIGIE 86

AAS7 EEGTEAAAATGIAMMLMCARF - Rmic-AHC98657 EEGTEAAAVTGVIGVNRIGIE 81
Amac-AE035320 EEGTEAAAATGMAFMLLSARF 81 AAS28 EEGTEAAAATGMVAMARCAII -
Ir-JAA66227 EEGTEAAAATAIPIMLMCARF 86 Rmic-AHC98669 EEGTEAAAATGMVAMARCASM 90

Ir-JAA72595 EEGTEAAAATAVPVMFCCAIF 66 AAS29 EEGTEAAAATAVTVVDGCMPR -
Rhaem-AFX65225 EEGTEAAAATAITMMTYCARF 81 Rmic-AHC98662 EEGTEAAAATAVMMVACCMSS 71

Rapp-AAK61376 EEGTEAAAATAITMMTYCARF 81 AAS31 EEGSEAAAVTGVVINTRTIGG -
Rpulc-JAA54312 EEGTEAAAATAITMMTYCARF 81 Rmic-AHC98667 EEGSEAAAVTGVTINTRTTTG 81

AAS12 EEGTIAAAVTGLSFVPISALH - Rmic-AHC98657 EEGTEAAAVTGVIGVNRIGIE 71

Amac-AE034279 EEGTIATAVTGLSFVALSALS 81 AAS37 EEGTEAAAATAVVMMCRSAAM -
Rapp-AAK61377 EEGTIATAVTGLGFVPLSAHY 71 Rpul-JAA54314 EEGTEAAAATAVMMMACCMSS 66
Rmic-AAP75707 EEGTIATAVTGLGFVPLSVHY 71 Rmic-AHC98662 EEGTEAAAATAVMMVACCMSS 66
Rmic-AHC98654 EEGTIATAVTGLGFVPLSAHY 71 Rmic-AHC98669 EEGTEAAAATGMVAMARCASM 71

Rmic-AHC98661 EEGTVAAAVTGLFVRPTAPLP 71 AAS38 EEGTIATAVTGLSFAPISALH -
Rmic-AHC98655 EEGTIAAAVTGLFVMPSSSLY 81 Amac-AE034279 EEGTIATAVTGLSFVALSALS 86
Hlong-BAD11156 EEGTVATAVTGISFIPLSAHY 67 Rapp-AAK61377 EEGTIATAVTGLGFVPLSAHY 81

AAS14 EEGTVATAVTGISLVALSALH - Rmic-AHC98654 EEGTIATAVTGLGFVPLSAHY 71

Amac-AE034279 EEGTIATAVTGLSFVALSALS 81 AAS42 EKGTEAAAATAVMMMACCMSA -
Rapp-AAK61377 EEGTIATAVTGLGFVPLSAHY 67 Rpulc-JAA54314 EEGTEAAAATAVMMMACCMSS 90
Rmic-AAP75707 EEGTIATAVTGLGFVPLSVHY 62 Amac-AE032774.1 EKGTEAAAATAVMMVACCLSI 86
Hlong-BAD11156 EEGTVATAVTGISFIPLSAHY Amac-AE032154.1 EKGTEAAAATAVMMVGCCLSI 81

AAS18 EEGSEAAGATAVIFFTRGGSS - Rmic-AHC98662 EEGTEAAAATAVMMVACCMSS 86
Amac-AE034313 EEGSEAAGATAVIFFTRGGIP 90 Rmic-AHC98652 EEGTEAAAATAVMMAACCLSS 81
Avar-DAA34478 EEGSEAAGATAVIFFTRGGAP 90 Rapp-AAK61375 EEGTEAAAATAVMMAACCLSS 81
Rmic-AHC98668 EEGTIAAAVTGLFVMPSSSLY 71 Is-XP_002405749 EEGTEAAAATAMVMLCCMSFP 76

AAS19 EEGSEAAAVTGFVIQLRTAAF - AAS44 EEGTEAAAATAIITTECCIMP -
Rpulc-JAA54307 EEGSEAAAVTGFVIQLRTAAF 100 Rmic-AHC98662 EEGTEAAAATAVMMVACCMSS 66

Amac-AE034218 EEGSEAAAVTGFVIQLRTAAF 100 AAS45 EEGTEAAAATGVVMMCDSLPM -
Amac-AE034217 EEGSEAAAVTGFVIQLRTAAF 100 Rmic-AHC98669 EEGTEAAAATGMVAMARCASM 71

Ir-JAA72548 EEGSEAAAVTGFVIQLRTAAF 100 AAS47 EEGTEAAAATAMPAANSCEMF -
Ir-ABI94058 EEGSEAAAVTGFVIQLRTAAF 100 Amac-AE034314 EEGTEAAAATAMLASNSCERF 86

Is-XP_002415308 EEGSEAAAVTGFVIQLRTAAF 100 AAS50 EEGSETDSATLMRISGKAAEE -
Rmic-TC22658 EEGSEAAAVTGFVIQLRTAAF 100 Rpulc-JAA54309 EEGSEADSATLLRISGKAAEE 90

AAS20 EVGTRAVAATEAQFVSKSLVH - Rmic-AHC98660 EEGSEADSATLLRISGKAAEE 90

Rpulc-JAA54167 EVGTRAVAATQAQFVSKSLVH 95 AAS52 ENGTVAAAASAAIGVGSAGPS -
Is-ISCW016489 EEGTRAVAATQAQFVSKSLVQ 90 Rpulc-JAA54315 EEGTEAAAATAAVGAGSAGPS 76

Rmic-AHC98664 EVGTRAVAATQAQFVSKSLVH 95 AAS54 EDGVEGLFLTPLIMMCYAGVS -

AAS21 EKGTEAVALSSGIIRHSKTPG - Rmic-AHC98663 EDGVEGLFLTPLIMMCYAGVS 100

Rpulc-JAA63258 EKGTEAVALSSGIIRHSKTPG 100 AAS58 EEGTEAAAATGMTLMMCGAMV -
Rmic-AHC98658 EKGTEAVALSSGIVRHSKTPG 95 Rmic-AHC98669 EEGTEAAAATGMVAMARCASM 71

Is-XP_002401986 EQGTEAVALSSGIVRHSRPPE 95 AAS65 EEGSETDSATLMRISGKA-CE -
Ir-JAB72483 EQGTEAVALSSGIVRHSRPPE 90 Rpulc-JAA54309 EEGSEADSATLLRISGKAAEE 81
Rmic-AHC98658 EKGTEAVALSSGIVRHSKTPG 95 Rmic-AHC98660 EEGSEADSATLLRISGKAAEE 88


AAS22 EEGSEAAGATGVIFYTKSAIV - AAS69 EEGTIAAAVTGLSFVATASFN -
Amac-AE034349 EEGSEAAGATGVIFYTKSMVV 90 Rmic-AHC98661 EEGTVAAAVTGLFVRPTAPLP 71

Rpulc-JAA54315 EEGSEAAAATAVIFYTKSAAV 86 AAS70 EEGTIATAVTGLSFVATASFN -
Rmic-AHC98668 EEGTIAAAVTGLFVMPSSSLY 90 Rmic-AHC98654 EEGTIATAVTGLGFVPLSAHY 66


AAS23 EEGTVAAAVTSIRMRMKSSRR -
Is-XP_002434763 EKGTVAAAVTSISMRMGSSLA 76

AAS25 EEGTEAAAATAVVMMCYSLPM -
Rmic-AHC98653 EEGTEAAAATAVTLMTYCARI 66
Rmic-AHC98662 EEGTEAAAATAVMMVACCMSS 66

Amac = A. maculatum, Is = I. scapularis, Ir = I. ricinus, Rapp= R. appendiculatus, Rmic = R. microplus, Rpulc = R. pulchellus, Rhaem = R. haemaphyssaloides, Avar = A. variegatum, Hlong= H. longicornis. Amino acid residues at P1 sites are bed.

Figure 5. Multiple sequence alignment of tick serpin reactive center loops (RCL).

Figure 5

Predicted RCLs in serpins found in this study and other tick serpins downloaded from GenBank were subjected to multiple sequence alignment using T-coffee in MacVector vrsion 12. Asterisk (*) sign denotes predicted amino acid residues at P1 sites: (A) polar acidic, (B) polar basic, (C) hydrophobic, and (D) polar uncharged. Identical amino acid residues are shaded gray. AAS = Amblyomma americanum serpins, Amac = A. maculatum, Avar = A. variegatum, Rmic = Rhipicephalus microplus, Rpulc = R. pulchellus, Rhaem= R. haemaphysalis, Rapp = R. appendiculatus, Hlong = Haemaphysalis longicornis, Is = Ixodes scapularis, Ir = I. ricinus.

DISCUSSION

This study provides an update of unique serpin-coding sequences expressed in unfed and fed A. americanum male and female ticks. While this is the first report of a very large number of serpin transcripts from male ticks, data presented here are not unusual. High numbers of serpin sequences were reported in ticks I. scapularis (Mulenga et al., 2009), R. pulchellus (direct submission), and A. maculatum (Karim et al., 2011), in mosquitoes Anopheles gambiae, Culex quinquefasciatus, and Aedes aegypti (Rawlings et al., 2012), in Bombyx mori (Zou et al., 2009), Tribolium (Zou et al., 2007), Drosophila (Reichhart, 2005), in mouse and human genomes (Puente and López-Otín, 2004; Gatto et al., 2013; Heit et al., 2013), and in Arabidopsis and Oryza sativa (Fluhr et al., 2012). Such large serpin counts across a great diversity of taxa indicate the importance of this protein family in regulating homeostasis in most branches of life. While serpin counts in other tick species do not approach the number for A. americanum as reported here, the discrepancy is likely due to the fact that this is the first analysis of tick transcriptomes of its scope with both male and female tissues at various time points having been explored.

Although based on the design of this study we are unable to conclude expression patterns, the observation of a greater diversity of AAS transcripts in male than female ticks is interesting. We speculate that the high diversity of AAS transcripts found in male ticks could be explained by the timing of reproductive physiological changes. Male metastriate tick species such as A. americanum feed for only a short time prior to completion of spermatogenesis and mating (Kiszewski et al., 2001). On the other hand, many female reproductive activities including vitellogenin synthesis and deposition into oocytes, ovulation, fertilization, and oviposition do not occur until after several days of feeding (Kiszewski et al., 2001; Sonenshine and Roe, 2013). The majority of AAS which were found exclusively in males in this study, were from male ticks fed for at least three days and were potentially ready to mate, while female ticks in this study were fed up to 120h with reproductive activities just beginning. As a result, it is possible that there are additional serpins important for female reproductive physiology, that are expressed at later feeding time points than those identified in this study. Although empirical data will be needed, it’s interesting to note that AAS50 and 121 both found in male ticks were abundant after feeding when the male tick has entered its physiologically reproductive state. It will be interesting to investigate expression of serpins in the immature stages, where there is a lack of sexual dimorphism. This will inform on which serpins are indeed involved in sex-specific physiology, and which are involved in other feeding-related physiology such as host-defense modulation. Evidence in insects, Drosophila and Aedes aegypti, indicated that serpins are among the male reproductive gland proteins transferred to females during mating (Coleman et al., 1995; Sirot et al., 2008). Although there are distinct differences between biology of ticks and insects, it is plausible that some male A. americanum serpins found in this study could serve as reproductive proteins.

The SG and MG represent two major organs through which the tick interacts with its host and with pathogens. Thus, the identification of 31 previously unknown AAS transcripts in SG and MG is interesting. Although relative expression analysis done in this study is limited, the expression patterns of AAS21 and AAS27 are notable. Based on relative abundance determined in this study AAS21 abundance is 1,000-fold higher at 48h MG compared to SG, while AAS27 increases 500 fold at 120h in MG more than SG. It will be interesting to investigate role(s) of AAS21 and AAS27 in tick feeding. Another important goal of this study was to identify A. americanum tick serpins that are expressed in both male and female metastriata ticks. We believe that these could represent those that are important to tick feeding regulation. Despite obvious differences in their biology, both male and female ticks must interact with host defense mechanisms before mating. Indeed, there is evidence that like females, male ticks express anti-inflammatory molecules such as histamine-binding proteins (Paesen et al., 1999; Bior et al., 2002), which are potentially involved in facilitating male tick feeding. Thus, the 16 AAS transcripts found in both male and female ticks, and in 48–120h SG and MG could represent those that are important to tick feeding success.

The high number of AAS sequences in this study could be explained by gene duplication and exon shuffling in the RCL region as suggested by high amino acid identity among some AAS sequences that clustered together on the phylogeny tree and those that showed differences restricted to the RCL region. Serpin diversity by gene duplication and subsequent divergence has been reported in a number of organisms including humans (Heit et al., 2013), mice (Borriello and Krauter, 1990; Hancock, 2005) and B. mori (Zou et al., 2009). In An. gambiae serpin genes clustering phylogenetically were found in clusters on the same chromosome, indicating that they could be duplicated genes (Suwanchaichinda and Kanost, 2009). Similarly, in I. scapularis 11 highly identical serpins were found on the same supercontig (Mulenga et al, 2009). The observation of differences restricted to the RCL has been observed in transcripts that are products of alternatively spliced exons. This phenomenon was reported in Manduca sexta (Jiang and Kanost, 1997), in B. mori (Zou et al., 2009), and in Ctenocephalides felis (Brandt et al., 2004). We also observed a very curious pattern between AAS4 and 12, and AAS13 and 15, in which RCLs were identical with differences restricted to outside of the RCL (Mulenga et al., 2007). Whether or not these observations are consistent with events in vivo requires further investigation. However, it is interesting to note that in this study we observed a similar pattern between serpin sequences in other tick species: A. maculatum AEO34217 and AEO34218, and R. pulchellus JAA62387 and JAA63611, where the RCL is the same and differences in sequences are outside of the RCL region. We speculate that if consistent with events in vivo, AAS transcripts that share the same RCL sequence could function as redundant proteins, or could regulate the same protease under different spatio-temporal conditions.

Within the RCL region, the amino acid at the P1 site is considered most important in determining the target protease(s) that is/are regulated by a candidate serpin (Gettins, 2002; Huntington, 2006; Huntington, 2011). Accordingly, the observation that 17 different amino acid residues were predicted at P1 sites of putative RCLs identified in this study indicates the potential diversity of proteases that may be regulated by these serpins. Our analysis of AAS RCL P1 sites showed a near-even distribution of residues across charge/polarity types with the exception of polar acidic similar to M. sexta, B. mori (Zou et al., 2009), plants (Roberts et al., 2004; Roberts and Hejgaard, 2008), and humans (Cassar and Hunter, 2013) where polar acidic P1 residues in serpins appear to be rare. Prediction of the P1 site amino acid residue is not in itself sufficient to determine the protease that may be regulated by a candidate serpin; empirical evidence is required. However, there is ample evidence that serpins with basic residues at the P1 site regulate trypsin and tryspin-like proteases (Gettins, 2002, Li et al., 1999, Leboulle et al., 2002a; Leboulle et al., 2002b; Prevot et al., 2006). Thus it is interesting that the most highly conserved AAS RCLs in this study have basic P1 residues. From the perspective of tick feeding biology, serpins with basic P1 residues could represent those that ticks use to evade trypsin-like protease-mediated host defense pathways such as blood clotting. We would like to caution the reader here that in both plants and in non-hematophagous organisms such as Drosophila, the majority of serpins have basic P1 amino acid residues (Reichhart, 2005; Fluhr et al., 2012). It is also interesting to note that two tick anti-coagulant serpins, HLS2 (orthologous to AAS12), in H. longicornis (BAD11156) (Imamura et. a., 2005), and Iris in I. ricinus (AJ269658) (Prevot et. al., 2006), contain hydrophobic residues at their P1 sites. Further experiments are therefore required in order to determine proteases/pathways that may be regulated by the serpins described in this study.

In nature, different tick species may infest the same animal host and implicitly, these different tick species will face the same host defense mechanisms. It is conceivable that different tick species could utilize conserved proteins to interact with the same host, such as the 50 cross-tick species conserved AAS sequences identified in this study. It is notable that only ~22% (11/50) were conserved in both metastriate and prostriate tick species. These could be particularly interesting candidates for further functional studies given that in general, amino acid identities between metastriate and prostriate tick proteins sequences are low. In particular, AAS25, which was detected in fed males and females, and in SG, is orthologous to Iris, found in SG and saliva of I. ricinus. Iris was demonstrated to inhibit lymphocyte proliferation, as well as the immune system cytokines IFN-γ and IL-6 (Leboulle et. al., 2002a; Leboulle et. al., 2002b). Similarly, Highly conserved AAS proteins represent very interesting candidates for development of universal anti-tick vaccines as advocated (Maritz-Olivier et al., 2007).

The RCL is important to serpin function (Gettins, 2002), and thus it is interesting to note that some AAS RCLs are 90–100% conserved in other tick species. These serpins could be involved in regulating target proteases that are important to all ticks. Based on our inter-species comparative sequence analysis, it is interesting to note that AAS sequences with polar basic P1 sites, followed by polar uncharged P1 sites, were likely to be conserved in other tick species. It is noteworthy that hydrophobic P1 residues, though prevalent, tended to show little conservation in other tick species. This suggests that proteases that can interact with hydrophobic Pl residues may be involved in species-specific physiology, and might be diverging at a fast rate. Although the present study is descriptive, it contributes significantly to the picture of serpin transcript diversity expressed by male and female A. americanum ticks. Additionally, based on the data in this analysis, the picture of serpin transcript diversity in other tick species is likely to be far from complete.

Supplementary Material

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ACKNOWLEDGEMENTS

This research was supported by National Institute Health grants (AI081093, AI093858, AI074789, AI074789-01A1S1) to AM. Authors would like to thank tick labs at Texas A&M and Oklahoma State universities for supplying ticks used in this research.

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