*5.2. Bioinformatics Analysis on Pedicted Dromaserpin Sequence*

The sialotranscriptome of the *H. dromedarii* tick was recently described and annotated [41]. Among the deposited sequences, a cDNA sequence that codes for Dromaserpin was selected for expression. The nucleotide sequence was translated into an amino acid sequence using a TransDecoder, confirmed later with Expasy translate (https://web.expasy. org/translate/ (accessed on 1 April 2020)), and the predicted sequence was used for the following in silico analysis. A homologous search of the full-length Dromaserpin sequence was performed using BLAST programs (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 5 October 2021)), through which the conserved domain was also identified. Localization of the identified RCL domain was done by SMART software (http://smart.embl-heidelberg.

de/ (accessed on 1 April 2020)). Later, we performed a comparison between the RCL amino acid sequence in Dromaserpin and the RCL sequence of the inhibitory serpins α-1 antitrypsin (P01009) and Antithrombin-III (P01008), and a non-inhibitory serpin Ovalbumin (P01012), available in Uniprot. Full-length amino acid sequence was submitted to the signal 4.1 server (http://www.cbs.dtu.dk/services/SignalP-4.1/ (accessed on 1 April 2020)) and the signal peptide was removed from the protein sequence for subsequent analysis. The predicted molecular weight (MW) and isoelectric point (pI) were determined using ProtParam tools from ExPASy server (https://web.expasy.org/protparam/ (accessed on 1 April 2020)). To determine potential N- or O-linked glycosylation sites, Dromaserpin amino acid was scanned using NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/ (accessed on 1 April 2020)) and NetOGlyc 4.0 servers (http://www.cbs.dtu.dk/services/NetOGlyc/ (accessed on 1 April 2020)). For phylogenetic analysis, protein sequences of 25 serpins from tick species and one human serpin were retrieved from GenBank. Accession numbers of these sequences, the percentage of identity and coverage with Dromaserpin are described in Supplementary Table S1. Multiple sequence alignments were performed using ClustalW algorithm (https://www.genome.jp/tools-bin/clustalw (accessed on 5 October 2021)) and sequences were edited manually. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [69]. Human Antithrombin III was utilized as outgroup to root the tree. The initial tree was obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances, estimated using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA7 [33].
