QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online August 31, 2007
Journal of Experimental Biology 210, 3188-3198 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.006494

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mulenga, A. Articles by Blandon, M. A. Search for Related Content PubMed PubMed Citation Articles by Mulenga, A. Articles by Blandon, M. A.

Molecular and expression analysis of a family of the Amblyomma americanum tick Lospins

Albert Mulenga^*, Rabuesak Khumthong and Maria A. Blandon

Department of Entomology, College of Agriculture and Life Sciences, Texas A & M University, TAMU 2475, College Station, TX 77843, USA

View larger version (28K): [in this window] [in a new window]   Fig. 1. Conservation of core serpin superfamily residues (Irving et al., 2000) in deduced Lospin proteins. The serpin superfamily archetype, 1-antitrypsin (1-ATT, accession no. AAB59495) was aligned with deduced Lospin (L) proteins using Vector NTI (Invitrogen) and conserved residues were manually aligned. L1–L17=Lospins 1–17. The minus sign (–) denotes conservation of the specific amino acid residues. Replaced residues are indicated. Numbering is based on 1-ATT. E%C denotes the expected % conservation at that position (Irving et al., 2000).

View larger version (25K): [in this window] [in a new window]   Fig. 2. (A) Predicted Lospin reactive center loops (RCL). Lospin RCLs were determined based on the eight-residue pattern (B) that characterizes inhibitory serpins (Hopkins et al., 1993). The residues in the RCL are numbered according to the standard nomenclature (Schechter and Berger, 1967), where residues on the amino-terminal side of the scissile (p1–p1') are not primed and those on the carboxy-terminal side are primed. Assuming that there are 17 residues between the base of RCL hinge and the scissile bond (Hopkins et al., 1993), bold-faced residues are predicted p1 residues with the broken arrow indicating the position of the scissile bond. L1–L17=Lospins 1–17.

View larger version (97K): [in this window] [in a new window]   Fig. 3. Structure-based sequence alignment of Lospins with native antithrombin (1AZX, chain I). Structure-based pairwise alignments between Lospins deduced and 1AZX amino acid sequences retrieved from the protein data bank (PDB), were done using Expresso (Armougom et al., 2006). Due to limitations on space, representative alignments only are shown here. Secondary structures were assigned based on 1AZXi (PDB). `H', -helix; `E', beta-strand. Helices are labeled from `hA' to `hI', ß-strands that constitute ß-sheet A are labeled as `sA', `sB' for ß-sheet B and `sC' for ß-sheet C. Highly conserved residues that correspond to the 51 core residues shown in Fig. 1 are indicated by an asterisk (*). Please note that s3c and s1B, s2C and s6A as well as s4B and s5B are merged and continuous, to denote boundaries; s1B, s6A and s5B are underlined.

View larger version (94K): [in this window] [in a new window]   Fig. 4. Comparative modeling and calculation of surface electrostatic potential for L5 and L13–16. Structure-based alignments from Fig. 3 were used as input in the MODELLER version 9v1 (Sali and Blundell, 1993) to develop Lospin models, which were subsequently verified as described in Materials and methods. Electrostatic potential of the template, 1AXZi (`A', native antithombin), 1BY7 (`B', plasminogen activator inhibitor-2, negative control) and Lospin models were calculated as described in Materials and methods and visualized using PyMol 0.99rev10 [(DeLano, 2002) www.pymol.org] at ±5kt/e of positive and negative contour fields. Structures D, E and F denote Lospins 5, 13 and 16 models. Basic patches are indicated and marked by a solid circle. C=Lospin 7 (control, has no basic residues on the -helix D). Structures are shown back to front.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Neighbor-joining guide phylogeny tree showing the relationship between 17 deduced Lospin polypeptides and 15 serpin sequences of other ticks. Deduced Lospin amino acid sequences were aligned with R. appendiculatus (Ras-1–4), H. longicornis (Hl) serpin, I. ricinus (Ir) serpin 1, 2, 4 and Iris=immunosuppressive Ixodes serpin, I. scapularis (Is) serpin and B. microplus (Bm) serpin 1–6. Note that, except for Bmserpin-5, which is annotated in GenBank, the other Bmserpins were obtained as ESTs from the TIGR database and translated in this study. Branch labels GA–GE, represent major groups of serpin sequences branching off from the outlier, the serpin superfamily archetype, 1-antitrypsin. *Accession numbers for Lospin sequences provided in the Results.

View larger version (44K): [in this window] [in a new window]   Fig. 6. (A) Transcription profiles and (B) normalized PCR band densities representing relative mRNA abundance of 15 selected Lospins. Total RNA extracted from salivary glands (SG), midgut (MG), ovary (OV) and carcass (CA, remnant of the tick following removal of SG, OV and MG) dissected from 5-day fed A. americanum ticks were subjected to semi-quantitative RT-PCR using gene specific primers shown in Table 1. The 16 s rRNA PCR fragment amplified from A. americanum 16 s rRNA primers (Table 1) was used as the endogenous control. PCR band densities were determined using ImageJ software (http://rsb.info.nih.gov/ij). Determined densities were normalized using the following formula: Y=V+V(H–X)/X, where Y=normalized mRNA density, V=observed Lospin PCR band density in individual tissues (MG, SG and OV), H=highest 16 s rRNA PCR band density among tested tissues (carcass in this case, CA), X=tissue (MG, SG and OV) 16 s rRNA PCR band density. (B) Normalized band densities were plotted as percent tissue distributions.