Characterization of a gene coding for the complement system component FB from loxosceles laeta spider venom glands

The human complement system is composed of more than 30 proteins and many of these have conserved domains that allow tracing the phylogenetic evolution. The complement system seems to be initiated with the appearance of C3 and factor B (FB), the only components found in some protostomes and cnidarians, suggesting that the alternative pathway is the most ancient. Here, we present the characterization of an arachnid homologue of the human complement component FB from the spider Loxosceles laeta. This homologue, named Lox-FB, was identified from a total RNA L. laeta spider venom gland library and was amplified using RACE-PCR techniques and specific primers. Analysis of the deduced amino acid sequence and the domain structure showed significant similarity to the vertebrate and invertebrate FB/C2 family proteins. Lox-FB has a classical domain organization composed of a control complement protein domain (CCP), a von Willebrand Factor domain (vWFA), and a serine protease domain (SP). The amino acids involved in Mg2+ metal ion dependent adhesion site (MIDAS) found in the vWFA domain in the vertebrate C2/FB proteins are well conserved; however, the classic catalytic triad present in the serine protease domain is not conserved in Lox-FB. Similarity and phylogenetic analyses indicated that Lox-FB shares a major identity (43%) and has a close evolutionary relationship with the third isoform of FB-like protein (FB-3) from the jumping spider Hasarius adansoni belonging to the Family Salcitidae

Characterization and phylogenetic analysis of genes coding for the components C3 and factor B of Complement System from Loxosceles spiders venom glands.

O sistema complemento parece ter surgido com o aparecimento de C3 e fator B (FB), os únicos componentes encontrados nos organismos mais primitivos, o que sugere que a via alternativa seja a mais antiga. Fragmentos de cDNA codificantes para FB (Lox-FB) e C3 (Lox-C3) foram sintetizados a partir do RNA total isolado da glândula de veneno de Loxosceles laeta e amplificados por técnicas de RACE-PCR. Lox-FB apresenta uma organização de domínios clássica, composta por domínios CCP, vWFA e de serino protease. Os aminoácidos envolvidos na ligação ao C3b são conservados, no entanto, a tríade catalítica clássica não foi encontrada. Lox-C3 apresenta uma configuração de domínios similar a do C3 humano, contendo dois sítios putativos de processamento: um entre as cadeias α e γ, e outro entre as cadeias α e β, indicando que Lox-C3 seja composto por três cadeias. As análises filogenéticas indicaram que Lox-C3 e Lox-FB são mais próximos evolutivamente aos componentes equivalentes da aranha Hasarius adansoni, com valores de identidade de 53% e 43%, respectivamente.The complement system seems to have arisen with the appearance of C3 and factor B (FB), the only components found in the most primitive organisms, suggesting that the alternative pathway is the oldest. cDNA fragments coding for the complement factor B (Lox-FB) and C3 (Lox-C3) were synthesized from total RNA Loxosceles laeta venom gland and amplified using RACE-PCR techniques. Lox-FB has a classical domain organization in mosaic, composed by CCPs, vWFA and serine protease domains. The amino acids involved in binding to C3b are conserved, however, the classical calatytic triad was not found. Lox-C3 presents a similar configuration of domains to C3 human and has two putative processing sites: the first one is located between α and γ chains and other between α and β chains, indicating that Lox-C3 is composed by three chains. The phylogenetic analyses indicated that Lox-C3 and Lox-FB are evolutionary closer to the equivalent components of Hasarius adansoni, with identity values of 53% and 43%, respectively

Alignment of Hu-FB and Lox-FB CCPs.

<p>The five CCPs are aligned to each other with the consensus amino acids shown in bold and at the bottom. Two disulfide bonds sustain the CCP domain and are formed between the first and third cysteine, and the second and the fourth cysteine. The alignment was done with CLUSTAL W using Bioedit v. 7.0.9.0 software.</p

cDNA sequence and deduced amino acid sequence of Lox-FB.

<p>The ORF predicts a protein of 651 amino acids with domains of known C2 and Bf proteins. The signal peptide is in italics and comprised of the initial 25 amino acids. The cysteines and the putative N-glycosylated sites are marked in black and grey, respectively.</p

Molecular model of Lox-FB.

<p>Construction of molecular model based on structure of human factor B (PDB 2ok5.1), using the tool SWISS-Model Workspace available on <a href="http://swissmodel.expasy.org/workspace/" target="_blank">http://swissmodel.expasy.org/workspace/</a>. The further analyses were performed using the software SwissPDBViewer.</p

Multiple alignment of the vWFA and serine protease domains from Lox-FB with sequences of FB/C2 proteins from other organisms.

<p>The alignment was performed using MUSCLE algorithm available in MEGA software. Amino acids that are highlighted in bold indicate identical regions. The amino acids residues that are functionally important at Factor D or C1s cleavage site; the metal ion dependent binding site (MIDAS) and the protease active sites are indicated by dark arrows; the three amino acids residues (T⁴³¹, A⁴³³, S⁶¹⁶) that are important on stabilization of catalytic triad are indicated by sign #; the conserved cysteines residues are indicated by asterisks and the two extra cysteines present in Lox-FB, Hd-FB3 and Rd-FB are highlighted in grey. <i>Loxosceles laeta</i> (Lox-FB), <i>Hasarius adansoni</i> (Hd-FB1; HD-FB2; Hd-FB3), <i>Tachypleus tridentatus</i> (Tt-FB1;Tt-FB-2), <i>Ruditapes decussatus</i> (Rd-FB), <i>Nematostella vectensis</i> (Nv-FB), <i>Branchiostoma belcheri</i> (Bb-FB), <i>Strongylocentrotus purpuratus</i> (Sp-FB), <i>Apostichopus japonicus</i> (Aj-FB), <i>Homo sapiens</i> (Hu-C2;Hu-FB).</p

Domain structure of Lox-FB deduced protein using Basic Local Alignment Tool (BLAST).

<p>Domain structure of Lox-FB deduced protein using Basic Local Alignment Tool (BLAST).</p

Overlap of human factor B (Hu-FB) (pink) and the <i>Loxosceles</i> factor B-like (Lox-FB) (grey).

<p>[A] vWA Domain [B] SP Domain [C] amino acids residues that comprise the catalytic triad of Hu-FB (H, D, S) and Lox-FB (S, N, P). The manipulation of models were performed using the software SwissPDBViewer.</p