The CD11a partner in Sus scrofa lymphocyte function-associated antigen-1 (LFA-1): mRNA cloning, structure analysis and comparison with mammalian homologues
© Vanden Bergh et al; licensee BioMed Central Ltd. 2005
Received: 06 July 2005
Accepted: 10 October 2005
Published: 10 October 2005
Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2), the most abundant and widely expressed beta2-integrin, is required for many cellular adhesive interactions during the immune response. Many studies have shown that LFA-1 is centrally involved in the pathogenesis of several diseases caused by Repeats-in-toxin (RTX) -producing bacteria.
The porcine-LFA-1 CD11a (alpha) subunit coding sequence was cloned, sequenced and compared with the available mammalian homologues in this study. Despite some focal differences, it shares all the main characteristics of these latter. Interestingly, as in sheep and humans, an allelic variant with a triplet insertion resulting in an additional Gln-744 was consistently identified, which suggests an allelic polymorphism that might be biologically relevant.
Together with the pig CD18-encoding cDNA, which has been available for a long time, the sequence data provided here will allow the successful expression of porcine CD11a, thus giving the first opportunity to express the Sus scrofa beta2-integrin LFA-1 in vitro as a tool to examine the specificities of inflammation in the porcine species.
Integrins constitute a large family of adhesion molecules with important roles in cell-extracellular matrix and cell-cell interactions which condition both the maintenance of tissue integrity and the promotion of cellular migration. They are heterodimeric membrane glycoproteins composed of non-covalently associated single-pass transmembrane α and β subunits, which are expressed on a wide range of cells . The biggest part of each integrin subunit is extracellular while transmembrane region and cytoplasmic tail are typically reduced. The N-terminal domains of the α and β subunits associate to form the integrin headpiece, which contains the ligand binding site. The C-terminal segments traverse the plasma membrane and mediate interactions with the cytoskeleton and with signalling proteins [2, 3].
Among the integrins, the leukocyte-specific β2-integrins (CD11/CD18) include four members: (i) CD11a/CD18 (LFA-1, αLβ2) on all leukocytes ; (ii) CD11b/CD18 (Mac-1, CR3, αMβ2) mainly on myeloid cells ; (iii) CD11c/CD18 (gp150/95, CR4, αXβ2, Leu-M5) and (iv) CD11d/CD18 (αDβ2) on monocytes and macrophages . The individuals lacking functional β2 integrins due to mutations in the β2 (CD18) subunit develop the LAD (lymphocyte adhesion deficiency) I syndrome characterized by repeated infections. This disease demonstrated that β2 integrins are of relevant importance in (i) leukocyte development and maturation, (ii) naïve cells circulation in secondary lymphoid tissues and (iii) leukocytes transendothelial migration to injured tissue [5–7].
Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, αLβ2), the most abundant and widespread in expression β2-integrin, binds to the membrane proteins termed intercellular adhesion molecules ICAM-1 to ICAM-5 [4, 8–12]. Several studies have shown that LFA-1 is centrally involved in the pathogenesis of diseases caused by Repeats-in-toxin (RTX) -producing bacteria. The virulence of both Actinobacillus actinomycetemcomitans (stomatitis in humans) and Mannheimia haemolytica (pneumonia in cattle) is clearly associated with the ligand-receptor interactions between their respective leukotoxin and CD11a/CD18, which triggers the synthesis and release of a wide array of cytokines and chemoattractants that exacerbate inflammation, and ultimately results in massive leukolysis [13, 14]. As Actinobacillus pleuropneumoniae, the main causative agent of pneumonia in pigs, also produces toxins of the RTX family (Apx toxins) , it is tempting to hypothesize that the pathogenesis of the disease similarly rely on an interaction with the porcine LFA-1. On a more practical point of view, increasing our knowledge about this putative interaction could help the pig industry in controlling the economical losses and antibiotics abuses that are currently associated with A. pleuropneumoniae pneumonia . The Sus scrofa CD18 (β2-) subunit has been well characterized , which is not the case of its partner in the LFA-1 heterodimer, CD11a. The purpose of this study is to report the cloning, sequencing and analysis of a cDNA encoding porcine CD11a, thus giving the first opportunity to produce recombinant LFA-1 for studies focused on interactions between Apx toxins and swine LFA-1.
Results and discussion
Characterization of PoCD11a-encoding cDNA and deduced amino-acid sequence
Among the seven positive clones sequenced, two presented a supplementary "cag" codon (2230–2232) that codes for a glutamine (Gln, Q) in position 744 [GenBank:DQ013284]. This addition is located in the extracellular domain of PoCD11a, outside of the I-domain and divalent cation-binding motifs and, according to the GORIV bioinformatic program, increased the length of an α-helix. The Gln-744 addition was also observed in the human [GenBank:NM_002209, GenBank:AY892236] and ovine  CD11a cDNAs. The Gln addition could thus have a biological importance for the mature CD11a. Studies of genomic sequences will permit to know if this addition represents two alleles or if it is generated by an alternative splicing.
Comparison among species
Between-species percent identities of CD11a constitutive blocks. Po, Hu, Mu, Bo and Ov : porcine, human, murine, bovine and ovine CD11a, respectively ; MIDAS: metal-ion dependent adhesion site ; vs : versus.
Po vs. Hu (%)
Po vs. Mu (%)
Po vs. Bo (%)
Po vs.Ov (%)
Putative signal peptide
Putative cation binding motif 1
Putative cation binding motif 2
Putative cation binding motif 3
Every cysteine residue in the mature porcine CD11a is present at the same location in human, murine, bovine and ovine CD11a, which is consistent with a role in maintaining the global structure of the protein. The mouse version distinguishes by an additional cysteine residue at position 199 within the extracellular portion. Of six potential Asn-glycosylation sites in porcine CD11a, the ones present at amino acids 186, 668, 724 and 860 are strictly conserved. Without predictable consequences on a functional point of view, one glycosylation site is only absent from porcine and murine CD11a (residue 897 of human sequence). The mouse sequence shows additional glycosylation sites at position 270 and 776. Furthermore, the porcine Asn-Xaa-Ser/Thr sites in position 87 and 728 are also found in human and murine homologues but not specially in the ruminant sequences, and the ovine sequence owns two supplementary sites at position 646 and 1000.
This study reports for the first time the isolation and sequencing of the porcine LFA-1 αL subunit (CD11a) cDNA, and demonstrates that, despite some focal differences, it shares all the main characteristics of its known mammalian homologues. Along with the porcine CD18-encoding cDNA which is available , the sequence data provided here allow the successful cloning of PoCD11a, thus giving the first opportunity to express porcine LFA-1 in vitro as a tool to examine the specificities of inflammation in the porcine species.
Total RNA from spleen of a freshly slaughtered pig (Sus scrofa domestica) was extracted with TRIzol (Invitrogen, USA) as described by the manufacturer.
Amplification of cDNA ends
We used SMART RACE technology (Clontech Laboratories Inc., USA) to obtain porcine CD11a (PoCD11a) 5'- and 3'- ends and RT-PCR to amplify full-length PoCD11a CDS. For first strand cDNA synthesis, and according to the bovine CD11a sequence available [GenBank:AY267467], gene-specific primers were designed which were expected to give nonoverlapping ~1,5 kb rapid amplification of cDNA ends (RACE) products : a sense primer for the 3'-RACE PCR 5'-tgcaatgtragctctcccatcttc-3' (corresponding to nt 2575–2598) and an antisense primer for the 5'-RACE PCR 5'-aagatgtacacrgccccctgctcctcca-3' (nt 1628–1655). Reverse transcription and polymerase chain reactions (PCR) were carried out according to the instruction manual of the SMART RACE cDNA Amplification Kit. The 5'- and 3'-RACE products were gel-purified using the Qiaquick Gel Extraction Kit (Qiagen), TA-cloned into pCRII-TOPO (Invitrogen, USA) and seeded on kanamycin IPTG plates. Minipreps were obtained from colonies grown in 5 ml LB-Kan broth and the clones were sequenced on the ABI-3100 Genetic Analyzer using the Big Dye terminator chemistry (Applied Biosystems, USA).
Molecular cloning of full-length cDNA
Total RNA from spleen cells was reverse transcribed using Improm II (Promega, USA). The full-length cDNA was then generated by long distance PCR using elongase amplification technology (Invitrogen, USA) with primers designed from the proximal part of 5'- and the distal part of 3'-RACE products: 5'-ggtatggtccctccagaagc-3' (forward) and 5'-tcaggcctgggcttcagtcg-3' (reverse). The following cycling parameters were applied: 5 min at 94°C, then 35 cycles including: (i) 30 s at 94°C, (ii) 45 s at 58°C, and (iii) 3 min 30 s at 68°C, followed by a final extension at 68°C for 10 min. Resulting PCR products were then processed for sequencing as aforementioned for the RACE products. The CD11a cDNA sequence was deduced from sequences obtained from seven independent clones. Sequence data have been deposited at GenBank under accession nos. DQ013285 and DQ013284.
Primers design was performed with Netprimer  and Primer 3 . Nucleotidic sequence and identity analyses were carried out using respectively Chromas v.2.21  and BLAST programs . Alignment of amino acids sequences was drawn by GeneDoc v.2.6.002  following the blosum 62 matrix. SignalP v.2.0.b2  and NetNGlyc v.1.0  provided peptide signal and N-glycosylation sites prediction, respectively. The secondary structures were resolved by the GOR secondary structure prediction method version IV .
List of abbreviations
cluster of differenciation
divalent-cation binding motif
intercellular adhesion molecule
lymphocyte function-associated antigen
metal-ion dependent adhesion site
rapid amplification of cDNA ends
Authors are grateful to Prof M. Georges for giving free access to all the facilities of the laboratory of molecular genetics. Philippe Vanden Bergh is the recipient of a studentship from the "Fonds pour la formation à la Recherche dans l'Industrie et l'Agriculture", rue d'Egmont 5, B-1000 Bruxelles. Thomas Fett and Laurent Zecchinon are supported by the belgian federal services for public health and security of the food chain and environment, grant S-6107. We thank Hélène Vandegaart for its technical assistance.
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