Molecular characterisation of the caprine (Capra hircus) lymphocyte function-associated antigen-1 alpha subunit-encoding cDNA
© Fett et al; licensee BioMed Central Ltd. 2005
Received: 04 July 2005
Accepted: 10 October 2005
Published: 10 October 2005
Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alpha L beta 2) is required for many cellular adhesive interactions during the immune response.
The Capra hircus CD11a-encoding cDNA was sequenced and compared with its human, murine, rat, bovine and ovine counterparts. Despite some focal differences, it shares all the main characteristics of its known mammalian homologues.
Therefore, along with the caprine CD18-encoding cDNA, which has been available for a few months, the sequence data revealed here will allow the Capra hircus LFA-1 expression in vitro as a tool to explore the specificities of inflammation in the caprine species.
Lymphocyte function-associated antigen-1 (LFA-1, αLβ2, CD11a/CD18) is a member of the β2-integrin subfamily of cell surface receptors. Integrins consist of a 120 to 180 kDa α subunit (CD11a in this case) and a 90 to 110 kDa β subunit that are noncovalently associated single-pass transmembrane proteins . The bulk of each integrin subunit is extracellular, where it typically functions as a receptor for extracellular matrix molecules or as a counterreceptor for surface proteins of apposed cells . The heterodimer CD11a/CD18 is expressed on all leukocytes and mediates high affinity adhesion to a variety of cell types that express one or more of the β2-integrins ligands, intercellular adhesion molecules (ICAM-1 to -5) [3–5]. The adhesion process mediated is a critical step of a wide range of immunological activities, including cytolysis of target cells, cross-interaction and cross-stimulation between lymphocytes, phagocytosis of complement-coated targets, neutrophils clearance from inflamed tissue, and the regulation of leukocyte traffic between the bloodstream and tissues [6–9]. As the relevance of the goat model for studying leukocyte traffic, diapedesis and pathologic tissue infiltration is well established in such important areas as mastitis [10–13] or lentivirus-associated diseases [14–16], increasing our knowledge about caprine β2 integrins is of great importance to offer new possibilities for research and to provide additional insights into those fields. Along with the caprine CD18-encoding cDNA, which is available for a few months , the sequence data provided here will allow the Capra hircus β2-integrin CD11a/CD18 expression in vitro as a tool to examine the specificities of inflammation in the caprine species.
Total RNA from phorbol myristate acetate (PMA)-stimulated (25 ng/ml for 15 min) caprine (Boer breed) peripheral blood mononuclear cells (PBMC) was extracted with TRIzol (Invitrogen, USA) as described by the manufacturer. The PBMCs were obtained by density gradient centrifugation with Ficoll-Paque Plus (Amersham, USA) and maintained in RPMI 1640 supplemented with 10% foetal bovine serum (Gibco BRL, USA), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C in a 5% CO2 atmosphere.
Amplification of cDNA ends
We used SMART RACE technology (Clontech Laboratories Inc., USA) to obtain caprine CD11a (CaCD11a) 5'- and 3'-ends and RT-PCR to amplify full-length CaCD11a CDS. For first-strand cDNA synthesis, and according to the sequence of bovine CD11a available [GenBank: AY267467], gene-specific primers were designed which were expected to give non overlapping ~1 kb RACE products: a sense primer for the 3'-RACE PCR : 5'-TGCAATGTRAGCTCTCCCATCTTC-3' (corresponding to nt 2572 to nt 2595) and an antisense primer for the 5'-RACE PCR : 5'-CCGGCCTCCTCTCTGCTCCCCATAG-3' (nt 1470 to nt 1446). 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 S.N.A.P.™ Gel Purification Kit (Invitrogen, USA), TA-cloned into pCRII-TOPO (Invitrogen, USA) and seeded on kanamycin IPTG plates. Miniprep were obtained from colonies grown in 5 ml LB-kanamycin broth and the clones were sequenced on the ABI-3730 Genetic Analyzer using the Big Dye terminator chemistry (Applied Biosystems, USA).
Molecular cloning of full-length cDNA
Total RNA from PMA-stimulated PBMCs was reverse transcribed using Improm II (Promega, USA). The full-length cDNA was then generated by long distance PCR using Platinum Taq DNA polymerase High Fidelity (Invitrogen, USA) with primers designed from the distal ends of both 5'- and 3'-RACE products : 5'-GTCGCCAGTAAATCCCAAGA-3' (sense, within the 5'-UTR) and 5'-GCACCTCAATCTCCACCACT-3' (antisense, 3'UTR). The procedures recommended by the manufacturer were followed, with these cycling parameters : 5 min at 94°C, then 35 cycles including (i) 30 s at 94°C, (ii) 30 s at 58°C and (iii) 3 min 30 s at 68°C, followed by a final extension at 68°C for 5 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 nine independent clones. Sequence data have been deposited at GenBank under accession No. AY773018 and AY773019.
Primers design was performed with Netprimer  and Primer 3 . Nucleotidic sequence and similarity analyses were carried out using respectively Chromas v.2.21  and BLAST programs . Alignment of amino acids sequences were drawn by GeneDoc v.2.6.002  following the BLOSUM62 matrix. SignalP v.2.0.b2  and NetNGlyc v.1.0  provided peptide signal and N-glycosylation sites prediction, respectively.
Results & discussion
Characterisation of CaCD11a-encoding cDNA and deduced aa sequence
General comparison among species
Between-species percent identities of CD11a constitutive blocks. Ca, Bo, Ov, Hu, Mu and Ra: caprine, bovine, ovine, human, murine and rat CD11a, respectively; MIDAS: metal-ion dependent adhesion site.
Ca vs. Bo (%)
Ca vs. Ov (%)
Ca vs. Hu (%)
Ca vs. Mu (%)
Ca vs. Ra (%)
Putative signal peptide
Putative cation binding motif 1
Putative cation binding motif 2
Putative cation binding motif 3
Every cysteine residue in the caprine extracellular portion of mature CD11a is present at the same location in bovine, ovine, human, murine and rat CD11a, which is consistent with a role in maintaining the global structure of the protein whereas two cysteine residues (positions 1009 and 1048) are absent from caprine CD11a and therefore do not seem to be indispensable. The mouse version distinguishes by an additional cysteine residue at position 199 (mouse numbering) within the extracellular portion. Of nine potential Asn-glycosylation sites in caprine CD11a, the ones present at amino acids 185, 667, 723 and 859 are strictly conserved, one is only absent from murine and rat CD11a (residue 894), without predictable consequences on a functional point of view.
Interestingly, as in sheep  and human [GenBank: NM_002209 and AY892236], an allelic variant with a triplet insertion resulting in an additional Glu744 in the extracellular domain was consistently identified, which suggests an allelic polymorphism that might be biologically relevant. Studies of genomic sequences will permit to know if this addition represents two alleles or not.
Finally, one has to note that the lowest between-species percent identities are observed with the rat CD11a sequence which has been derived from an annotated genomic sequence. Cloning and characterisation of rat CD11a from rat PBMCs would probably give a higher identity.
This study reports for the first time the isolation and sequencing of the caprine LFA-1 α subunit (CD11a) cDNA, and demonstrates that, despite some focal differences, it shares all the main characteristics of its known mammalian homologues. Along with the caprine CD18-encoding cDNA which is now available , the sequence data provided here will allow the successful expression of caprine LFA-1 in vitro as a tool to examine the specificities of inflammation in the caprine species.
This study is supported by the Belgian federal services for public health and security of the food chain and environment, grant S-6107. The authors are grateful to Prof M. Georges for giving free access to all the facilities of the laboratory of molecular genetics and to Prof. J.-F. Beckers for the caprine PBMCs.
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