Molecular characterisation of the caprine (Capra hircus) lymphocyte function-associated antigen-1 alpha subunit-encoding cDNA

RNA isolation

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.

Bioinformatics

Primers design was performed with Netprimer [18] and Primer 3 [19]. Nucleotidic sequence and similarity analyses were carried out using respectively Chromas v.2.21 [20] and BLAST programs [21]. Alignment of amino acids sequences were drawn by GeneDoc v.2.6.002 [22] following the BLOSUM62 matrix. SignalP v.2.0.b2 [23] and NetNGlyc v.1.0 [24] provided peptide signal and N-glycosylation sites prediction, respectively.

Characterisation of CaCD11a-encoding cDNA and deduced aa sequence

Two alleles have been identified for the CaCD11a cDNA. The sequence contains ~4200 bp with an ORF of 3498 [Genbank: AY773019] or 3495 bp [Genbank: AY773018] depending on the allele that codes for 1165 or 1164 aa followed by ~600 bp in the 3'-UTR (Fig. 1). The mature CaCD11a contains a 23-aa putative leader peptide, an extracellular domain of 1061 or 1062 residues (24-1084/1085), a single hydrophobic transmembrane region of 24 residues (1085/1086–1108/1109) and a cytoplasmic tail of 57 residues (Fig. 1). Nine N-linked putative glycosylation sites (Asn-X-Thr/Ser) are present in the extracellular domain (Fig. 2). The mature protein contains 19 cysteine residues among which one is located into the cytoplasmic tail (Fig. 2). The extracellular domain also contains an inserted (I) domain of 172 amino acids (residues 153–324) quite similar to those found in all the leukocyte integrin α subunits sequenced to date and located between the β sheets 2 and 3 of a seven bladed β-propeller region [25]. The I-domain is homologous with repeated domains found in von Willebrand factor and cartilage matrix protein [1] and can be expressed as an isolated domain. Its three-dimensional structure consists of a five-stranded parallel β-sheet core surrounded on both faces by α-helices, with a short antiparallel strand occurring on one edge of this sheet [26]. The I-domain contains a metal ion-dependent adhesion site (MIDAS) [27] (residues 159–163, 228, 261) (Fig. 2) and an I-domain allosteric site (IDAS) that plays a functional role in ICAM-1 binding [28–30]. Three repeats with a divalent cation binding motif are found at amino acid residues 465–473, 527–535 and 587–595 (Fig. 2). All the conserved cysteines and all but one N-glycosylation sites are found outside the I region and divalent cation binding motifs (Fig. 2), consistent with the hypothesis that these regions may undergo conformational changes important in ligand binding [31, 32].

General comparison among species

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 [34] 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.