Sequence-based appraisal of the genes encoding neck and carbohydrate recognition domain of conglutinin in blackbuck (Antilope cervicapra) and goat (Capra hircus)

Abstract

Conglutinin, a collagenous C-type lectin, acts as soluble pattern recognition receptor (PRR) in recognition of pathogens. In the present study, genes encoding neck and carbohydrate recognition domain (NCRD) of conglutinin in goat and blackbuck were amplified, cloned, and sequenced. The obtained 488 bp ORFs encoding NCRD were submitted to NCBI with accession numbers KC505182 and KC505183. Both nucleotide and predicted amino acid sequences were analysed with sequences of other ruminants retrieved from NCBI GenBank using DNAstar and Megalign5.2 software. Sequence analysis revealed maximum similarity of blackbuck sequence with wild ruminants like nilgai and buffalo, whereas goat sequence displayed maximum similarity with sheep sequence at both nucleotide and amino acid level. Phylogenetic analysis further indicated clear divergence of wild ruminants from the domestic ruminants in separate clusters. The predicted secondary structures of NCRD protein in goat and blackbuck using SWISSMODEL ProtParam online software were found to possess 6 beta-sheets and 3 alpha-helices which are identical to the result obtained in case of sheep, cattle, buffalo, and nilgai. However, quaternary structure in goat, sheep, and cattle was found to differ from that of buffalo, nilgai, and blackbuck, suggesting a probable variation in the efficiency of antimicrobial activity among wild and domestic ruminants.

Figure 1

PCR amplification of cDNA encoding conglutinin NCR domain (Lane M: 100 bp plus DNA ladder, (a) Lane 1: goat PCR amplicon of ~500 bp, and (b) Lane 1: blackbuck PCR amplicon of ~500 bp).

Figure 2

Restriction digestion of recombinant plasmids by Pst I restriction digestion (Lane M: 100 bp plus DNA ladder, (a) insert release of 220 bp, 406 bp, and 2.8 kb from goat recombinant plasmids (pJET-GCGN) Lane 1-2, and (b) insert release of 261 bp, 566 bp, and 2.6 kb from blackbuck recombinant plasmids (pJET-BBCGN) Lane 1-2).

Figure 3

Alignment of nucleotide sequences of goat and blackbuck conglutinin NCRD with sequences of other ruminants (cattle, buffalo, sheep, and nilgai) and collectins (cattle collectin-43, cattle collectin-46, cattle surfactant protein-D, recombinant human surfactant protein-D (rhSP-D), and recombinant rat surfactant protein-A (rrSP-A)). Identity of the sequences is indicated by dots and the differences by the corresponding nucleotide symbols. Gaps introduced for optimal alignment are indicated by dashes. The GenBank accessions are given in parentheses.

Figure 4

Alignment of predicted amino acid sequences of goat and blackbuck conglutinin NCRD with other wild and domestic ruminants (cattle, buffalo, sheep, and nilgai) and collectins (cattle collectin-43, cattle collectin-46, cattle surfactant protein-D, recombinant human surfactant protein D (rhSP-D), and recombinant rat surfactant protein A (rrSP-A)). Identity of the sequences is indicated by dots and the differences by corresponding one letter symbol of amino acids. Gaps introduced for optimal alignment are indicated by dashes. Four conserved cysteine residues are indicated by vertical (green) boxes. Predicted regions of secondary structures are indicated by horizontal (blue) boxes (A1-alpha helical region-1, A2-alpha helical region-2, A3-alpha helical region-3, B1-beta sheet region-1, B2-beta sheet region-2, B3-beta sheet region-3, B4-beta sheet region-4, B5-beta sheet region-5, and B6-beta sheet region-6). Three regions, collagen triplet, coiled neck, and lectin domain, are distinctly demarcated by the sequences confined within the red arrow marks. The signature sequences within lectin domain are indicated by red horizontal box.

Figure 5

Phylogenetic relationship between the predicted amino acid sequences for the NCR domain of conglutinin from different species (cattle, buffalo, nilgai, blackbuck, sheep, and goat) and collectins (cattle collectin-43, cattle collectin-46, cattle surfactant protein-D, recombinant human surfactant protein D (rhSP-D), and recombinant rat surfactant protein A (rrSP-A)) using Mega version 5.1 Clustal W method. Numbers outside the branches indicate the bootstrap values obtained using 1,000 replicates and values above 50% are shown. The scale bar at the bottom measures the amino acid substitutions per site for a unit branch length.

Figure 6

Phyre2 software predicted secondary structure from partial conglutinin amino acid sequences, (a) blackbuck and (b) goat.

Figure 7

(a) SWISS MODEL predicted structure of NCRD region of conglutinin of various wild and domestic ruminant species (cattle, buffalo, nilgai, blackbuck, sheep, and goat). (b) SWISS MODEL predicted and PDB modeled structure of SP-D.