Figure 1.
Identification of the EchAMP transcript.
Relative abundance of the EchAMP transcript in echidna milk cells as compared to other major milk proteins. A novel sequence was found to be one of the highest expressed transcripts as determined by cDNA sequencing of echidna milk cells [15]; the corresponding gene was named as EchAMP (GenBank Accession no. KC148542).
Figure 2.
Expression profile of EchAMP in different echidna tissues.
Expression of EchAMP relative to GAPDH as determined by Reverse- Transcriptase PCR in different echidna tissues. Abundant expression of EchAMP was seen in milk cells while a low level of expression was detected in intestine, liver, testes and penis.
Figure 3.
Multiple alignment of signal peptides of monotreme caseins and EchAMP protein.
The EchAMP signal peptide shares two identical (*), three conserved (:) and two semi-conserved amino acids (.) with other monotreme casein signal peptides. E: Echidna; P: Platypus; CSN1: α-casein; CSN2: β-casein; CSN3: -casein. Colors indicate the physicochemical properties of residues. Red: Small+Hydrophobic; Magenta: Basic; Green: Hydroxyl+Sulfhydryl+Amine.
Table1. GRAVY of EchAMP protein.
Figure 4.
Kyte and Doolittle hydropathicity plot for EchAMP protein.
Amino acid position is presented on the X-axis. Kyte and Doolittle hydropathicity scores (window size n = 7) for individual amino acids are on the Y-axis. The N-terminal region of EchAMP protein is hydrophobic while the central and C-terminal portions are hydrophilic. A steep increase in hydrophilicity is observed around the signal peptide cleavage site of the protein.
Figure 5.
Identification of EchAMP protein in echidna milk.
A1, A2 and B1, B2 represent milk samples collected from two lactating echidnas at two different time points during their late-lactation phase. 70 µg protein of each sample was electrophoresed for 3 hours at 100V using a 12% SDS-Polyacrylamide gel. Bands E3 and E4 were excised from the gel, subjected to in-gel trypsin digestion and anlaysed by LTQ Orbitrap Velos. The spectra of peptides from these bands showed a significant match with the predicted EchAMP protein with high confidence levels.
Figure 6.
Deleage-Roux apha-helicity plot for EchAMP protein.
The amino acid positions are indicated on the X-axis. The alpha-helicity scores are indicated on the Y- axis. The cut-off score was taken as 0.99. The EchAMP protein has significant alpha helical structure in the N-terminal region, followed by a steep decrease in the region spanning the amino acids 20–23. A second dip in alpha helicity is seen between the amino acids 55–65.
Figure 7.
Mucin type O- glycosylation sites in EchAMP protein.
NetOGlyc 3.1 server predicted the presence of six sites for mucin type O-glycosylation in EchAMP protein. The amino acid positions are indicated on the X-axis. The O-glycosylation potential is indicated on the Y-axis.
Figure 8.
Identification of EchAMP gene in platypus genome.
(A) Schematic representation of BLAST analysis of echidna EchAMP sequence against platypus genome. The sequences on platypus supercontig that showed homology with the echidna EchAMP were intervened with non-matching sequences and corresponded to exon and intron sequences respectively. The predicted exon/intron junctions in the platypus genome were in consensus with eukaryotic splice junctions. The last coding exon of the echidna EchAMP sequence did not show any homology to the platypus genome. The platypus orthologue of EchAMP was designated as PlatAMP. (B) Alignment of Genscan predicted partial PlatAMP peptide sequence with EchAMP protein sequence. The PlatAMP peptide shared 94% identity with the EchAMP protein. The underlined amino acids in the PlatAMP indicate the sites of potential mucin-type GalNAc O-glycosylation. Identical amino acid (*); conserved amino acid (:); semi-conserved amino acid (.). Colors indicate the physicochemical properties of residues. Red: Small+Hydrophobic; Magenta: Basic; Blue: Acidic; Green: Hydroxyl+Sulfhydryl+Amine.
Figure 9.
Detection of EchAMP protein in conditioned media.
(A) The EchAMP protein present in conditioned media (CM) was detected by silver staining. No corresponding band was seen in vector conditioned or control (Ctrl) HEK293T conditioned media. The EchAMP protein was found to be higher in conditioned media collected 48 hours post transfection as compared to the one collected at 24 hours. (B) Purification of EchAMP protein using Anti-Flag M2 Affinity Gel. EchAMP protein was purified from the conditioned media collected 48 hours post transfection using an Anti- Flag M2 Affinity Gel column. Silver staining of samples from each stage of the purification procedure run on a 15% SDS- polyacrylamide gel show the presence of the purified EchAMP protein in the eluates.
Figure 10.
(A) Bacteriostatic activity using E.coli 2348/69: EchAMP showed significant inhibition of growth as compared to the empty vector (pcDNA3) P<0.05 (B) Bacteriostatic activity using Salmonella enterica 43971: EchAMP showed significant inhibition of growth as compared to the empty vector (pcDNA3) P<0.05 (C) Bacteriostatic activity using Staphylococcus aureus 29213: EchAMP showed significant inhibition of growth as compared to the empty vector (pcDNA3) P<0.05 (D) Bacteriostatic activity using Staphylococcus aureus 25923 : EchAMP showed significant inhibition of growth as compared to the empty vector (pcDNA3) P<0.05 (E) Bacteriostatic activity using Staphylococcus epidermidis : EchAMP showed highly significant inhibition of growth as compared to the empty vector (pcDNA3) P<0.05 (F) Bacteriostatic activity using Pseudomonas aeruginosa 27853: EchAMP showed highly significant inhibition of growth as compared to the empty vector (pcDNA3) P<0.05 (G) Bacteriostatic activity using Enterococcus faecalis 10100: EchAMP showed no inhibition of growth as compared to the empty vector and bacitracin P>0.05 (* Statistically significant result P<0.05). Each assay was performed in triplicate and the experiments were repeated at least thrice. Standard error bars are indicated.