A functional polymorphism influencing the promoter activity of alpaca α-lactalbumin gene (LALBA)

Alpha-lactalbumin (α-La), encoded by LALBA gene, is a Ca2+ binding whey-protein whose key function is to facilitate lactose synthesis by the galactosyltransferase component, serving as a regulatory subunit. Other biological functions have been demonstrated including immune modulation, cell growth regulation, antimicrobial activity, etc. Gene promoters have transcription factor (TF) binding sites necessary for gene expression regulation. Mutations in the promoters may modify the transcription rates or the mRNA stability, thus affecting the protein yield. This study aims to sequence the LALBA promoter in alpacas, identify putative TFs and detect genetic diversity affecting gene expression. A DNA fragment (800 bp) spanning the gene promoter until the exon 1 was amplified and sequenced for 20 alpacas. Multiple alignments and SNP discovery were accomplished by DNAsis software, whereas Transfact 7.0 was used for the TF sites search. Three independent gene reporter assays were achieved by pGL3 specific contructs to test luciferase expression in HEK 293T cells. Data elaboration was performed using JASP software (p < 0.05, students’s t-test). TF binding sites analysis evidenced 16 putative consensus sequences, including 3 C/EBPα, 3 Sp1, one NF-1, etc. Seven polymorphic sites were found. Taking as reference the first nucleotide of the exon 1, one SNP (g.15C > G) was found in the signal peptide, but it is a silent mutation. The other 6 SNPs were detected in the promoter (g.-553A > G, g.-428C > T, g.-308C > G, g.-236A > T, g.-73C > G, g.-51A > G). The SNP g.-553A > G creates a putative binding site of the TF Sp1. This motif is a well-known enhancer element for the basal expression of many genes, including milk proteins. To assess the SNP effect on the LALBA promoter, we amplified and cloned a DNA region of 178bp in the pGL3-basic vector from four homozygous individuals (two g.-553AA and two g.-553GG). The two different constructs (g.553A and g.-553G), with the pGL3 vector as a control were used to transiently transfect HEK293T cells. After 48 h, the reporter activity of the variants was measured using the luciferase assay system. The G variant of this SNP enhances the promoter activity of the alpaca LALBA (p < 0.01). Therefore, we suppose an effective role of this binding site in the expression of the α-La in alpaca milk that, consequently, may affect the functional roles of the protein.