Mutations in lipopolysaccharide-binding protein (LBP) gene change the susceptibility to clinical mastitis in Chinese Holstein

Mastitis is an unsolved human challenge all dairy farms facing with, which leads to immeasurable economic loss to the farmers. LBP gene plays a vital role in the innate immune recognition of Gram-negative bacterium that is a major cause of bovine clinical mastitis, but little is known about LBP mutations and their effects on cows' susceptibility to clinical mastitis. In this study, PCR-SSCP method was adopted to analyze SNPs of LBP gene in Chinese Holstein for the first time. 17 SNPs were found in the promoter core region, exon1, exon2, exon3, exon4 and exon8. The mutation g.-81C -> T in promoter leads to an AP-2 binding site lost. Two mutations, g.11T -> C (4 Leu -> Ser) and g.68G -> C (23Gly -> Ala) in signal peptide brought about molecular secondary structural change, meanwhile, g.11T -> C made a Big-1 domain lost, and there was an N-myristoylation site at the g.68G/C locus. The three mutations above were in complete linkage disequilibrium in allele A. In mature LBP protein, five mutations were found: g.3034G -> A(36Asp -> Asn), g.3040A -> G(38Asn -> Asp), g.3056T -> C(43Ile -> Thr) in allele D; g.4619G -> A(67Ala -> Thr) in allele F; 19975G -> A (282Val -> Met) in allele J. And SNPs in allele D and F were in complete linkage disequilibrium, also in which 38Asn -> Asp and 67Ala -> Thr influenced the protein secondary structure. Prediction of the 3-D structure shows mutations 36Asp -> Asn, 38Asn -> Asp and 43 Ile -> Thr were on the concave surface of LBP protein at barrel-N, 67Ala -> Thr was in the apolar pocket at barrel-N. Motif analysis shows 36Asp -> Asn causes loss of a CK2 phosphorylation site, 67 Ala -> Thr forms a new PKC phosphorylation site. And 43Ile -> Thr, 67Ala -> Thr made hydrophobic amino acids to be hydrophilic amino acids. Interestingly, the morbidity of AB (mixed type g.-81C/T, g.11T/C, g.68G/C), CD (mixed type g.3034G/A, g.3040A/G, g.3056T/C) and EF (mixed type g.4619G/A) genotype cows are significant higher than others in this study (P < 0.01), and it can be deduced that the SNPs in these 3 genotypes might affect the secretion of LBP protein and regulate the binding ability of LBP protein to LPS. Taken together, it is revealed that these SNPs may hold the secret of susceptibility to clinical mastitis in Chinese Holstein.