Mutations In Functional Domain Of Lipopolysacchairde-binding Portein (LBP) Significantly Change The Susceptibility To Clinical Mastitis In Holstein

Clinical mastitis is an unsolved human challenge all dairy farms facing with,which leads to immeasurable economic loss to the farmers. Gram-negativebacterium is an important cause for the clinical cases of bovine mastitis, and whichoften result in peracute mastitis or even life-threatening. LPS is a highly conservedglycolipid component of the cell wall of all GN bacteria, which is also the keyelement that causes bovine mastitis. LBP gene plays a vital role in the innateimmune recognition of Gram-negative bacterium and locates in the upstream ofTLR signaling pathway, but little is known about LBP mutations and their effectson cows’ susceptibility to clinical mastitis.In this study, PCR-SSCP method was adopted to analyze SNPs of LBP inChinese Holstein for the first time.17SNPs were found in the promoter core region,exon1, exon2, exon3, exon4and exon8. The mutation g.-81C→T in promoter leadsto an AP-2binding site lost. Two mutations, g.11T→C (4Leu→Ser) and g.68G→C (23Gly→Ala) in signal peptide in exon1brought about molecular secondarystructural change, meanwhile, g.11T→C made a Big-1domain lost, and there wasan N-myristoylation site at the g.68G/C locus. The three mutations above were incomplete linkage disequilibrium in allele A. In mature LBP protein, five mutationswere found: g.3034G→A(36Asp→Asn), g.3040A→G(38Asn→Asp), g.3056T→C(43Ile→Thr) in allele D in exon2; g.4619G→A(67Ala→Thr) in allele F in exon3;19975G→A (282Val→Met) in allele J in exon8. And SNPs in allele D and F werein complete linkage disequilibrium, also in which38Asn→Asp and67Ala→Thrinfluenced the protein secondary structure. Prediction of the3-D structure showsmutations36Asp→Asn,38Asn→Asp and43Ile→Thr were on the concavesurface of LBP protein at barrel-N,67Ala→Thr was in the apolar pocket atbarrel-N，282Val→Met was at the distal tip of barrel-C. Motif analysis shows36Asp→Asn causes loss of a CK2phosphorylation site,67Ala→Thr forms a new PKC phosphorylation site. And43Ile→Thr,67Ala→Thr made hydrophobicamino 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), andit can be deduced that the SNPs in these3genotypes might affect the secretion ofLBP 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 clinicalmastitis in Holstein.