期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：46
页码：13186-13190
DOI：10.1073/pnas.1613428113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceContrary to the paradigm, the signal peptide of CD18, an adhesive protein expressed on the surface of leukocytes of cattle and other ruminants, is not cleaved. Intriguingly, leukotoxin produced by Mannheimia haemolytica binds to the intact signal peptide and causes lysis of ruminant leukocytes, resulting in acute pneumonia. In this proof-of-principle study, we used precise gene editing to induce a single amino acid substitution in CD18, which resulted in the cleavage of the signal peptide and abrogation of leukotoxin-induced cytolysis of leukocytes of the gene-edited bovine fetus. This report demonstrates the feasibility of developing lines of cattle genetically resistant to M. haemolytica-caused pneumonia, which will have significant impact on the sustainability of food-animal production in the United States and elsewhere. Signal peptides of membrane proteins are cleaved by signal peptidase once the nascent proteins reach the endoplasmic reticulum. Previously, we reported that, contrary to the paradigm, the signal peptide of ruminant CD18, the {beta} subunit of {beta}2 integrins, is not cleaved and hence remains intact on mature CD18 molecules expressed on the surface of ruminant leukocytes. Leukotoxin secreted by Mannheimia (Pasteurella) haemolytica binds to the intact signal peptide and causes cytolysis of ruminant leukocytes, resulting in acute inflammation and lung tissue damage. We also demonstrated that site-directed mutagenesis leading to substitution of cleavage-inhibiting glutamine (Q), at amino acid position 5 upstream of the signal peptide cleavage site, with cleavage-inducing glycine (G) results in the cleavage of the signal peptide and abrogation of leukotoxin-induced cytolysis of target cells. In this proof-of-principle study, we used precise gene editing to induce Q(-5)G substitution in both alleles of CD18 in bovine fetal fibroblast cells. The gene-edited fibroblasts were used for somatic nuclear transfer and cloning to produce a bovine fetus homozygous for the Q(-5)G substitution. The leukocyte population of this engineered ruminant expressed CD18 without the signal peptide. More importantly, these leukocytes were absolutely resistant to leukotoxin-induced cytolysis. This report demonstrates the feasibility of developing lines of cattle genetically resistant to M. haemolytica-caused pneumonia, which inflicts an economic loss of over $1 billion to the US cattle industry alone.
