title: Intact signal peptide of CD18, the beta-subunit of beta2-integrins, renders ruminants susceptible to Mannheimia haemolytica leukotoxin.
abstract: Signal peptides of membrane proteins are cleaved by endoplasmic reticulum-resident signal peptidase, and thus, are not present on mature membrane proteins. Here, we report that, contrary to the paradigm, the signal peptide of ruminant CD18, the beta-subunit of beta(2)-integrins, is not cleaved. Intriguingly, the intact signal peptide of CD18 is responsible for the susceptibility of ruminant leukocytes to Mannheimia (Pasteurella) haemolytica leukotoxin (Lkt). Inhibition of Lkt-induced cytolysis of ruminant leukocytes by CD18 peptide analogs revealed that the Lkt-binding site is formed by amino acids 5-17 of CD18, which, surprisingly, comprise most of the signal sequence. Flow cytometric analysis of ruminant leukocytes indicated the presence of the signal peptide on mature CD18 molecules expressed on the cell surface. Analysis of transfectants expressing CD18 containing the FLAG epitope at the putative cleavage site confirmed that the signal peptide of bovine CD18 is not cleaved. Analysis of the signal sequence of CD18 of eight ruminants and five nonruminants revealed that the signal sequence of CD18 of ruminants contains "cleavage-inhibiting" Q, whereas that of nonruminants contains "cleavage-conducive" G at position -5 relative to the cleavage site. Site-directed mutagenesis of Q to G at position -5 of the signal peptide of bovine CD18 resulted in the cleavage of the signal peptide and abrogation of cytolysis of transfectants expressing bovine CD18 carrying the Q(-5)G mutation. We propose that engineering cattle and other ruminants to contain this mutation would provide a novel technology to render them less susceptible to pneumonic pasteurellosis and concomitant economic losses.
authors.affiliation: Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USA.
authors.forename: Sudarvili
authors.initials: S
authors.lastname: Shanthalingam
authors.forename: Subramaniam
authors.initials: S
authors.lastname: Srikumaran
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D018821:CD18 Antigens / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D016978:Mannheimia haemolytica / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics*; D012418:Ruminants / Q000378:metabolism* / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D013045:Species Specificity
chemical_list: D018821:CD18 Antigens; D005098:Exotoxins; D021382:Protein Sorting Signals; C018579:leukotoxin
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