Cloning, nucleotide sequence, and expression of the Pasteurella haemolytica A1 glycoprotease gene

Abstract

Pasteurella haemolytica serotype Al secretes a glycoprotease which is specific for 0-sialoglycoproteins such as glycophorin A. The gene encoding the glycoprotease enzyme has been cloned in the recombinant plasmid pPHl, and its nucleotide sequence has been determined. The gene (designated gcp) codes for a protein of 35.2 kDa, and an active enzyme protein of this molecular mass can be observed in Escherichia coli clones carrying pPHl. In vivo labeling of plasmid-encoded proteins in E. coli maxicells demonstrated the expression of a 35-kDa protein from pPHl. The amino-terminal sequence of the heterologously expressed protein corresponds to that predicted from the nucleotide sequence. The glycoprotease is a neutral metalloprotease, and the predicted amino acid sequence of the glycoprotease contains a putative zinc-binding site. The gene shows no significant homology with the genes for other proteases of procaryotic or eucaryotic origin. However, there is substantial homology between gcp and an E. coli gene, orJX, whose product is believed to function in the regulation of macromolecule biosynthesis.

Identification and Characterization of CAMP Cohemolysin as a Potential Virulence Factor of Riemerella anatipestifer

“…Analysis of the hydropathicity of the sequence by the TMpred program (http://www.ch .embnet.org) showed it to be predominantly hydrophilic, suggesting that Cam is a cytoplasmic or peripheral protein. An examination of the amino terminus showed no signal sequence, indicating that the mechanism of transport across the double membrane of bacteria for secretion was unconventional, similar to that of sialoglycoproteases found in other organisms (1,30).…”

Effects on Transcription of Mutations in ygjD , yeaZ , and yjeE Genes, Which Are Involved in a Universal tRNA Modification in Escherichia coli

“…The functions of YgjD orthologs have been investigated in many microbial species. In Mannheimia (formerly Pasteurella) haemolytica, the YgjD ortholog was initially identified as an Osialoglycoprotein endopeptidase (Gcp), suggesting that YgjD family members function as endopeptidases (1). However, the yeast YgjD ortholog, kinase-associated endopeptidase 1 (Kae1), has been reported to be a component of a complex known as KEOPS (kinase, endopeptidase, and other small proteins), which is involved in the regulation of telomere length (3).…”

Mannheimia haemolytica

“…Glycoprotease obtained from the culture supernatant of M haemolytica can selectively hydrolyze IgG1, thereby reducing opsonization-induced phagocytosis and bacterial killing; it can also selectively degrade host mucosal sialoglycoproteins. 1,72,94 Shewen et al 106 used this glycoprotease to demonstrate that vaccination of calves with the recombinant glycoprotease alone and in combination with M haemolytica culture supernatant vaccine enhances protection against experimental disease. The pathogenic function of this enzyme is unknown; however, adherence of bacteria to host epithelial cells and aggregation of platelets in the alveoli have been demonstrated, whereas glycoprotease activity can be potentiated by coincubation with the LKT.…”