Pastuerella haemolytica glycoprotease gene and the purified enzy

Details Pastuerella haemolytica glycoprotease gene and the purified enzy Pastuerella haemolytica glycoprotease gene and the purified enzy

1993-08-12

1996-08-06

Wax, Robert A.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

4352402, 4352523, 4353201, 536 232, 536 2432, C12N 121, C12N 952, C12N 1557, C12P 2100

Patent

active

055433120

DESCRIPTION:

BRIEF SUMMARY
This invention relates to the cloning, sequencing and expression of DNA of a gene for Pasteurella haemolytica glycoprotease. The genetic information and expression products may be used in a variety of types of assays. The purified glycoprotease enzyme may be use in a variety of chemical and biochemical modifications of glycoproteins.
P. haemolytica is the principal microorganism associated with bovine pneumonic pasteurellosis, a major cause of sickness and death in feedlot cattle in North America. Martin et al. 1980. "Factors associated with mortality in feedlot cattle: The Bruce County beef cattle project." Can.J.Comp.Med.; Yates, W. D. G. 1982. "A review of infectious bovine rhinotricheitis, shipping fever pneumonia and viral-bacterial synergism in respiratory disease of cattle." Can.J.Comp.Med. P. haemolytica has been divided into sixteen serotypes base on soluble or extractable surface antigens. Biberstein, E. L. 1978. "Biotyping and serotyping of Pasteurella haemolytica." Methods Microbiol. Among the sixteen serotypes, serotype A1 is the predominant microorganism isolated from pneumonic lungs. Smith, P. C. 1983. "Prevalence of Pasteurella haemolytica in transported calves." Am. J. Vet. Res.; Yates, W. D. G. 1982. "A review of infectious bovine rhinotricheitis, shipping fever pneumonia and vital-bacterial synergism in respiratory disease of cattle." Can. J. Comp. Med. P. haemolytica A1 produces a number of antigens which are secreted into the culture supernatant during its growth. These antigens include a heat-labile cytotoxin specific for ruminant leukocytes, Shewen et al. 1988. "Vaccination of calves with leukotoxic culture supernatant form Pasteurella haemolytica." Can. J. Vet. Med., a serotype-specific outer-membrane protein, Gonzalez et al. 1986. "Cloning of Serotype-Specific Antigen form Pasteurella haemolytica A1." Infect. Immun., a glycoprotease specific for sialoglycoproteins, Otulakowski et al. 1983. "Proteolysis of Sialoglycoprotein by Pasteurella haemolytica Cytotoxic Culture Supernatant." Infect. Immun. and neuraminidase Frank, G. H. 198. "Neuraminidase Activity of Pasteurella haemolytica Isolates." Infect. Immun. Vaccination of calves with bacterial-free culture supernatant form logarithmic phase cultures induces resistance to experimental challenge. and a vaccine based on the culture supernatant has been developed (Presponse.TM.) Shewen et al. 1988. "Efficacy testing a Pasteurella haemolytica extract vaccine." Vet.Med.; Shewen et al. 1988. "Vaccination of calves with leucotoxic culture supernatant from Pasteurella haemolytica." Can.J.Vet.Med.
The glycoprotease of P. haemolytica A1 is highly specific for O-glycosylated glycoproteins, so that proteins which lack extensive O-sialoglycopeptides residues are not cleaved by the glycoprotease. The best characterized glycoprotein substrate for the glycoprotease is glycophorin A from human erythrocytes. We have found that cleavage by the enzyme occurs either in situ on the surface of erythrocyte plasma membrane, or when the substrate glycoprotein is in solution. This enzyme is a neutral metallo-protease and is non-toxic to cultured mammalian cells including bovine pulmonary macrophages, bovine endothelial cells and erythrocytes Otulakowski et al. 1983. "Proteolysis of Sialoglycoprotein by Pasteurella haemolytica Cytotoxic Culture Supernatant." Infect. Immun. The role of the glycoprotease in pathogenesis and in the induction of an immune response is unknown. A homogeneous enzyme preparation for the glycoprotease is difficult to isolate by conventional biochemical techniques.
We have discovered and cloned a gene for the P. haemolytica glycoprotease. We have cloned, sequenced and expressed the gene. The expression product of the gene can be isolated to provide the glycoprotease in homogeneous form. The enzyme has restricted substrate specificity, which has a variety of chemical and biochemical uses in modifying glycoproteins. Such uses include, characterization of glycoproteins through specific sites of cleavage by the glycoprotease, the glycoprotea

REFERENCES:
patent: 4503035 (1985-03-01), Pestka
patent: 4906571 (1990-03-01), Mellors et al.
patent: 5082785 (1992-01-01), Manning
Proteolysis of Sialoglycoprotein by Pasteurella haemolytica Cytotoxic Culture Supernatant, Otulakowski et al, Infection and Immunity, vol. 42 (1983) pp. 64-70.
Neuraminidase Activity of Pasteurella haemolytica Isolates, Frank et al, Infection and Immunity, vol. 32 (1987) pp. 1119-1122.
Distribution of glycoprotease activity and the glycoprotease gene among serotypes of Pasteurella haemolytica, Abdullah, et al, Biochem. Soc. Transactions, vol. 18 (1990) pp. 901-903.
Vaccination of Calves with Leukotoxic Culture Supernatant from Pasteurella haemolytica, Shewen et al, Can. J. Veterinary Res., vol. 52 (1988) pp. 30-35.
Efficacy testing a Pasteurella haemolytica extract vaccine, Shewen, et al., Veterinary Med (Oct. 1988) pp. 1078-1083.
Cloning, Nucleotide Sequence, and Expression of the Pasteurella haemolytica A1 Glycoprotease Gene, Abdullah, et al., J. Bacteriol., vol. 173 (1991) pp. 5597-5603.
Cloning and Expression of the Leukotoxin Gene of Pasteurella haemolytica A1 in Excherichia coli K-12, Lo, et al., Infection and Immunity, vol. 50 (1985) pp. 667-671.
Wozney et al (1990) Meth Enzymol. 182, 738-749.
Sofer et al. (1983) Bio Techniques Nov./Dec., 198-203.

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