Proteolysis of sialoglycoprotein by Pasteurella haemolytica cytotoxic culture supernatant

Abstract

Proteolytic enzyme activity releasing sialo glycopeptides from 3H-labeled human erythrocyte ghosts was detected in cytotoxic (leukotoxic) culture supernatants from 9 of 12 Pasteurella haemolytica serotypes. Microcrystalline cellulose thin-layer chromatograms of radioactive water-soluble products showed the following two radioactive peaks: a high-mobility minor peak (Rf, 0.54 to 0.74), identified as sialic acid, and a low-mobility major peak (Rf, 0.18 to 0.21), partially characterized as a trichloroacetic acid-soluble, sialic acid-rich fragment with a molecular weight of greater than 3,500, not extractable by chloroform. The sialic acid content of this fragment after treatment with Clostridium perfringens neuraminidase was estimated to be 7.2 X 10(-2) mumol mg-1. The presence of neuraminidase as a separate activity in some culture supernatants was confirmed. It is considered to be responsible for the observed release of free sialic acid. Preliminary studies with the crude enzyme showed that it has a broad pH optimum around pH 7.0 and that activity is not affected by inhibitors of trypsin, chymotrypsin, thermolysin, thio and serine enzymes, nor by an inhibitor of neuraminidase, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. Activity was, however, inhibited by o-phenanthroline at a high concentration after prolonged treatment. The enzyme hydrolyzed glycophorin at a rate four times higher than the rate for casein. Free glycophorin inhibited the enzyme-induced release of radioactive products from 3H-labeled ghosts. It is speculated that the novel enzyme is a neutral protease, probably metal-dependent, with specificity for sialoglycopeptides. The possible relationship of this protease to the previously reported host species-specific leukotoxicity of P. haemolytica and its potential role in virulence is discussed.

Genomic Distribution of a Serotype 1‐Specific Antigen‐Coding DNA Fragment of Pasteurella Haemolytica

“…However, subsequent studies as well as related studies in other bacteria have suggested that outer membrane protein antigens may play a significant role in serotype-associated immunogenicity and host-resistance to virulent challenge (8, 13). A number of soluble and cell associated antigens are potential virulence determinants of P. huemolyticu Al, but most are also found in other serotypes that are not pathogenic to cattle (18,24,29). Relative to this, it has been observed that vaccination with live logarithmic-phase P. huernolyticu A1 organisms or its culture supernatant induced protection against the disease (30,34,39), whereas vaccination with a formalinized bacterin had deleterious effects (27,28).…”

Molecular studies of Ssa1, a serotype-specific antigen of Pasteurella haemolytica A1

“…Vaccination of calves with live P. haemolytica serotype 1 organisms or with bacteria-free culture supernatants from logarithmic-phase cultures induces resistance to experimental challenge (33). These culture supernatants contain a number of soluble antigens, including a leukotoxin specific for ruminant cells (19,32), glycoprotease (1,2,28), neuraminidase (12,28), and other soluble materials shed during bacterial growth. Vaccination of calves with the culture supernatant stimulates the production of leukotoxin-neutralizing antibodies as well as serotype-specific agglutinating antibodies (33), both of which are apparently necessary for protection.…”

Critical assessment of the efficiency of CD34 and CD133 antibodies for enrichment of rabbit hematopoietic stem cells

“…After magnetic selection of CD34 1 cells/magnetic beads, the cells were detached from the beads by incubation with the Pasteurella haemolytica glycoprotease, which selectively cleaves glycoproteins rich in O-linked glycans. 71 The purity of the released cells was assessed by immunofluorescence techniques using either of the class III CD34 antibodies, TUK3 or 115.2, or the nonworkshop class III antibody 8G12, 72 whose epitopes are not removed by the glycoprotease. 54 Nowadays, the enrichment of CD34 1 cells applying magnetic sorting and using CD34 MicroBeads Kit (clone QBEnd10; Miltenyi Biotec) is well established and commercially available.…”