Triton X-114 phase fractionation of an integral membrane surface protein mediating monoclonal antibody killing of Mycoplasma hyorhinis

Abstract

A previously defined immunoglobulin M(K) monoclonal antibody reacting with a surface epitope of Mycoplasma hyorhinis is shown in this report to mediate specific, complement-dependent mycoplasmacidal activity. Immunoblot analysis of mycoplasma components and their tryptic cleavage products showed that the epitope recognized was present on a protein with an apparent molecular weight of 23,000 (p23) and on a limit tryptic fragment of this protein with an apparent molecular weight of 18,000 (p18). Both p23 and p18 are shown by Triton X-114 phase fractionation to partition efficiently into the hydrophobic detergent phase. Other antigens bearing epitopes not expressed at the cell surface were present among the numerous hydrophilic proteins found in the aqueous phase. The external orientation and membrane association of the p23 antigen were further established by demonstrating that trypsin treatment of intact mycoplasmas generated the antigenic p18 fragment, which remained tightly associated with the organism. These results localize an epitope responsible for antibody-mediated mycoplasma killing onto a specific, surface-exposed region of an integral membrane protein of this organism. Since the monoclonal antibody used in this study does not bind to the surface of all strains of M. hyorhinis, the epitope identified also defines a structural marker of antigenic surface variation within this species, a feature previously observed during serological classification of the organism. Analysis of the antigenic and structural features of the p23 surface antigen may therefore be useful in establishing mechanisms of surface antigen variation among integral membrane proteins of mycoplasmas that could dictate important antigenic characteristics recognized during chronic disease caused by these agents.

Characterization of membrane surface proteins of Mycoplasma agalactiae during natural infection

“…Extraction of integral membrane proteins was carried out through TX‐114 phase partitioning as described by Bordier [18] and adapted with some modifications [19]. Extracted proteins were processed for SDS‐PAGE and immunoblotting.…”

Comparison of Mycoplasma agalactiae isolates by pulsed field gel electrophoresis, SDS-PAGE and immunoblotting

“…Extraction of integral membrane proteins was subjected to TX-114 phase partitioning as described by Bordier [19] and adapted with some modifications [20]. Briefly, pellets of A4.…”

Comparison of Mycoplasma bovis Strains Based on SDS‐PAGE and Immunoblot Protein Patterns

“…After three further washes, the blots were incubated with peroxidase-conjugated Protein A (Amersham Buchler, Braunschweig, Germany) as secondary antiserum at room temperature for 2 h. After the final washes, reactive protein bands were visualized according to the procedure of TAKETA (1987) using nitro blue tetrazolium (Serva) as dye.Triton X-114 phase partitioning Viable M . bovis cells were subjected to Triton X-114 (Serva) phase partitioning as described byRIETHMANN et al (1987).…”