Molecular size variations in an immunoprotective protein complex among isolates of Anaplasma marginale

Abstract

A major surface protein complex from the Florida isolate of Anaplasma marginale has been previously shown to induce protection in immunized cattle and has been proposed as the basis of a subunit vaccine against anaplasmosis. This complex in the Florida isolate is composed of two noncovalently associated polypeptides with molecular masses of 105 and 100 kilodaltons (kDa). The analogous protein complex from four geographically different isolates of A. marginale was immunoprecipitated and compared with the protein complex of the Florida isolate. The polypeptides of the complex varied in apparent molecular mass among the isolates. By using antibodies recognizing epitopes on each polypeptide of the Florida isolate, the antigenic identity of the polypeptides in the analogous complexes was determined. The polypeptides recognized by the neutralizing monoclonal antibody 22B1, which recognizes a 105-kDa polypeptide in the Florida isolate, ranged from 70 to 100 kDa in the other isolates. Those polypeptides recognized by rabbit antiserum R911, which recognizes a 100-kDa polypeptide in the Florida isolate, ranged from 97 to 100 kDa. The surface-exposed peptides in the complexes were compared by limited enzymatic digestion to assess structural homology among isolates. Despite the marked variations in molecular weight, there were conserved peptides between the 22B,-reactive polypeptides and between the R911-reactive peptides. Determination of the role of the conserved peptides in inducing immunity will be critical in the application of these polypeptides as the basis of a subunit vaccine for bovine anaplasmosis.

The CD4+ T cell immunodominant Anaplasma marginale major surface protein 2 stimulates γδ T cell clones that express unique T cell receptors

“…To prepare antigen for in vitro assays, the sonicate was diluted in phosphate-buffered saline (PBS; pH 7.4) containing 25 g/ml of the protease inhibitors, antipain, and E-64 (Boehringer Mannheim, Indianapolis, IN) and 300 g/ml phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis, MO). Native MSP2 was purified essentially as described [20,21]. Briefly, pelleted A. marginale were resuspended in electrophoresis sample buffer and separated on multiple preparative sodium dodecyl sulfate gradient (10 -20%) polyacrylamide gels.…”

Intermolecular relationships of major surface proteins of Anaplasma marginale

“…MSP-5 was originally identified in outer membrane preparations of the Norton Zimbabwe strain and subsequently shown to be conserved in other strains, including Florida (31). MSP-1 is a complex of two polypeptides with molecular masses of 105 and 100 kDa (4), designated MSP-la and MSP-lb, respectively (18). MSP-la exhibits marked size polymorphism among different strains of A. marginale (18) and is encoded by a single-copy gene (2).…”

Anaplasmosis Control: Past, Present, and Future

“…MSP 1 through 5 have been identified and information on the gene sequences, recombinant protein analogs, monospecific and monoclonal antibodies, isolate variability, and potential value in diagnostic assays and vaccines are available. [28][29][30][31][32][33]19,[34][35][36][37][38][39][40][41][42] MSP1 has been shown to confer partial protection in immunized cattle against A. marginale challenge, and to contain an epitope recognized by monoclonal antibodies capable of in vitro neutralization of isolated rickettsia. 38 MSP1a varies in size among different geographic isolates, but the neutralization-sensitive epitope is conserved.…”

“…38 MSP1a varies in size among different geographic isolates, but the neutralization-sensitive epitope is conserved. 35 Sequencing of the MSP1a gene of different isolates and characterization of this epitope using synthetic peptides demonstrated that the neutralization-sensitive epitope was located on a tandem 28 or 29 amino acid repeat of the MSP1a. 30 The number of tandem repeats varies between isolates of A. marginale .…”