Immunization with Anaplasma marginale membranes containing major surface proteins (MSPs) induces protective immunity against clinical disease (N. Tebele, T. C. McGuire, and G. H. Palmer, Infect. Immun. 59:3199-3204, 1991). For use in design of a recombinant antigen subunit vaccine for A. marginak, intermolecular relationships of known A. marginale MSPs were analyzed. Under nonreducing conditions, MSP-2 and MSP-5 occur as multimers. A large (>300-kDa-molecular-mass), nonreduced protein complex

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... ntained MSP-la linked by disulfide bonds to MSP-lb and by noncovalent bonds to MSP-5. MSP-2 was also noncovalently bound to this complex. The nearest neighbor membrane proteins were identified by cross-linking reactions followed by immunoblotting with anti-MSP antibodies. A cross-linked aggregate retained in the stacking gel contained MSP-la, MSP-lb, MSP-2, MSP-3, MSP-4, and MSP-5. Collectively, the data indicate that MSP-2 and MSP-5 occur as monomers and disulfide-bonded multimers. The MSP-1 complex occurs as both disulfide-bonded and noncovalently associated MSP-la and MSP-lb, and MSP-2 and MSP-5 are noncovalently associated with MSP-1. Also, MSP-1, MSP-2, MSP-3, and MSP-4 are nearest neighbors, and MSP-5 is noncovalently associated with this cross-linked complex.