Characterization of antigenic sites of human prostatic acid phosphatase

Human prostatic acid phosphatase [PAP] is antigenically uniquely different from acid phosphatases of other tissue origins. Nevertheless, a small degree of antigenic cross-reactivity between PAP and other lysosomal acid phosphatase(s) [LAP] has been suspected. In order to resolve this question, we have adopted two approaches: one involving structural studies by peptide mapping, and the other involving topological mapping through the use of uniquely defined antibodies. Purified PAP was dissociated into subunits and was further cleaved by chemical and enzymological methods. The limited digestion of PAP by submaxillary protease yielded three fragments [Sp-1, 2, and 3]. One of the fragments, Sp-3 [Mr = 11,000-12,000], was shown to regain catalytic activity after interaction with anti-PAP antibodies. This along with other data suggested that the active site is localized in the Sp-3 fragment. These submaxillary protease fragments were also used in the antigenic studies. For the detailed antigenic mapping studies, we prepared 12 monoclonal anti-PAP antibodies. These monoclonal anti-PAP antibodies exhibited a remarkably specific binding to PAP, particularly to the Sp-1 fragment, without binding to other acid phosphatase preparations. We also prepared lysosomal acid phosphatase [LAP] and raised anti-LAP antibodies in rabbits. The anti-LAP antibodies were fractionated into subpopulations by the preparative isoelectric focusing method. Three anti-LAP antibody subpopulations [pI 5.2, 6.9, and 7.5] exhibited specific binding to LAP. However, two anti-LAP subpopulations [pI 5.3 and and 6.8] showed binding to the Sp-3 fragment, an active site fragment of PAP. Thus, the PAP molecule seems to consist of three domains, namely, Sp-1, Sp-3, and Sp-2. Sp-3, which is the active site domain, is an antigenically cross-reactive region. The Sp-1 domain represents an antigenically unique region of PAP, whereas none of the antibodies studied thus far bind to the Sp-2 fragment.